mDia1/3-Rho and Rac1 signaling facilitates presynaptic endocytosis.(A) Schematic of the interplay between RhoA and Rac1 signaling via GTPase regulatory proteins (e.g. GTPase activating proteins (GAPs) among others) common for RhoA and Rac1. (B) Analysis of Rac1 activity by Rac1-GTP pulldown (PD) from whole-cell lysates (input) of mouse hippocampal neurons expressing shCTR or shmDia1 +3 utilizing immobilized PAK as a bait. Samples were analyzed by immunoblotting for mDia1, mDia3, Rac1, and Tubulin using specific antibodies. Input, 10% of material used for the pulldown. The contrast of pulldown and input blots was seperately adjusted for visualization purposes. (C) Densitometric quantification of Rac1-GTP normalized to total Rac1 levels (input) in lysates from neurons transduced with shCTR or shmDia1 +3 (2.2±0.2; p<0.05, one sample t-test) from immunoblots exemplified in (B). Values for shCTR were set to 1. Data are expressed as mean ± SEM from N=3 independent experiments. (D) Representative three-channel time-gated stimulated emission depletion (STED) image of synapses from hippocampal cultures, fixed and immunostained for Bassoon (magenta), Rac1 (cyan), and Homer1 (green). Scale bar, 250 nm. (E) Averaged normalized line profiles for synaptic distribution of Rac1 and Homer1 relative to Bassoon (Maximum set to 0 nm). Data represent mean ± SEM. N=3 independent experiments from n=79 synapses. (F) Averaged normalized vGAT-CypHer fluorescence traces for neurons transduced with shCTR or shmDia1 +3 in response to 200 AP (40 Hz, 5 s) stimulation. Cells were acutely treated with 0.1% DMSO or 10 µM Rac1 Inhibitor (EHT 1864) in the imaging buffer. Data shown represent the mean ± SEM. N=8 independent experiments from nshCTR + DMSO = 46 videos, nshmDia1+3 + DMSO = 45 videos, nshCTR + EHT 1864 = 42 videos, nshmDia1+3 + EHT 1864 = 43 videos. (G) Endocytic decay constants of vGAT-CypHer traces in F: τshCTR + DMSO = 14.7±0.9 s, τshmDia1+3 + DMSO=27.5±2.3 s, τshCTR + EHT 1864 = 30.3±6.7 s, τshmDia1+3 + EHT 1864 = 41.0±4.3 s; pshCTR + DMSO vs shmDia1+3 + DMSO<0.05, pshCTR + DMSO vs shmDia1+3 + EHT 1864 < 0.0001, Kruskal-Wallis test with Dunn’s post-test. Data represent mean ± SEM. (H) Endocytic decay constants of Synaptophysin-pHluorin traces (Figure 6—figure supplement 1E) of neurons transduced with shCTR (τshCTR = 12.0±0.7 s) or shmDia1 +3 (τshmDia1+3 = 22.7±2.0 s) and transfected with constitutively active Rac1 (Rac1-CA; Q61L variant; τshCTR + Rac1-CA=13.6±1.2 s, τshmDia1+3 + Rac1-CA=13.3±1.4 s) or dominant negative Rac1 (Rac1-DN; T17N variant; τshCTR + Rac1-DN = 27.8±1.3 s, τshmDia1+3 + Rac1-DN = 33.4±1.6 s) in response to 200 AP (40 Hz, 5 s) stimulation (pshCTR vs shmDia1+3 < 0.01; pshCTR vs shCTR + Rac1-DN<0.0001, pshCTR vs shmDia1+3 + Rac1-DN<0.01, pshmDia1+3 vs shmDia1+3 + Rac1-DN<0.01, one-way ANOVA with Tukey’s post-test). Data are expressed as mean ± SEM. N=3 independent experiments from nshCTR = 12 videos, nshmDia1+3 = 23 videos; nshCTR + Rac1-CA=10 videos, nshmDia1+3 + Rac1-CA=14 videos, nshCTR + Rac1-DN = 9 videos; nshmDia1+3 + Rac1-DN = 13 videos.Figure 6—source data 1.Original scans for the anti-mDia3, anti-Tubulin, and anti-Rac1 immunoblots from Figure 6B.Figure 6—source data 2.Original scan for the anti-mDia1 immunoblot from Figure 6B.Figure 6—source data 3.Original scans for immunoblots from Figure 6B with highlighted bands and sample labels.Figure 6—source data 4.Numerical source data for Figure 6C, E, F, G and H.Figure 6—source data 5.Original scans for the anti-Rac1 immunoblots used for analysis are shown in Figure 6C.Figure 6—source data 6.Original scans for immunoblots used for analysis are shown in Figure 6C with highlighted bands and sample labels.Original scans for the anti-mDia3, anti-Tubulin, and anti-Rac1 immunoblots from Figure 6B.Original scan for the anti-mDia1 immunoblot from Figure 6B.Original scans for immunoblots from Figure 6B with highlighted bands and sample labels.Numerical source data for Figure 6C, E, F, G and H.Original scans for the anti-Rac1 immunoblots used for analysis are shown in Figure 6C.Original scans for immunoblots used for analysis are shown in Figure 6C with highlighted bands and sample labels.Cooperative action of mDia1/3 and Rac1 pathways in presynaptic endocytosis.(A) Analysis of Rac1 activity by Rac1-GTP pulldown (PD) from whole-cell lysates (input) of mouse hippocampal cultures upon inhibition of Rho activity utilizing immobilized PAK as bait. Cells were treated with 0.1% DMSO or 10 µM Rho Inhibitor (Rhosin) for 2 hr before harvest. Samples were analyzed by immunoblotting for Rac1 and Tubulin using specific antibodies. Input, 10% of material used for the pulldown. The contrast of pulldown and input blots was seperately adjusted for visualization purposes. (B) Representative three-channel time-gated STED images of synapses from hippocampal cultures treated with 0.1% DMSO or 10 µM Rac1 Inhibitor (EHT 1864) for 2 hr. Cells were fixed and stained for Bassoon (magenta), F-Actin (cyan), and Homer1 (green). Scale bar, 250 nm. (C) Presynaptic F-Actin levels in synapses of neurons treated with 0.1% DMSO (100±8.5) or 10 µM Rac1 Inhibitor (EHT 1864; 58.6±6.5; p<0.0001, one sample Wilcoxon test) for 2 hr. Line profiles of F-Actin overlapping with Bassoon (presynapse) distribution were integrated. Data shown are normalized to DMSO (set to 100) and expressed as mean ± SEM. nDMSO = 30, nEHT 1864 = 46 from two independent experiments. (D) Minima of background-corrected vGAT-CypHer fluorescence traces (surface normalized) for neurons treated with 0.1% DMSO (1.0±0.2 for shmDia1 +3) or 10 µM Rac1 Inhibitor (EHT 1864; 0.8±0.1 for shCTR; 0.8±0.1 for shmDia1 +3) in response to 200 AP stimulation (40 Hz, 5 s). Data represent mean ± SEM. Values were normalized to DMSO-treated shCTR (set to 1). N=8 independent experiments from nshCTR + DMSO = 46 videos, nshmDia1+3 + DMSO=45 videos, nshCTR + EHT 1864 = 42 videos, nshmDia1+3 + EHT 1864 = 43 videos. (E) Averaged normalized Synaptophysin-pHluorin fluorescence traces from stimulated (200 APs; 40 Hz, 5 s) hippocampal neurons transduced with lentiviruses encoding shCTR or shmDia1 +3 and transfected with plasmids for expression of constitutively-active Rac1 (Rac1-CA; Q61L variant) or dominant-negative Rac1 (Rac1-DN; T17N variant). Data represent mean ± SEM. N=3 independent experiments from nshCTR = 12 videos, nshmDia1+3 = 23 videos, nshCTR + Rac1-CA=10 videos, nshmDia1+3 + Rac1-CA=14 videos, nshCTR + Rac1-DN = 9 videos; nshmDia1+3 + Rac1-DN = 13 videos. The corresponding endocytic decay constants are shown in Figure 6H. (F) Maxima of background-corrected Synaptophysin-pHluorin fluorescence traces (surface normalized maximum values of traces shown in E) from stimulated (200 APs; 40 Hz, 5 s) hippocampal neurons transduced with lentiviruses encoding shCTR (Fmax/F0=1.3±0.0) or shmDia1 +3 (Fmax/F0=1.5±0.0) and transfected with plasmids encoding CA (Fmax/F0 shCTR + Rac1-CA=1.4±0.2; Fmax/F0 shmDia1+3 + Rac1-CA=1.5±0.1) or DN versions (Fmax/F0 shCTR + Rac1-DN = 1.2±0.1; Fmax/F0 shmDia1+3 + Rac1-DN = 1.3±0.1) of Rac1. Data represent mean ± SEM. (G) Densitometric quantification of Cdc42-GTP normalized to total Cdc42 levels in lysates from shmDia1 +3 transduced neurons (2.7±0.6; p<0.05, one sample t-test). Values for shCTR were set to 1. Data are expressed as mean ± SEM from N=3 independent experiments. (H) Representative three-channel time-gated stimulated emission depletion (STED) image of synapses from hippocampal mouse cultures, fixed and immunostained for Bassoon (magenta), Cdc42 (cyan), and Homer1 (green). Scale bar, 250 nm. (I) Averaged normalized line profiles for synaptic distribution of Cdc42 and Homer1 relative to Bassoon (Maximum set to 0 nm). Data are expressed as mean ± SEM (N=3; n=96 synapses). (J) Averaged normalized vesicular glutamate transporter 1 (vGAT)-CypHer fluorescence traces for neurons transduced with shCTR or shmDia1 +3 in response to 200 AP (40 Hz, 5 s) stimulation. Cells were acutely treated with 0.1% DMSO or 10 µM Cdc42 Inhibitor (ML141) in the imaging buffer. Data shown represent the mean ± SEM. N=6 independent experiments from nshCTR + DMSO = 31 videos, nshmDia1+3 + DMSO=33 videos, nshmDia1+3 + ML141=32 videos. (K) Endocytic decay constants of vGAT-CypHer traces in J: τshCTR + DMSO = 15.6±1.0 s, τshmDia1+3 + DMSO=28.0±3.1 s, τshCTR + ML141=17.6±1.6 s, τshmDia1+3 + ML141=33.1 ± 7.7 s; pshCTR + DMSO vs shmDia1+3 + DMSO<0.01, Kruskal-Wallis test with Dunn’s post-test. Data shown represent the mean ± SEM. N=6 independent experiments from nshCTR + DMSO = 31 videos, nshmDia1+3 + DMSO=33 videos, nshCTR + ML141=29 videos, nshmDia1+3 + ML141=32 videos.Figure 6—figure supplement 1—source data 1.Original scan for the anti-Rac1 immunoblots from Figure 6—figure supplement 1A.Figure 6—figure supplement 1—source data 2.Original scan for the anti-Tubulin immunoblot from Figure 6—figure supplement 1A.Figure 6—figure supplement 1—source data 3.Original scans for immunoblots in Figure 6—figure supplement 1A with highlighted bands and sample labels.Figure 6—figure supplement 1—source data 4.Numerical source data of Figure 6—figure supplement 1C, D, E, F, G, I, J, K.Figure 6—figure supplement 1—source data 5.Original scans for anti-Cdc42 immunoblots used for analysis are shown in Figure 6—figure supplement 1G.Figure 6—figure supplement 1—source data 6.Original scans for anti-Cdc42 immunoblots used for analysis are shown in Figure 6—figure supplement 1G with highlighted bands and sample labels.Original scan for the anti-Rac1 immunoblots from Figure 6—figure supplement 1A.Original scan for the anti-Tubulin immunoblot from Figure 6—figure supplement 1A.Original scans for immunoblots in Figure 6—figure supplement 1A with highlighted bands and sample labels.Numerical source data of Figure 6—figure supplement 1C, D, E, F, G, I, J, K.Original scans for anti-Cdc42 immunoblots used for analysis are shown in Figure 6—figure supplement 1G.Original scans for anti-Cdc42 immunoblots used for analysis are shown in Figure 6—figure supplement 1G with highlighted bands and sample labels. less

Cooperative action of mDia1/3 and Rac1 pathways in presynaptic endocytosis.(A) Analysis of Rac1 activity by Rac1-GTP pulldown (PD) from whole-cell lysates (input) of mouse hippocampal cultures upon inhibition of Rho activity utilizing immobilized PAK as bait. Cells were treated with 0.1% DMSO or 10 µM Rho Inhibitor (Rhosin) for 2 hr before harvest. Samples were analyzed by immunoblotting for Rac1 and Tubulin using specific antibodies. Input, 10% of material used for the pulldown. The contrast of pulldown and input blots was seperately adjusted for visualization purposes. (B) Representative three-channel time-gated STED images of synapses from hippocampal cultures treated with 0.1% DMSO or 10 µM Rac1 Inhibitor (EHT 1864) for 2 hr. Cells were fixed and stained for Bassoon (magenta), F-Actin (cyan), and Homer1 (green). Scale bar, 250 nm. (C) Presynaptic F-Actin levels in synapses of neurons treated with 0.1% DMSO (100±8.5) or 10 µM Rac1 Inhibitor (EHT 1864; 58.6±6.5; p<0.0001, one sample Wilcoxon test) for 2 hr. Line profiles of F-Actin overlapping with Bassoon (presynapse) distribution were integrated. Data shown are normalized to DMSO (set to 100) and expressed as mean ± SEM. nDMSO = 30, nEHT 1864 = 46 from two independent experiments. (D) Minima of background-corrected vGAT-CypHer fluorescence traces (surface normalized) for neurons treated with 0.1% DMSO (1.0±0.2 for shmDia1 +3) or 10 µM Rac1 Inhibitor (EHT 1864; 0.8±0.1 for shCTR; 0.8±0.1 for shmDia1 +3) in response to 200 AP stimulation (40 Hz, 5 s). Data represent mean ± SEM. Values were normalized to DMSO-treated shCTR (set to 1). N=8 independent experiments from nshCTR + DMSO = 46 videos, nshmDia1+3 + DMSO=45 videos, nshCTR + EHT 1864 = 42 videos, nshmDia1+3 + EHT 1864 = 43 videos. (E) Averaged normalized Synaptophysin-pHluorin fluorescence traces from stimulated (200 APs; 40 Hz, 5 s) hippocampal neurons transduced with lentiviruses encoding shCTR or shmDia1 +3 and transfected with plasmids for expression of constitutively-active Rac1 (Rac1-CA; Q61L variant) or dominant-negative Rac1 (Rac1-DN; T17N variant). Data represent mean ± SEM. N=3 independent experiments from nshCTR = 12 videos, nshmDia1+3 = 23 videos, nshCTR + Rac1-CA=10 videos, nshmDia1+3 + Rac1-CA=14 videos, nshCTR + Rac1-DN = 9 videos; nshmDia1+3 + Rac1-DN = 13 videos. The corresponding endocytic decay constants are shown in Figure 6H. (F) Maxima of background-corrected Synaptophysin-pHluorin fluorescence traces (surface normalized maximum values of traces shown in E) from stimulated (200 APs; 40 Hz, 5 s) hippocampal neurons transduced with lentiviruses encoding shCTR (Fmax/F0=1.3±0.0) or shmDia1 +3 (Fmax/F0=1.5±0.0) and transfected with plasmids encoding CA (Fmax/F0 shCTR + Rac1-CA=1.4±0.2; Fmax/F0 shmDia1+3 + Rac1-CA=1.5±0.1) or DN versions (Fmax/F0 shCTR + Rac1-DN = 1.2±0.1; Fmax/F0 shmDia1+3 + Rac1-DN = 1.3±0.1) of Rac1. Data represent mean ± SEM. (G) Densitometric quantification of Cdc42-GTP normalized to total Cdc42 levels in lysates from shmDia1 +3 transduced neurons (2.7±0.6; p<0.05, one sample t-test). Values for shCTR were set to 1. Data are expressed as mean ± SEM from N=3 independent experiments. (H) Representative three-channel time-gated stimulated emission depletion (STED) image of synapses from hippocampal mouse cultures, fixed and immunostained for Bassoon (magenta), Cdc42 (cyan), and Homer1 (green). Scale bar, 250 nm. (I) Averaged normalized line profiles for synaptic distribution of Cdc42 and Homer1 relative to Bassoon (Maximum set to 0 nm). Data are expressed as mean ± SEM (N=3; n=96 synapses). (J) Averaged normalized vesicular glutamate transporter 1 (vGAT)-CypHer fluorescence traces for neurons transduced with shCTR or shmDia1 +3 in response to 200 AP (40 Hz, 5 s) stimulation. Cells were acutely treated with 0.1% DMSO or 10 µM Cdc42 Inhibitor (ML141) in the imaging buffer. Data shown represent the mean ± SEM. N=6 independent experiments from nshCTR + DMSO = 31 videos, nshmDia1+3 + DMSO=33 videos, nshmDia1+3 + ML141=32 videos. (K) Endocytic decay constants of vGAT-CypHer traces in J: τshCTR + DMSO = 15.6±1.0 s, τshmDia1+3 + DMSO=28.0±3.1 s, τshCTR + ML141=17.6±1.6 s, τshmDia1+3 + ML141=33.1 ± 7.7 s; pshCTR + DMSO vs shmDia1+3 + DMSO<0.01, Kruskal-Wallis test with Dunn’s post-test. Data shown represent the mean ± SEM. N=6 independent experiments from nshCTR + DMSO = 31 videos, nshmDia1+3 + DMSO=33 videos, nshCTR + ML141=29 videos, nshmDia1+3 + ML141=32 videos.Figure 6—figure supplement 1—source data 1.Original scan for the anti-Rac1 immunoblots from Figure 6—figure supplement 1A.Figure 6—figure supplement 1—source data 2.Original scan for the anti-Tubulin immunoblot from Figure 6—figure supplement 1A.Figure 6—figure supplement 1—source data 3.Original scans for immunoblots in Figure 6—figure supplement 1A with highlighted bands and sample labels.Figure 6—figure supplement 1—source data 4.Numerical source data of Figure 6—figure supplement 1C, D, E, F, G, I, J, K.Figure 6—figure supplement 1—source data 5.Original scans for anti-Cdc42 immunoblots used for analysis are shown in Figure 6—figure supplement 1G.Figure 6—figure supplement 1—source data 6.Original scans for anti-Cdc42 immunoblots used for analysis are shown in Figure 6—figure supplement 1G with highlighted bands and sample labels.Original scan for the anti-Rac1 immunoblots from Figure 6—figure supplement 1A.Original scan for the anti-Tubulin immunoblot from Figure 6—figure supplement 1A.Original scans for immunoblots in Figure 6—figure supplement 1A with highlighted bands and sample labels.Numerical source data of Figure 6—figure supplement 1C, D, E, F, G, I, J, K.Original scans for anti-Cdc42 immunoblots used for analysis are shown in Figure 6—figure supplement 1G.Original scans for anti-Cdc42 immunoblots used for analysis are shown in Figure 6—figure supplement 1G with highlighted bands and sample labels. less

Knockdown of RAC1b decreases TAp73 expression but not vice versa. (A) PANC-1 cells were subjected to two rounds of transfection (on two consecutive days) with 50 nM each of TAp73 siRNA or control siRNA and processed for immunoblotting of RAC1b (upper band) and RAC1 (lower band). The blot was also probed with an antibody to HSP90 to test for equal protein loading. Successful knockdown of p73 was verified by qPCR analysis as shown in (B) and earlier by immunoblotting [15]. (B) PANC-1 cells were transiently transfected with 50 nM of siRNA to either RAC1b, TAp73, or a scrambled Co siRNA, and subsequently subjected to qPCR analyses of RAC1b (left two graphs) or TAp73 (right two graphs). (C,D) RAC1b increases TAp73 mRNA abundance but not vice versa. (C) PANC-1 cells transiently transfected with either empty vector (V) or HA-tagged versions of either TAp73α or TAp73β were analyzed by qPCR for expression of RAC1b. (D) As in (C), except that PANC-1 cells stably expressing either empty pCGN vector (V) or HA-RAC1b in pCGN (HA-R1b) were analyzed by qPCR for expression of TAp73. In (C,D), data represent the mean ± SD (n = 3). Asterisks (*) indicate significance relative to the Co siRNA (B) or the empty vector (C,D) (p < 0.05, Wilcoxon rank-sum test); ns, non-significant. less

Leishmania infection in human macrophages reduces the expression of proteins involved in actin dynamics. Human macrophages embedded in a collagen I matrix, infected or not by L. amazonensis, L. braziliensis or L. infantum, were immunostained with anti-Rac1, anti-Cdc42, and anti-RhoA antibodies. (A) Expression of Rac1 fluorescence in human macrophages in a 3D environment. (B) Fluorescence expression of Cdc42 in human macrophages in a 3D environment. (C) Expression of RhoA fluorescence in human macrophages in a 3D environment. Images obtained using a Leica SP8 confocal microscope. (D) Significantly reduced Rac1 fluorescence intensity in infected human macrophages compared to controls in a 3D environment. (E) Significantly reduced Cdc42 fluorescence intensity in infected human macrophages compared to controls in a 3D environment. (F) Significantly reduced RhoA fluorescence intensity in infected human macrophages compared to controls in a 3D environment. Fluorescence intensity quantified by averaging 25 to 30 cells per experimental group using FIJI software. Blue: Dapi. ***, p < 0.001 (Kruskal–Wallis; ANOVA). Representative results from three independent experiments. less

Protein and phosphorylation levels of three C/EBPβ variants in the late stage of differentiation of adipose progenitor cells into adipocytes in vitro. Protein (A) and phosphorylation (B) levels of three C/EBPβ variants (LAP*, LAP, and LIP) in cells derived from control and adipo-rac1-KO mice were determined by immunoblot analysis using antibodies against C/EBPβ (A) and phosphorylated C/EBPβ (B), respectively. α-tubulin served as a loading control. Intensities of bands for LAP* (C), LAP (D), LIP (E), phosphorylated LAP* (p-LAP*) (F), phosphorylated LAP (p-LAP) (G), and phosphorylated LIP (p-LIP) (H) in immunoblots were quantified. Data are shown as means ± S.E. (n = 3). ** p < 0.01, *** p < 0.001 (Student’s t test). less

Protein and phosphorylation levels of three C/EBPβ variants in the late stage of differentiation of adipose progenitor cells into adipocytes in vitro. Protein (A) and phosphorylation (B) levels of three C/EBPβ variants (LAP*, LAP, and LIP) in cells derived from control and adipo-rac1-KO mice were determined by immunoblot analysis using antibodies against C/EBPβ (A) and phosphorylated C/EBPβ (B), respectively. α-tubulin served as a loading control. Intensities of bands for LAP* (C), LAP (D), LIP (E), phosphorylated LAP* (p-LAP*) (F), phosphorylated LAP (p-LAP) (G), and phosphorylated LIP (p-LIP) (H) in immunoblots were quantified. Data are shown as means ± S.E. (n = 3). ** p < 0.01, *** p < 0.001 (Student’s t test). less

Differentiation of 3T3-L1 cells into adipocytes in vitro. (A) Schematic representation of the differentiation protocol. The day when cells reached confluence was referred to as day 0. (B) sh-RNA-mediated knockdown of Rac1 in 3T3-L1 cells. The protein expression level of Rac1 in 3T3-L1 cells that expressed control or Rac1-targeting shRNA was determined by immunofluorescent microscopy. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Scale bar, 200 μm. (C) Fluorescent staining of Rac1 as shown in (B) was quantified. Data are shown as means ± S.E. (n = 36). *** p < 0.001 (Student’s t test). (D) Fluorescent staining of Rac1 and lipid droplets with an anti-Rac1 antibody and LipiDye, respectively, in differentiated 3T3-L1 adipocytes that expressed control or Rac1-targeting shRNA at day 8. Nuclei were stained with 4′,6-diamidino-2-phenylindole. For the staining of lipid droplets, the high-magnification image (right) of the boxed area in the low magnification image (left) is also shown. Scale bar, 100 μm. (E) The percentage of lipid droplet-containing cells in differentiated 3T3-L1 adipocytes that expressed control or Rac1-targeting shRNA at day 8 was calculated. Data are shown as means ± S.E. (n = 36). *** p < 0.001 (Student’s t test). less

Methamphetamine (METH) regulated Rac1 and Cdc42 activity in hippocampus and cortex. (A) Representative immunoblots of Rac1, Rac1-GTP, Cdc42, Cdc42-GTP and β-actin in PFC and hippocampus. (B) Quantification of Rac1 and Cdc42 normalized to β-actin, Rac1-GTP normalized to Rac1, and Cdc42-GTP normalized to Cdc42 in PFC. One-way ANOVA, Rac1/β-actin, F (2, 8) = 0.393, p = 0.691, Rac1-GTP/Rac1, F (2, 26) = 15.933, p = 0.004, Cdc42/β-actin, F (2, 8) = 0.178, p = 0.841, Cdc42-GTP/Cdc42, F (2, 8) = 0.178, p < 0.001. (C) Quantification of Rac1 and Cdc42 normalized to β-actin, Rac1-GTP normalized to Rac1, and Cdc42-GTP normalized to Cdc42 in hippocampus. One-way ANOVA, Rac1/β-actin, F (2, 8) = 1.439, p = 0.309, Rac1-GTP/Rac1, F (2, 8) = 68.719, p < 0.001, Cdc42/β-actin, F (2, 8) = 0.451, p = 0.657, Cdc42-GTP/Cdc42, F (2, 8) = 209.847, p < 0.001. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA and Bonferroni’s post hoc analysis. less

Nuclear Vav3 interacts with components of PRC1 complex.A Schema depicting the assays performed in Fig. 3. B–D Western blot analyses of input (B), α−Vav3 Ab immunoprecipitated (C), and α−Bmi1 Ab Immunoprecipitated (D) cytoplasmic and nuclear fractions of empty vector and p190-BCR-ABL transduced Ba/F3 cells. E Confocal microscopic images of PLA between Vav3 and Bmi1 in empty vector or p190-BCR-ABL transduced Ba/F3 cells. F Quantification of the mean fluorescence intensity of the PLA signals depicted in D (n = 16–43 per group). G Confocal microscopic images of PLA between Vav3 and Bmi1 in empty vector or p190-BCR-ABL transduced murine B-cell progenitors. H Quantification of the mean fluorescence intensity of the PLA signals depicted in G (n = 12 per group). Nuclear Vav3 and Bmi1 reside in close proximity and p190-BCR-ABL expression enhances PLA signal. I, J Confocal microscopic images of PLA between Vav3 and Bmi1 (I) and quantification (J) in empty vector transduced WT leukemic murine B-cell progenitors and empty vector/Vav3 FL/Vav3 GEF inactivating mutant lentiviral vector transduced Vav3−/−- leukemic murine B-cell progenitors (n = 11–17 per group). K, L Confocal microscopic images of PLA between Vav3 and Bmi1 (K) and quantification (L) in WT and Rac2−/− leukemic murine B-cell progenitors (n = 12–15 per group). Scale bar, 10 μm. Data are presented as mean ± SD of a 2 or 3 independent experiments. Statistical significance was determined using the unpaired Student-t or Anova test when more than two groups were compared. Differences in survival were examined using the log-rank P test. *p <  0.05; **p <  0.01; ***p <  0.001. less

Nuclear Vav3 interacts with components of PRC1 complex.A Schema depicting the assays performed in Fig. 3. B–D Western blot analyses of input (B), α−Vav3 Ab immunoprecipitated (C), and α−Bmi1 Ab Immunoprecipitated (D) cytoplasmic and nuclear fractions of empty vector and p190-BCR-ABL transduced Ba/F3 cells. E Confocal microscopic images of PLA between Vav3 and Bmi1 in empty vector or p190-BCR-ABL transduced Ba/F3 cells. F Quantification of the mean fluorescence intensity of the PLA signals depicted in D (n = 16–43 per group). G Confocal microscopic images of PLA between Vav3 and Bmi1 in empty vector or p190-BCR-ABL transduced murine B-cell progenitors. H Quantification of the mean fluorescence intensity of the PLA signals depicted in G (n = 12 per group). Nuclear Vav3 and Bmi1 reside in close proximity and p190-BCR-ABL expression enhances PLA signal. I, J Confocal microscopic images of PLA between Vav3 and Bmi1 (I) and quantification (J) in empty vector transduced WT leukemic murine B-cell progenitors and empty vector/Vav3 FL/Vav3 GEF inactivating mutant lentiviral vector transduced Vav3−/−- leukemic murine B-cell progenitors (n = 11–17 per group). K, L Confocal microscopic images of PLA between Vav3 and Bmi1 (K) and quantification (L) in WT and Rac2−/− leukemic murine B-cell progenitors (n = 12–15 per group). Scale bar, 10 μm. Data are presented as mean ± SD of a 2 or 3 independent experiments. Statistical significance was determined using the unpaired Student-t or Anova test when more than two groups were compared. Differences in survival were examined using the log-rank P test. *p <  0.05; **p <  0.01; ***p <  0.001. less

Altered protein prenylation, lipid peroxidation and increased intracellular ROS upon statin treatment. (A) Western analysis of tree main prenylated proteins (RHOA, RAS, and RAC1) of the mevalonate pathway after treatment with Atorvastatin (0.5 µM) and with rescue by Mevalonic acid (MEV) (100 µM) in the A673 cell line. MHC1 protein membrane (HLA-A) was used as control of the partitioning of membrane proteins. (B) Schematic representation of statins impact on mevalonate pathway, intracellular ROS and lipid peroxidation. (C–E) Atorvastatin induced increased intracellular ROS in a dose-dependent manner through mevalonate pathway. (C) The graph represents specific MFI using CellRox probe in the A673, POE, and TC71 Ewing cell lines treated with 2 doses of Atorvastatin (A673 0.5 µM and 1 µM, POE 2.4 µM and 4.8 µM, TC71 2.6 µM and 5.2 µM) (n = 3 biological replicates). (D) The A673 Ewing cell line was treated for 72 h with Atorvastatin at the IC50 (0.5 µM) alone or with mevalonic acid (MEV) (100 µM), Farnesyl pyrophosphate (Fpp) (10 µM), or GeranylGeranyl-pyrophosphate (GGpp) (10 µM) metabolites (n = 3 biological replicates). (E) Measurement of cell viability (Top) and intracellular ROS (Bottom) after treatment with Atorvastatin (IC50 × 2 = 1 µM) for 48 h associated with NAC (3 mM), an inhibitor of intracellular ROS in the A673 cell line (n = 3 biological replicates). (F,G) Analysis and quantification of γH2Ax expression by Western blot of the A673 cell line treated with 0.5 µM Atorvastatin for 72 h and rescued by MEV (100 µM), Fpp (10 µM), or GGpp (10 µM) (F), or by treatment with NAC (3 mM) (G). (H) The graph represents specific MFI using a Bodipy probe in the A673, POE, and TC71 Ewing cell lines treated with 2 doses of Atorvastatin (n = 3 biological replicates). (I) The A673 cell line was treated with 0.5 µM Atorvastatin alone or with MEV (100 µM), Fpp (10 µM), or GGpp (10 µM) (n = 3 biological replicates). Data are presented as mean +/− SD. **** p value < 0.0001, *** p value < 0.001, ** p value < 0.01, * p value < 0.05 versus DMSO (C,H) or versus Ato treatment (D,E–I), unpaired Student’s t-test. less

Ax inhibits the enzyme activity of TcdB in vitro. (A) Whole cell lysate (20 µg) as source for Rac1 was supplemented with TcdB (10 nM) in the presence or absence of Ax (150 µM) or DMSO (solvent control) for 2 h at 37°C. Afterwards, samples were subjected to SDS-PAGE followed by Western blotting. Non-glucosylated Rac1 signals are normalized to Hsp90 loading control. Relative signals to toxin control are given as mean ± SD (n = 4). A representative Western blot image is depicted. Ordinary one-way ANOVA was performed with Dunnett’s multiple comparison test against toxin-only control. Asterisks indicate significance levels for toxin containing samples, with ns (not significant), *p < 0.05 and **p < 0.01. (B) C2I (1 ng) was incubated with whole cell lysate (40 µg) with two different concentrations of Ax (100 µM, 1,000 µM) and DMSO as solvent control for 30 min at 37°C. ADP-ribosylated and thereby biotin-labeled actin (actinADP-ribose) signals are normalized to Hsp90 loading control. Relative signals to toxin control are given as mean ± SD (n = 3). A representative Western blot is depicted. Ordinary one-way ANOVA was performed with Dunnett’s multiple comparison test against C2I-only control. Asterisks indicate significance levels for toxin containing samples, with ns (not significant). (C) TcdB (200 pM) and recombinant Rac1 (5 µM) were incubated with increasing concentrations of Ax and castanospermine (Cast) to analyze glucosyltransferase activity of TcdB by UDP-Glo™ glycosyltransferase assay. Nonlinear fit was applied with Graphpad Prism via log(inhibitor) vs. normalized response function. The estimated IC50 value for Ax (∼1.5 mM) is displayed in the graph. (D) TcdB (50 nM) was incubated with increasing concentrations of Ax and castanospermine (Cast) to analyze glucosylhydrolase activity of TcdB by UDP-Glo™ glycosyltransferase assay. Nonlinear fit was applied with Graphpad Prism via log(inhibitor) vs. normalized response function. The estimated IC50 value for Ax (∼1.6 mM) is displayed in the graph. less

Ax prevents TcdB-induced intracellular Rac1 glucosylation. (A) Vero cells were intoxicated with TcdB (10 pM) in the presence or absence of increasing concentrations of Ax or DMSO as solvent control. After 5 h, cells were harvested, lysed and transferred to Western blotting. Non-glucosylated Rac1 signal was normalized to Hsp90 loading control. Relative signal intensities are given as mean ± SD (n = 4). Ordinary one-way ANOVA was performed with Dunnett’s multiple comparison test against toxin-only control. Asterisks indicate significance levels for toxin containing samples, with ns (not significant), *p < 0.05 and **p < 0.01. (B) Vero cells were treated with TcdB (10 pM), Ax (150 µM) and DMSO as solvent control. After 5 h, cells were fixated and immunofluorescence staining was performed. Nuclei (blue), actin cytoskeleton (green) and non-glucosylated Rac1 (red) were stained. Representative images of the individual channels and the merge of all three are depicted. Scale bars correspond to 50 µm. less

mGBP-2 inhibits the activation of Rac1 and promotes the activation of CDC42 and RhoA. (A) Control, KD #1, and KD #2 67NR cells were serum starved for 12 h and then incubated with 20% FBS in DMEM for 30 min, lysed, and analyzed for active Rac1, CDC42, and RhoA as described in Methods. (B–D) Immunoblots from PBD pulldowns were quantified and results for levels of each active Rho protein were normalized to their total cellular level and then set to 1 for control 67NR cells (*, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to control cells; n = 3). Representative western blots are shown. (E) Control, KD #1, and KD #2 67NR cells were serum-starved for 18 h and then incubated with 20% FBS in DMEM for 30 min. Cell lysates were analyzed for phospho-Akt and total Akt. A representative blot is shown. (F) Scanned X-ray films were uploaded into Image J to measure densitometric values of pAkt and total Akt. The ratio of pAkt and total Akt densitometric values were calculated and represented on the graph as the average pAkt ± SD relative to control, which was assigned an arbitrary value of 1 (p = 0.7617 compared to control cells, n = 2). n.s.= not signficant. less

Rac1 activity is regulated by the ubiquitin-proteasome system.A) Representative western blot analysis of Rac1-GTP by CRIB pulldown (PD). HUVECs were treated for 2 hrs with the indicated inhibitors and pulldown of endogenous active Rac1 was performed as described in materials and methods. B) Quantification of Rac1 activity by densitometric analysis of western blot after CRIB pulldown (n = 4). * p<0.05 compared to control in Holm-Sidak’s post-hoc test of one-way ANOVA. less

α-Defensin-5 decreases the cytotoxic effects of TcdA, TcdB and of the combination of both toxins. (A) Vero cells were either treated with TcdA (10 pM, left panel) or TcdB (10 pM, right panel) in the presence or absence of increasing concentrations of α-defensin-5 (1, 3, 6 µM), and the percentage or rounded cells was determined. For comparison, Vero cells were treated with native TcdA (10 pM, left lower panel) in the presence or absence of α-defensin-5 (6 µM). Values are given as mean ± SD (n = 3). Significance was determined using the one-way ANOVA test (n.s. = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (B) Vero cells (upper panel) were treated with TcdA (10 pM) and α-defensin-5 with increasing concentrations (1, 3, 6 µM) for 3.5 h. Caco-2 cells (lower panel) were treated with either TcdA (10 pM), TcdB (10 pM) or the combination of both toxins (each 10 pM) with or without α-defensin-5 (6 µM) for 8 h. Afterward, cells were lysed and subjected to Western blot analysis. Non-glucosylated Rac1 was detected using a specific antibody. Hsp90 or GAPDH were used as controls for equal protein loading. (C) Caco-2 cells were treated with the combination of TcdA (10 pM) plus TcdB (10 pM) and with/without α-defensin-5 (6 µM) for 8.5 h. For control, cells were left untreated. After incubation, cells were fixed and permeabilized. Non-glucosylated Rac1 was detected using a specific anti-Rac1-antibody, F-actin was stained with phalloidin-FITC, nuclei were stained with Hoechst33342. less

α-Defensin-5 has no influence on the enzymatic activity of TcdA and CDTa but leads to precipitation of TcdA and inhibition of the cytotoxic pore-forming activity of CDTb. (A) Caco-2 lysate (40 µg) was incubated with TcdA (300 ng) and varying concentrations of α-defensin-5 (6, 12, 24 µM) for 1 h (left panel) at 37°C. Caco-2 lysate (40 µg) was incubated with CDTa (1 ng), 10 µM biotinylated NAD+ and varying concentrations of α-defensin-5 (1, 3, 6 µM) for 30 min at 37°C. Next, samples were subjected to SDS-PAGE and Western Blotting. Non-glucosylated Rac1 was detected with a specific antibody, biotin-labeled, e.g., ADP-ribosylated actin was detected using streptavidin-peroxidase. Hsp90 and GAPDH were used to confirm equal protein loading. Values are given as mean ± SD (n = 2). Significance was determined using the one-way ANOVA test (n.s. = not significant). (B) TcdA (1 µg) was incubated with and without α-defensin-5 (6 µM) for 15 min at 37°C in serum-free medium. Afterward, samples were centrifuged and separated fractions were subjected to SDS-PAGE (left panel). Densitometric analyses from individual experiments are shown as bar graph (right panel). Values are given as mean ± SD (n = 2). Significance was determined using the one-way ANOVA test (n.s. = not significant, **p < 0.01). (C) Vero cells were treated with CDTb (5.3 nM) in the absence of CDTa with or without increasing concentrations of α-defensin-5 (1, 3, 6 µM) for 4 h at 37°C. Representative images are shown in the left panel. After the incubation time, a MTS cell viability assay was performed. Values are given as mean ± SD (n=3). Significance was determined using the one-way ANOVA test (n.s. = not significant, ****p < 0.0001). (D) Caco-2 cells were seeded in an 8-well ibidi plate and pretreated with Fura-2AM (3 µM) for 45 min. Next, baseline was measured for 2 min, and the cells were then treated with CDTb (13 nM) in the presence or absence of α-defensin-5 (6 µM) as indicated. less

Inhibition of learning-induced Rac1 activity causes memory enhancement. a Behavioral schedule (top) for contextual fear conditioning (CFC)-induced memory retention curve (D, day) in wild-type (WT) mice (bottom). WT mice were trained using one-shock CFC and then separate group mice were tested for memory retention at various periods (1 h, day 1, day 7, day 14, or day 36). *P < 0.05 and **P < 0.01 (from one-way ANOVA); n = 41, 28, 17, 22, and 21 mice. b Representative image of western blottings (left) and data (right) showing total Rac1 levels and Rac1-GTP in hippocampal extracts from naive (Na) and trained (one shock) at various retention intervals (1 h, day 1, day 2, day 7, day 11, or day 14). *P < 0.05 and **P < 0.01 (from one-way ANOVA); n = 12, 7, 7, 3, 4, 4, and 4 mice. c Representative immunostaining of Rac1-GTP (left) and data (right) showing percentages of Rac1-GTP+ cells in the hippocampal CA1 region of Na mice and CFC mice at various periods (day 1, day 2, day 7, day 11, or day 14). Coronal sections of the hippocampal CA1 region of Na mice and CFC mice with the anti-Rac1-GTP (green) and anti-nuclei (blue). Scale bar, 500 μm. ****P < 0.0001 (from one-way ANOVA); n = 15-17 slices from 7 mice for each group. d Behavioral effects of the bidirectional manipulation of hippocampal Rac1 activity on contextual fear memory. After AAV injection for 2 weeks, mice were subjected to one-shock CFC and then separate group mice were tested for 4 min at different time periods (1 h or day 1). *P < 0.05 and **P < 0.01 (from two-way ANOVA); n = 8, 10, 6, 7, 4, and 10 mice. e Time courses of freezing behavior (bottom) in WT mice with injection of Ehop016 or saline (top). *P < 0.05 and ****P < 0.0001 (from two-way ANOVA); n = 10, 14 mice. Data are presented as means ± SEM. Also see Supplementary Table 1. less

Q165P mutation partially impairs SPOP interaction with BRD4 and undermines BRD4 ubiquitinationA, BLNCaP cells were transfected with the indicated plasmids for 48 h and harvested for Western blot analysis with the indicated antibodies to detect the interaction between BRD4 and SPOP (A) and BRD4 ubiquitination (B).CC4‐2 cells were stably infected with the lentivirus expressing empty vector (EV) or SPOP mutants and harvested for Western blot analysis with the indicated antibodies.DC4‐2 xenograft tumors expressing EV or F133V and SPOP WT and Q165P PDX tumors were harvested for Western blot analysis with the indicated antibodies. T1: tumor 1; T2: tumor 2.ERepresentative IHC images of PTEN protein in SPOP WT and Q165P PDX tumors. Scale bars: 100 μm for 20× fields; 50 μm for 40× fields.Source data are available online for this figure. less

Q165P mutation partially impairs SPOP interaction with BRD4 and undermines BRD4 ubiquitinationA, BLNCaP cells were transfected with the indicated plasmids for 48 h and harvested for Western blot analysis with the indicated antibodies to detect the interaction between BRD4 and SPOP (A) and BRD4 ubiquitination (B).CC4‐2 cells were stably infected with the lentivirus expressing empty vector (EV) or SPOP mutants and harvested for Western blot analysis with the indicated antibodies.DC4‐2 xenograft tumors expressing EV or F133V and SPOP WT and Q165P PDX tumors were harvested for Western blot analysis with the indicated antibodies. T1: tumor 1; T2: tumor 2.ERepresentative IHC images of PTEN protein in SPOP WT and Q165P PDX tumors. Scale bars: 100 μm for 20× fields; 50 μm for 40× fields.Source data are available online for this figure. less

JQ1‐resistant SPOP hotspot mutant organoids are sensitive to NEO2734A, BSix organoid lines including four SPOP WT (BM1, BM5, ST1, and 313HR) and two MUT (573R and ASC1) were harvested for Western blot analysis with the indicated antibodies (A) and IFC with p‐AKT‐S473 antibody (B). Asterisk, SRC3 at expected molecular mass. E‐cadherin antibody was used to indicate the cell membrane (red) and DAPI for nucleus (blue). Scale bars: 25 μm.C, DSix organoid lines including four SPOP WT (BM1, BM5, ST1, and 313HR) and two MUT (573R and ASC1) were cultured for 5 days, followed by the treatment with JQ1 (2 μM), CPI‐637 (2 μM), JQ1 (2 μM) + CPI‐637 (2 μM) or NEO2734 (2 μM) for five more days. The representative images of organoids after the treatment are shown in (C) and the quantified data of the organoid diameter are shown in (D). Scale bars: 25 μm. All data shown are means ± SEM, n ≥ 38. The P value was calculated by the unpaired two‐tailed Student's t‐test; n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001. See Appendix Table S4 for the detailed comparison, P values and sample number (n).E, F313HR (SPOP WT) and 573R (Q165P) organoids were cultured for 5 days, followed by the treatment with the indicated inhibitors for 5 days. The organoids were stained with caspase‐3 antibody (green) to detect apoptotic cells. E‐cadherin antibody staining was used to define the cell membrane (red) and the nucleus was counterstained with DAPI (blue). The representative images of cleaved caspase‐3 IFC staining are shown in (E). Scale bars: 25 μm. All data shown are means ± SEM, n = 10. The P value was calculated by the unpaired two‐tailed Student's t‐test; *P < 0.05, ***P < 0.001. See Appendix Table S4 for the detailed comparison, P values, and sample number (n).Source data are available online for this figure. less

The formation of GTP-bound active Rac1 (Rac1·GTP) by ectopic expression of constitutively activated Akt2. (a) 3T3-L1 adipocytes were infected with the control (-) or Myr-Akt2-expressing virus. Endogenous Rac1 was visualized by immunofluorescent microscopy using an anti-Rac1 antibody. Myr-Akt2 was visualized by immunofluorescent microscopy using an anti-HA antibody. Rac1·GTP was visualized by immunofluorescent microscopy using an anti-V5 antibody after treatment with glutathione S-transferase (GST)-POSH(251–489)-V5×3, but not GST-V5×3. Scale bar, 50 μm. (b) The formation of Rac1·GTP shown in (a) was quantified. Data are shown as means ± S.E. (n = 10). * p < 0.05; ** p < 0.01. less

Effect of AI-XII on insulin-induced formation of Rac1·GTP. (a) Endogenous Rac1 was visualized by immunofluorescent microscopy using an anti-Rac1 antibody. Rac1·GTP was visualized by immunofluorescent microscopy using an anti-V5 antibody after treatment with GST-POSH(251–489)-V5×3, but not GST-V5×3. Scale bar, 50 μm. (b) The formation of Rac1·GTP shown in (a) was quantified. Data are shown as means ± S.E. (n = 10). * p < 0.05. less

Study of compensatory mechanisms and downstream signaling pathway analysis. (A–C) Representative images of western blot analysis (A) and quantification (B,C) of RhoB, RhoC expression, MLC and cofilin activation (B) and Rac1 and Cdc42 expression (C) in HUVECs after transfection with siRNA control (siNEG) and two sequences of siRNA for RhoA (siRhoA#1 and siRhoA#2) (50 nM) (n = 3) and mouse lung ECs from RhoAΔEC mice and littermate controls (n = 2). Full-length blots are presented in Supplementary Fig. 7. *P < 0.05; **P < 0.01; ***P < 0.001. less

Akt2-dependent activation of Rac1 in mouse gastrocnemius muscle as detected by immunofluorescent microscopy of cross sections of gastrocnemius muscle.(a) The expression vector for Myr-p110α, together with either one of two siRNA duplexes against mouse Akt2 (#1 and #2) and a mixture of NC siRNA duplexes, was introduced into gastrocnemius muscle of wild-type mice. Insulin (175.5 μg/kg body weight) was administered intravenously. Endogenous Rac1 and Akt2 were visualized by immunofluorescent staining with anti-Rac1 and anti-Akt2 antibodies, respectively. Myr-p110α was visualized by immunofluorescent staining with an anti-HA antibody. Activated Rac1 (Rac1·GTP) was visualized by immunofluorescent staining with an anti-V5 antibody after treatment with GST-POSH(251–489)-V5×3. Scale bar, 20 μm. (b) Activation of Rac1 shown in (a) was quantified. Gray, orange, and blue bars represent the treatment with NC, #1, and #2 siRNA duplexes, respectively. Data are shown as means ± S.E. (n = 6). *P < 0.001. less