Journal: Frontiers in Pharmacology

Article Title: PACSIN2 regulates mercaptopurine cytotoxicity and cellular biomechanics through Rac1 modulation in intestinal epithelial cells

doi: 10.3389/fphar.2026.1860702

Figure Lengend Snippet: The effect of PACSIN2 on mercaptopurine (MP) and the Rac1 inhibitor NSC23766 cytotoxicity. (A) Cytotoxicity MTT dose-response curve of LS180 MOCK, LS180 PACSIN2 KD cells, LS180 MOCK EMPTY, and LS180 MOCK PACSIN2 OVEREXPRESSION cells treated with MP (0.3 µM 160 µM) for 72 h. A significant difference of MP sensitivity between LS180 MOCK and LS180 PACSIN2 KD cells was detected at 0.3 µM (P = 0.001), 0.6 µM (P = 0.001), 1.25 µM (P = 0.0001), 2.5 µM (P = 0.0001). A significant difference of MP sensitivity between LS180 MOCK EMPTY and LS180 MOCK PACSIN2 cells was found at 1.25 µM (P < 0.05), 2.5 µM (P = 0.001), 5 µM (P = 0.0001), 10 µM (P = 0.001) and 20 µM (P = 0.01). Data are expressed as mean ± standard deviation from three independent experiments (Two-way ANOVA, post-test Tukey). (B) Cytotoxicity MTT dose-response curves of LS180 MOCK, LS180 PACSIN2 KD cells, LS180 MOCK EMPTY, and LS180 MOCK PACSIN2 OVEREXPRESSION cells treated with the Rac1 inhibitor NSC23766 (0.3 µM 160 µM) for 72 h. A significant difference from the Rac1 inhibitor NSC23766 was found between LS180 MOCK and KD cells (P < 0.0001)) and between LS180 EMPTY cells and LS180 PACSIN2 OVEREXPRESSION cells (P < 0.0001). Data are expressed as mean ± standard deviation from three independent experiments (Two-way ANOVA, post-test Tukey). (C) Cytotoxicity MTT dose-response curve LS180 MOCK, LS180 PACSIN2 KD cells, LS180 MOCK EMPTY and LS180 MOCK PACSIN2 OVEREXPRESSION cells co-treated with MP (from 0.3 µM to 160 µM) and the Rac1 inhibitor NSC23766 2.5 µM for 72 h. There was a significant difference between LS180 MOCK and LS180 KD cell lines (P < 0.0001)) and between LS180 MOCK EMPTY and LS180 MOCK PACSIN2 OVEREXPRESSION cell lines (P < 0.0001). Data are expressed as mean ± standard deviation from three independent experiments (Two-way ANOVA, post-test Tukey).

Article Snippet: The Rac1 G-LISA kit ( Cytoskeleton, Denver, CO, United States) was used to specifically detect active GTP-bound Rac1 and distinguish it from Rac2 and Rac3 isoforms in incubated cell/tissue lysates at a concentration of 1 mg/mL.

Techniques: Over Expression, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: PACSIN2 regulates mercaptopurine cytotoxicity and cellular biomechanics through Rac1 modulation in intestinal epithelial cells

doi: 10.3389/fphar.2026.1860702

Figure Lengend Snippet: The effect of PACSIN2 on Rac1 expression and activity levels. (A) Immunoblotting results of Rac1 protein levels in LS180 MOCK and LS180 PACSIN2 KD cells normalized against β‐actin (data expressed as mean ± standard deviation, n = 3, t‐test). (B) Rac1 protein activity evaluated as Rac1‐GTP amount in LS180 MOCK and LS180 PACSIN2 KD cell lines (data expressed as mean ± standard deviation, n = 3, t‐test). (C) immunoblotting results of Rac1 protein levels in LS180 MOCK cells transfected with an empty plasmid (LS180 EMPTY) used as control and LS180 cells presenting the PACSIN2 overexpression (LS180 PACSIN2 OVEREXPRESSION) cells at 48 after transfection (48 h) and after additional 72 h (72h post) normalized against β‐actin (data expressed as mean ± standard deviation, n = 3, t‐test). (D) Rac1 protein activity evaluated as Rac1‐GTP amount in LS180 EMPTY and LS180 PACSIN2 OVEREXPRESSION cells at 48 after transfection (48 h) and after additional 72 h (72h post, data expressed as mean ± standard deviation, n = 3, t‐test).

Article Snippet: The Rac1 G-LISA kit ( Cytoskeleton, Denver, CO, United States) was used to specifically detect active GTP-bound Rac1 and distinguish it from Rac2 and Rac3 isoforms in incubated cell/tissue lysates at a concentration of 1 mg/mL.

Techniques: Expressing, Activity Assay, Western Blot, Standard Deviation, Transfection, Plasmid Preparation, Control, Over Expression

Journal: Frontiers in Pharmacology

Article Title: PACSIN2 regulates mercaptopurine cytotoxicity and cellular biomechanics through Rac1 modulation in intestinal epithelial cells

doi: 10.3389/fphar.2026.1860702

Figure Lengend Snippet: Analysis of Rac1 protein levels under basal condition and after mercaptopurine (MP) treatment. Western Blot analysis of Rac1 protein levels in LS180 MOCK and LS180 PACSIN2 KD cell lines untreated and after 24 h (A) , 48 h (B) or 72 h (C) of MP treatment at the IC20, IC50 and IC80. Western Blot analysis of Rac1 protein levels in LS180 EMPTY and LS180 PACSIN2 OVEREXPRESSION cell lines untreated and after 24 h (D) , 48 h (E) or 72 h (F) of MP treatment at the IC20, IC50 and IC80. Rac1 levels were normalized using GAPDH or β‐actin and expressed in logarithmic scale (data expressed as mean ± standard deviation, n = 3, Two‐way ANOVA, post‐test Tukey). The β‐actin membranes of Panel (D–F) are the same presented in Panel (D–F) , respectively.

Article Snippet: The Rac1 G-LISA kit ( Cytoskeleton, Denver, CO, United States) was used to specifically detect active GTP-bound Rac1 and distinguish it from Rac2 and Rac3 isoforms in incubated cell/tissue lysates at a concentration of 1 mg/mL.

Techniques: Western Blot, Over Expression, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: PACSIN2 regulates mercaptopurine cytotoxicity and cellular biomechanics through Rac1 modulation in intestinal epithelial cells

doi: 10.3389/fphar.2026.1860702

Figure Lengend Snippet: Analysis of Rac1 protein activity after mercaptopurine (MP) treatment. G‐LISA analysis of Rac1 protein activity in LS180 MOCK and LS180 PACSIN2 KD cell lines untreated and after 24 h (A) , 48 h (B) or 72 h (C) of treatment at IC20, IC50 and IC80 of MP. G‐LISA analysis of Rac1 protein activity in LS180 EMPTY and LS180 PACSIN2 OVEREXPRESSION cell lines untreated and after 24 h (D) , 48 h (E) or 72 h (F) of treatment at IC20, IC50 and IC80 of MP (data expressed as mean ± standard deviation, n =3, Two‐way ANOVA, post‐test Tukey).

Article Snippet: The Rac1 G-LISA kit ( Cytoskeleton, Denver, CO, United States) was used to specifically detect active GTP-bound Rac1 and distinguish it from Rac2 and Rac3 isoforms in incubated cell/tissue lysates at a concentration of 1 mg/mL.

Techniques: Activity Assay, Over Expression, Standard Deviation

Journal: bioRxiv

Article Title: Morphogenesis of the muscular ventricular septum of the mouse heart is driven by retinoic acid signalling to myocardium

doi: 10.64898/2026.08.12.744354

Figure Lengend Snippet: (a) UMAP of compact myocardial cells from R26 RARα403 and Mef2c-AHF-Cre Tg+ ; R26 RARα403 IVS subsetted and -clustered into left- and right ventricular compact myocardium. (b) Feature plot showing Cre expression in left- and right ventricular compact myocardial cells from control and mutant hearts. Cre is expressed only in mutants and predominantly in right ventricular cardiomyocytes. (c) Feature plots of selected DEGs. RA target genes Rarb and Mdk are downregulated only in RV compact myocardial cells in the mutant. Similarly, the genes encoding the lysophosphatidic acid receptor Lpar3 and the modulator of Wnt signalling Sfrp1 are downregulated in RV but not LV cells. (d, e) RNAscope ISH showing Lpar3 (d) and Sfrp1 (e) expression in sections of E11.5 R26 RARα403 and Mef2c-AHF-Cre Tg+ ; R26 RARα403 embryos. Full arrowheads point to expression in the septum, while empty arrowheads in mutants indicate loss of expression. (f) Feature plot showing the expression of Rac1 in control and mutant subsetted myocardium at E11.5. (g) Normalized read counts of Rac1 transcript in bulk RNA sequencing data of E13.5 control and mutant IVS; P-value (adjusted p-values, DEseq2, n = [3 controls; 3 mutants]). See . (h) Quantifications of relative GTP-bound activated RAC1 levels by G-LISA (normalized to controls) of microdissected and pooled E11.5 and E13.5 IVS regions; P-value (Welch’s t-test) is shown. N [controls; mutants]: E11.5 [5;4], E13.5 [4;4]. IVS, interventricular septum; LV, left ventricle; RA, retinoic acid; RV, right ventricle; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: To score the levels of RAC1::GTP, we used the Rac1 G-LISA GTPase Activation Assay kit (Cytoskeleton Inc., #BK126).

Techniques: Expressing, Control, Mutagenesis, RNAscope, RNA Sequencing

Journal: Cells

Article Title: Macropinocytosis of Amyloid Precursor Protein Is Regulated by the Recruitment and Activity of Fe65, Arf6 and Rho GTPases

doi: 10.3390/cells15151366

Figure Lengend Snippet: Rac1, Cdc42 and RhoA demonstrate rapid and sustained recruitment to bound/crosslinked APP and membrane ruffles and increased GTP-bound levels. ( A ) N2a cells transfected with Rac1-EGFP (green), PLCδPH-mRFP (magenta) and APP695. Cells were incubated with tagged anti-APP or anti-NCAM antibodies (red) on ice and then were either immediately fixed on ice as a baseline (00:00) or incubated for 30 s (00:30), 2 min (02:00), 5 min (5:00), and 10 min (10:00) at 37 °C prior to fixation. Colocalization between signals is indicated by white pixels. ( B , C ) The quantification of colocalization data ( n = 3 independent experiments; 15 images acquired for each time point in both conditions per replicate) is represented as the difference between the mean percent colocalized or MCC at each timepoint relative to baseline (XX:XX−00:00). N2a cells transfected with ( D ) Cdc42-EGFP or ( G ) RhoA-EGFP, PLCδPH-mRFP and APP695. The experiment from ( A ) was repeated with Cdc42- or RhoA-expressing cells. ( E , F , H , I ) Quantification of colocalization data ( n = 3 independent experiments; 15 images acquired for each time point in both conditions per replicate). Data representation and analysis from ( B , C ) was repeated for N2a cells expressing Cdc42-EGFP. * denotes significant difference from baseline within group; # denotes significant difference between groups at the indicated timepoint. Significance was measured by a two-way ANOVA with a Tukey’s test using a single pooled variance. ( J ) G-LISA assay for Rac1-GTP from lysates of untreated N2a cells or cells incubated on ice with anti-NCAM, anti-APP or EGF (positive control), then incubated for 2 min. Lysates were immediately acquired after and used for Rac1 G-LISA activation assays. To compare the relative amount of GTP-bound Rac1 in each condition, the absolute optical density (OD) was measured from each condition’s lysate loaded into G-LISA assay wells. Experiments were run in technical triplicates across three independent experiments ( n = 3). Significant differences from untreated samples were calculated by a one-way ANOVA with Tukey’s test. This same experiments and analyses were performed for ( K ) Cdc42-GTP levels and ( L ) RhoA-GTP levels. Data are presented as mean ± SEM; */# p < 0.05. Representative images are single planes of whole-cell z-stacks; white bars represent the scale bar = 5 μm .

Article Snippet: We also examined the activation of Rac1, Cdc42, and RhoA following antibody binding using a G-LISA kit (Cytoskeleton) to quantify active GTP-bound Rac1, Cdc42 and RhoA.

Techniques: Membrane, Transfection, Incubation, Expressing, Positive Control, Activation Assay

Journal: Cells

Article Title: Macropinocytosis of Amyloid Precursor Protein Is Regulated by the Recruitment and Activity of Fe65, Arf6 and Rho GTPases

doi: 10.3390/cells15151366

Figure Lengend Snippet: Rac1, Cdc42 and RhoA demonstrate rapid and sustained recruitment to bound/crosslinked APP and membrane ruffles and increased GTP-bound levels. ( A ) N2a cells transfected with Rac1-EGFP (green), PLCδPH-mRFP (magenta) and APP695. Cells were incubated with tagged anti-APP or anti-NCAM antibodies (red) on ice and then were either immediately fixed on ice as a baseline (00:00) or incubated for 30 s (00:30), 2 min (02:00), 5 min (5:00), and 10 min (10:00) at 37 °C prior to fixation. Colocalization between signals is indicated by white pixels. ( B , C ) The quantification of colocalization data ( n = 3 independent experiments; 15 images acquired for each time point in both conditions per replicate) is represented as the difference between the mean percent colocalized or MCC at each timepoint relative to baseline (XX:XX−00:00). N2a cells transfected with ( D ) Cdc42-EGFP or ( G ) RhoA-EGFP, PLCδPH-mRFP and APP695. The experiment from ( A ) was repeated with Cdc42- or RhoA-expressing cells. ( E , F , H , I ) Quantification of colocalization data ( n = 3 independent experiments; 15 images acquired for each time point in both conditions per replicate). Data representation and analysis from ( B , C ) was repeated for N2a cells expressing Cdc42-EGFP. * denotes significant difference from baseline within group; # denotes significant difference between groups at the indicated timepoint. Significance was measured by a two-way ANOVA with a Tukey’s test using a single pooled variance. ( J ) G-LISA assay for Rac1-GTP from lysates of untreated N2a cells or cells incubated on ice with anti-NCAM, anti-APP or EGF (positive control), then incubated for 2 min. Lysates were immediately acquired after and used for Rac1 G-LISA activation assays. To compare the relative amount of GTP-bound Rac1 in each condition, the absolute optical density (OD) was measured from each condition’s lysate loaded into G-LISA assay wells. Experiments were run in technical triplicates across three independent experiments ( n = 3). Significant differences from untreated samples were calculated by a one-way ANOVA with Tukey’s test. This same experiments and analyses were performed for ( K ) Cdc42-GTP levels and ( L ) RhoA-GTP levels. Data are presented as mean ± SEM; */# p < 0.05. Representative images are single planes of whole-cell z-stacks; white bars represent the scale bar = 5 μm .

Article Snippet: G-LISA kits for GTP-bound Rac1 (BK128), Cdc42 (BK127) and RhoA (BK124) were all purchased from Cytoskeleton (Denver, CO, USA).

Techniques: Membrane, Transfection, Incubation, Expressing, Positive Control, Activation Assay

Journal: Cells

Article Title: Macropinocytosis of Amyloid Precursor Protein Is Regulated by the Recruitment and Activity of Fe65, Arf6 and Rho GTPases

doi: 10.3390/cells15151366

Figure Lengend Snippet: The mutation of the YENPTY site reduces Fe65 and GTPase recruitment to bound/crosslinked APP and membrane ruffles. ( A ) N2a cells transfected with WT-APP or APP-AENATA, Fe65-EGFP (green), and PLCδPH-mRFP (magenta). APP was bound/crosslinked by a tagged APP antibody (red) while on ice, then incubated for 30 s. Colocalization was measured between Fe65 and bound/crosslinked WT-APP or APP-AENATA, as well as between Fe65 and PLCδPH (white pixels). ( B ) The quantification of the mean % of Fe65 colocalized with either bound/crosslinked APP (top) or PLCδPH (bottom) in N2a cells expressing WT-APP or APP-AENATA. The data presented are from three independent experiments ( n = 3; 15 images acquired for each condition per replicate) with cells expressing WT-APP or APP-AENATA. Significant differences between cells expressing WT-APP or APP-AENATA mutant were analyzed by a two-tailed unpaired t -test. This experiment and analysis were repeated with N2a cells expressing ( C , D ) Arf6-GFP, ( E , F ) Rac1-GFP, ( G , H ) Cdc42-GFP, and ( I , J ) RhoA-GFP. Data are presented as mean ± SEM; * p < 0.05. Representative images are single planes of whole-cell z-stacks; white bars represent the scale bar = 5 μm .

Article Snippet: G-LISA kits for GTP-bound Rac1 (BK128), Cdc42 (BK127) and RhoA (BK124) were all purchased from Cytoskeleton (Denver, CO, USA).

Techniques: Mutagenesis, Membrane, Transfection, Incubation, Expressing, Two Tailed Test

Journal: Cells

Article Title: Macropinocytosis of Amyloid Precursor Protein Is Regulated by the Recruitment and Activity of Fe65, Arf6 and Rho GTPases

doi: 10.3390/cells15151366

Figure Lengend Snippet: Small GTPase inhibition and siRNA knockdown of Arf6 inhibits the macropinocytosis of APP. ( A ) N2a cells transfected with APP695 and LAMP1-mCh (red) that were treated with DMSO or 5 μM NAV-2729 (Arf6 inhibitor). APP was then bound/crosslinked by fluorescent tagged APP antibodies and imaged after a 15 min incubation. Colocalization was assessed between crosslinked APP and LAMP1 (white pixels). ( B ) Quantification of the mean % of APP colocalized with LAMP1 ( n = 3 independent experiments; 10 images per replicate), with significance calculated by a two-tailed unpaired t -test. ( C ) N2a cells transfected with APP695, LAMP1-mCh and either non-targeting scramble siRNA (control; CTRL) or ARF6 siRNA. Following transfection and differentiation, APP was bound/crosslinked by fluorescent-tagged APP antibodies and imaged after 15 min incubation. Colocalization was assessed between and LAMP1 (white pixels). ( D ) Quantification of the mean % of APP colocalized with LAMP1 ( n = 3 independent experiments; 10 images per replicate), with significance calculated by a two-tailed unpaired t -test. ( E ) N2a cells transfected with APP695 and LAMP1-mCh (red) that were treated with DMSO or 35 μM Rhosin (RhoA inhibitor). APP was then bound/crosslinked by fluorescent-tagged APP antibodies and imaged after a 15 min incubation. Colocalization was assessed between crosslinked APP and LAMP1 (white pixels). ( F ) Quantification of the mean % of APP colocalized with LAMP1 ( n = 3 independent experiments; 10 images per replicate), with significance calculated by a two-tailed unpaired t -test. ( G ) N2a cells transfected with APP695 and LAMP1-mCh (red) that were treated with DMSO, 10 μM EHT 1864 (Rac1 inhibitor), or 10 μM ML 141 (Cdc42 inhibitor). APP was then bound/crosslinked by fluorescent-tagged APP antibodies and imaged after 15 min incubation. Colocalization was assessed between crosslinked APP and LAMP1 (white pixels). ( H ) Quantification of the mean % of APP colocalized with LAMP1 ( n = 3 independent experiments; 10 images per replicate), with significance calculated by a one-way ANOVA with Tukey’s test. Data are presented as mean ± SEM; * p < 0.05. Representative images are single planes of whole-cell z-stacks; white bars represent the scale bar = 5 μm .

Article Snippet: G-LISA kits for GTP-bound Rac1 (BK128), Cdc42 (BK127) and RhoA (BK124) were all purchased from Cytoskeleton (Denver, CO, USA).

Techniques: Inhibition, Knockdown, Transfection, Incubation, Two Tailed Test, Control

Journal: Cells

Article Title: Macropinocytosis of Amyloid Precursor Protein Is Regulated by the Recruitment and Activity of Fe65, Arf6 and Rho GTPases

doi: 10.3390/cells15151366

Figure Lengend Snippet: Effects of GTPase inhibition on the recruitment of Fe65 and Arf6 to bound/crosslinked APP and membrane ruffles at 30 s. ( A ) N2a cells transfected with Fe65-EGFP (green), PLCδPH-mRFP (magenta) and APP695. Cells were treated with 0.1% DMSO, 5 μM NAV-2729, 10 μM EHT 1864, 10 μM ML 141, and 35 μM Rhosin. After treatment, cells were incubated with tagged anti-APP antibodies (red) on ice and then fixed following an incubation for 30 s. Colocalization was assessed between Fe65 and antibody bound/crosslinked APP or PLCδPH (white pixels). ( B ) Quantification of the mean % of Fe65 colocalized with APP (left) or PLCδPH (right) from three independent experiments ( n = 3; 15 images acquired for each condition per replicate). ( C ) N2a cells transfected with Arf6-EGFP (green), PLCδPH-mRFP and APP695. Cells were treated with 0.1% DMSO, 5 μM NAV-2729, 10 μM EHT 1864, 10 μM ML 141, and 35 μM Rhosin. After treatment, cells were incubated with tagged anti-APP antibodies (red) on ice and then fixed following a 30 s incubation. Colocalization was assessed between Arf6 and bound/crosslinked APP or PLCδPH (white pixels). ( D ) Quantification of the mean % of Arf6 colocalized with APP (left) or PLCδPH (right) from three independent experiments ( n = 3; 15 images acquired for each condition per replicate). Significant changes in the percentage colocalized between treatments were assessed by a one-way ANOVA with Tukey’s test. Data are presented as mean ± SEM; * p < 0.05. Representative images are single planes of whole-cell z-stacks; white bars represent the scale bar = 5 μm .

Article Snippet: G-LISA kits for GTP-bound Rac1 (BK128), Cdc42 (BK127) and RhoA (BK124) were all purchased from Cytoskeleton (Denver, CO, USA).

Techniques: Inhibition, Membrane, Transfection, Incubation

Journal: Cells

Article Title: Macropinocytosis of Amyloid Precursor Protein Is Regulated by the Recruitment and Activity of Fe65, Arf6 and Rho GTPases

doi: 10.3390/cells15151366

Figure Lengend Snippet: Effects of GTPase inhibition on the recruitment of the Rho GTPases Rac1, Cdc42 and RhoA to bound/crosslinked APP and membrane ruffles at 30 s. ( A ) N2a cells transfected with Rac1-EGFP (green), PLCδPH-mRFP (magenta) and APP695. Cells were treated with 0.1% DMSO, 5 μM NAV-2729, 10 μM EHT 1864, 10 μM ML 141, and 35 μM Rhosin. After treatment, cells were incubated with tagged anti-APP antibodies (red) on ice and then fixed following a 30 s incubation. Colocalization was assessed between Rac1 and bound/crosslinked APP or PLCδPH (white pixels). ( B ) Quantification of the mean % of Rac1 colocalized with APP (left) and PLCδPH (right) from three independent experiments ( n = 3; 15 images acquired for each condition per replicate). N2a cells transfected with ( C ) Cdc42-EGFP or ( E ) RhoA-EGFP, PLCδPH-mRFP and APP695. Cells were treated as described above, then incubated with tagged anti-APP antibodies and fixed following a 30 s incubation. Colocalization with APP or PLCδPH was measured (white pixels). ( D , F ) Quantification of the mean % of Cdc42 or RhoA colocalized with APP (left) and PLCδPH (right) from three independent experiments ( n = 3; 15 images acquired for each condition per replicate). Significant changes in the percentage colocalized between treatments were assessed by a one-way ANOVA with Tukey’s test. Data are presented as mean ± SEM; * p < 0.05. Representative images are single planes of whole-cell z-stacks; white bars represent the scale bar = 5 μm .

Article Snippet: G-LISA kits for GTP-bound Rac1 (BK128), Cdc42 (BK127) and RhoA (BK124) were all purchased from Cytoskeleton (Denver, CO, USA).

Techniques: Inhibition, Membrane, Transfection, Incubation

Journal: bioRxiv

Article Title: WNT11 suppresses tumor initiation and invasion by inactivating RAC1

doi: 10.64898/2026.07.13.738348

Figure Lengend Snippet: (A–C) Pulldown assays assessing GTP-bound (active) RAC1 in response to WNT11 overexpression in the WNT11 responsive WNT11-low cancer cell lines. Each panel includes: Lane 1 – lysate incubated with GTPγS (positive control); Lane 2 – lysate incubated with GDP (negative control); Lane 3 – untreated lysate (endogenous RAC1 activity). Lower panels show input blots for WNT11 and RAC1. (D–E) RAC1 activation in response to WNT11 knockdown in WNT11 responsive WNT11-high cell lines. The numeric label indicates the lysates were prepared from cells expressing control shRNA (1); Wnt11 shRNA#1 (2) and WNT11 shRNA#2 (3). At the bottom of the panel is a blot of the lysate without a pull-down. α-Tubulin was the loading control. (F-G) RAC1 activation in response to WNT11 modulation in WNT11 unresponsive cell lines. EV, Empty Vector control.

Article Snippet: RAC1 activation assays were performed using a pull-down assay kit to detect GTP-bound RAC1, following the manufacturer’s protocol from Cytoskeleton, Inc. (BK035; Denver, CO).

Techniques: Over Expression, Incubation, Positive Control, Negative Control, Activity Assay, Activation Assay, Knockdown, Expressing, Control, shRNA, Plasmid Preparation

Journal: bioRxiv

Article Title: WNT11 suppresses tumor initiation and invasion by inactivating RAC1

doi: 10.64898/2026.07.13.738348

Figure Lengend Snippet: (A) Representative images from matrigel invasion assays on OVCAR5 cells stably overexpressing WNT11, with or without the expression of a dominant-negative RAC1 mutant (RAC1 DN ). Quantification of invading cells is shown below the images and presented as mean ± SEM, with corresponding p values indicating statistical significance. (B) Representative images of third generation spheres in serial replating sphere formation assays using the same cell lines as in (A) . (C) Quantification of third-generation (Gen-3) spheres from the experiment shown in (B) . (D) qPCR analysis of Yamanaka stemness and NANOG gene expression using the cells in (A–C) . Data are presented as mean ± SEM (n ≥ 3, n is number of biological replicates). *p < 0.05 compared to control cells, set as baseline. (E) Representative images from matrigel invasion assays on U87 cells with stable WNT11 knockdown, with or without co-expression of constitutively active RAC1 (RAC1 CA ). Quantification of invading cells is shown below the images and presented as mean ± SEM, with corresponding p values indicating statistical significance. (F) Representative images of third generation spheres from serial replating sphere formation assays using the same cell line as (E) . (G) Quantification of third-generation (Gen-3) spheres from the experiment shown in (F) . (H) qPCR analysis of Yamanaka stemness gene and NANOG expression in the cells as in (E-G) . Data are presented as mean ± SEM (n ≥ 3, n is the number of biological replicates). *p < 0.05 compared to control cells, set baseline. EV, Empty Vector control.

Article Snippet: RAC1 activation assays were performed using a pull-down assay kit to detect GTP-bound RAC1, following the manufacturer’s protocol from Cytoskeleton, Inc. (BK035; Denver, CO).

Techniques: Stable Transfection, Expressing, Dominant Negative Mutation, Mutagenesis, Gene Expression, Control, Knockdown, Plasmid Preparation

Journal: STAR Protocols

Article Title: Protocol to identify covalent inhibitors targeting RhoA Cys16

doi: 10.1016/j.xpro.2026.104494

Figure Lengend Snippet: Identification of CL16 binding to RhoA by gel-based ABPP experiment (A) Rationale of gel-based ABPP for screening compound binding to RhoA in vitro . Purified RhoA protein was pre-treated with solvent control or library compounds followed by labelling with fluorescent activity-based probes. (B) Expected outcomes in gel-based ABPP. In the 1 st round of screening, compound binding to RhoA results in a decrease in in-gel fluorescence (FL) intensity. Further validation of the binding can be performed by experiments using varying concentrations of the hit compounds. In the 2 nd round of screening, selected hits are examined for their binding to Cdc42 and Rac1 to determine their selectivity for RhoA.

Article Snippet: Rac1 Pull-Down Activation Assay Biochem Kit , Cytoskeleton , Cat# BK035.

Techniques: Binding Assay, In Vitro, Purification, Solvent, Control, Activity Assay, Fluorescence, Biomarker Discovery

Journal: STAR Protocols

Article Title: Protocol to identify covalent inhibitors targeting RhoA Cys16

doi: 10.1016/j.xpro.2026.104494

Figure Lengend Snippet: Biochemical assays to Investigate RhoA inhibition by CL16 (A) Expected results of RhoGTPase activity assay in which CL16 selectively impairs the activation of RhoA and not Rac1 and Cdc42. (B) Schematic diagram illustrating the workflow of the co-immunoprecipitation experiment. (C) Expected results of co-immunoprecipitation whereas CL16 interferes with interactions between RhoA and ARHGEF1.

Article Snippet: Rac1 Pull-Down Activation Assay Biochem Kit , Cytoskeleton , Cat# BK035.

Techniques: Inhibition, Activity Assay, Activation Assay, Immunoprecipitation