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

Journal: bioRxiv

Article Title: Sphingosine-1-phosphate cross-talks to Notch via a S1PR1-Dll4-MPDZ complex to regulate endothelial barrier function

doi: 10.64898/2026.05.20.726610

Figure Lengend Snippet: a , Immunoblots showing cleaved Notch1 (c-Notch1) and total Notch1 in MVECs treated with S1P for 1-hour ± γ-secretase inhibitor DAPT; quantification below (mean ± SEM). b , Fluorescent micrographs of YFP-Notch1 reporter intensity after S1P treatment; quantification below. Scale bar, 50 μm. c-d , Fold change in HES1 and HEY1 gene expression levels in (c) MVECs after S1P ± DAPT treatment, and in (d) mouse tissues after intravenous injection of S1P ± DAPT. e , Evans blue dye leakage in mouse dermis 1-hour post-injection with DMSO, S1P, or S1P + DAPT. Scale bar, 5 mm. f-i , Quantified dye leakage in (f) dermis, (g) lung, (h) liver, and (i) kidney, measured via absorbance. j , Heat maps of dextran dye diffusion in engineered microvessels ± S1P. Scale bar, 50 μm. k-l , Diffusive permeability in microvessels lined with (k) DAPT-vs. DMSO-treated or (l) NOTCH1 KO vs. SCR KO MVECs (mean ± SD). m , Micrographs of VE-cadherin (magenta), actin (green), and nuclei (blue) staining in microvessels. Scale bar, 50 μm. n-o , Mean corrected intensities of junctional (n) VE-cadherin and (o) actin from (m). p , Rac1 activity (PBD pull-down). Active/total Rac1 quantification below. q , Schematic of canonical Notch signaling. r , HES1 / HEY1 gene expression levels in dominant negative-MAML-GFP (DN-MAML-GFP) normalized to GFP expressing cells. s , Permeability in DN-MAML-GFP vs GFP-lined microvessels ± S1P. t , Schematic of Notch cortical pathway mediated by its transmembrane domain (TMD). u , Permeability in TMD-RFP vs RFP-lined microvessels ± S1P. v , Permeability in TMD-RFP vs RFP-lined microvessels ± clinical S1P receptor degrader fingolimod.

Article Snippet: PAK-PBD beads to bind active Rac1 (PAK-02) were purchased from Cytoskeleton and reconstituted according to the manufacturer’s instructions.

Techniques: Western Blot, Gene Expression, Injection, Diffusion-based Assay, Permeability, Staining, Activity Assay, Dominant Negative Mutation, Expressing

Journal: Nature Communications

Article Title: Endocrine therapy reprogramming of breast cancer facilitates metastatic escape via upregulation of P-Rex1/Rac1 signalling

doi: 10.1038/s41467-026-70683-x

Figure Lengend Snippet: A Gene set enrichment analysis on Fast-growing compared to parental cells ( n = 288 genes) and Endocrine Tolerant resistant cells compared to parental cells ( n = 288 genes) using Curated.MSigDB. B / C Steiner Forest signalling network analysis in upregulated genes ( n = 288) of B Fast-growing and C Endocrine Tolerant cells. Node size indicates number of attached edges, with nodes with >8 in bold. Node colours: Brown=genes, light blue=phenotype, dark blue=protein complex, pink=protein family. D Multiplexed analysis of EGFR phosphorylation in: basal cells, 30 min 100 ng/mL EGF, 30 min 100 nM 17 β-estradiol, or 30 min serum stimulation. Analysed by two-way ANOVA with Bonferroni’s multiple comparison test from n = 3 biological replicates, error bars=SD. E Western blot of EGFR, pEGFR (Y1045) and β-actin in Fast and Endocrine Tolerant cells after 100 ng/mL EGF treatment for indicated times, representative of 3 biological replicates. F Multiplexed analysis of HER2 phosphorylation following treatment described in D . Analysed by two-way ANOVA with Bonferroni’s multiple comparison test. G Western blot of HER2, pHER2 (Y1221/1222) and β-actin in Fast and Endocrine Tolerant cells after 100 ng/mL EGF stimulation for indicated times, representative of 3 biological replicates. (H) PREX1 transcripts from scRNAseq analysis of MCF-7 cells treated for 48 hours with fulvestrant ( n = 340 cells), and in Fast-growing ( n = 578 cells) and Endocrine Tolerant ( n = 659 cells). I Western blot of P-Rex1, Rac1, Pak2 and β-actin in Fast and Endocrine Tolerant cells, representative of 3 biological replicates. J Densitometry of P-Rex1 normalised to β-actin from n = 3 biological replicates. Analysed by two-sided t-test, error bars=SD. K Western blot of P-Rex1, ER and GAPDH in short-term fulvestrant and estrogen treated MCF-7 cells. Representative of 5 replicates. L Densitometry of P-Rex1 normalised to β-actin from n = 5 biological replicates, analysed by one-way ANOVA with Tukey’s multiple comparisons, error bars=SEM. M / N Multiplexed analysis of M pIGF1R and N pIR phosphorylation following treatment described in D . Analysed by two-way ANOVA with Bonferroni’s multiple comparisons. O Western blot of pIR/pIGF1R and β-actin in Fast and Endocrine Tolerant cells after 30 mins insulin. Representative of 3 replicates. P Western blot for P-Rex1 and GAPDH in Fast and Endocrine Tolerant cells expressing non-targeting (NT) and PREX1 shRNA, representative of n = 2 biological replicates. Q Quantitation of transwell migration assay of Fast and Endocrine Tolerant cells that express NT and PREX1 shRNA. Analysed by unpaired two-sided t-test from n = 3 biological replicates, error bars=SEM. R / S Correlation between PREX1 and EGFR in the (R) TCGA (RNAseq RSEM; n = 812 cancers) and S Metastatic Breast Cancer Project (RNAseq RSEM; n = 67 cancers). r=Spearman’s correlation. Orange line is least squares fit. Source data provided in Source Data file.

Article Snippet: Primary antibodies were cyclin A (1:1000, #sc-239), cyclin D1 (1:500, #sc-20044), estrogen receptor-α (human) (1:500, #sc-543), β-actin (1:15,000, #sc-69879), and GAPDH (1:15,000, #sc-32233) from Santa Cruz Biotechnology; p21 (1:1000, #610234), and total Rb (1:500, #554136) from BD Biosciences; P-Rex1 (1:1000, #HPA001927) from Sigma-Aldrich; Rac1 (1:1000, #ARC03) from Cytoskeleton Inc; PAK2 (1:2000, #A4553) from AB Clonal; estrogen receptor-α (mouse) (1:1000, #ab32063) from Abcam; EGFR (1:1000, #H00001956-M02) from Abnova; pHER2 (Tyr1221/1222) (1:1000, #2243); pEGFR (Tyr1045) (1:1000, #2237), HER2 (1:1000, #2165) and pERK (Thr202/Tyr204) (1:1000, #9101) from Cell Signalling Technology; and pIR/IGF1R (Tyr1162/Tyr1163) (1:1000, #44-804 G) from Invitrogen.

Techniques: Phospho-proteomics, Comparison, Western Blot, Expressing, shRNA, Quantitation Assay, Transwell Migration Assay, RNA sequencing

Journal: Nature Communications

Article Title: Endocrine therapy reprogramming of breast cancer facilitates metastatic escape via upregulation of P-Rex1/Rac1 signalling

doi: 10.1038/s41467-026-70683-x

Figure Lengend Snippet: A Schematic of spontaneous tumour development and dissemination in the MMTV-PyMT model. Expression of Rac1 (A_51_P513254), Prex1 (A_51_P348372) and Pak2 (A_51_P172323) in stages of MMTV-PyMT development ( GSE43566 ) of adenoma ( n = 5), carcinoma ( n = 6), dissemination ( n = 6) and metastases ( n = 5). Data analysed by one-way ANOVA with Tukey’s multiple comparisons. B Schematic of derivation of MMTV-PyMT Rac1-FRET biosensor mice with biosensor in active (blue) and inactive (red) conformations, and derivation of a chronic tamoxifen biosensor cell line. C MMTV-PyMT Rac1-FRET parental (PyMT) and chronic tamoxifen (PyMT-Tam) cell lines western blotted for ER, P-Rex1, Pak2 and Rac1. β-actin loading control is shown for each western blot. Representative of three replicates. D Densitometry of P-Rex1 expression normalised to β-actin from triplicate biological replicates, analysed by two-sided t-test, and error bars represent SEM. E Representative image of Rac1-FRET activity in MMTV-PyMT cells chronically treated with tamoxifen with matched parental control. Scale bar = 100 µm. F Quantitation of Rac1-FRET activity of cells in E . Triplicate biological replicates analysed by two-sided t-test; error bars represent SEM. G Schematic of MMTV-PyMT Rac1-FRET biosensor mouse with titanium optical imaging window implanted over a mammary tumour. H Tumour growth of control ( n = 11) and tamoxifen ( n = 9) treated MMTV-PyMT Rac1-FRET mice. Data analysed by two-way ANOVA with a mixed effect model. Error bars represent SEM. I Image of Rac1-FRET activity in control and tamoxifen treated mammary tumours, representative of three biological replicates. Scale bar = 50 µm. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were cyclin A (1:1000, #sc-239), cyclin D1 (1:500, #sc-20044), estrogen receptor-α (human) (1:500, #sc-543), β-actin (1:15,000, #sc-69879), and GAPDH (1:15,000, #sc-32233) from Santa Cruz Biotechnology; p21 (1:1000, #610234), and total Rb (1:500, #554136) from BD Biosciences; P-Rex1 (1:1000, #HPA001927) from Sigma-Aldrich; Rac1 (1:1000, #ARC03) from Cytoskeleton Inc; PAK2 (1:2000, #A4553) from AB Clonal; estrogen receptor-α (mouse) (1:1000, #ab32063) from Abcam; EGFR (1:1000, #H00001956-M02) from Abnova; pHER2 (Tyr1221/1222) (1:1000, #2243); pEGFR (Tyr1045) (1:1000, #2237), HER2 (1:1000, #2165) and pERK (Thr202/Tyr204) (1:1000, #9101) from Cell Signalling Technology; and pIR/IGF1R (Tyr1162/Tyr1163) (1:1000, #44-804 G) from Invitrogen.

Techniques: Expressing, Western Blot, Control, Activity Assay, Quantitation Assay, Optical Imaging

Journal: Nature Communications

Article Title: Endocrine therapy reprogramming of breast cancer facilitates metastatic escape via upregulation of P-Rex1/Rac1 signalling

doi: 10.1038/s41467-026-70683-x

Figure Lengend Snippet: A The Rac1 pathway can be targeted by small molecule inhibitors; NSC23766, 1A-116, R-ketorolac. B / C Endocrine Tolerant and Fast-growing cells were treated with DMSO, 100 µM R-ketorolac, 25 µM NSC23766 and 6.25 µM 1A-116 for 1 week, and colony formation detected with 10% Diff Quik Stain 2. Colony formation (% colony area) ( n = 5 biological replicates) was quantitated using Image J. Data analysed by one-way ANOVA with multiple comparisons. Error bars are SEM. D / E Wound closure in Endocrine Tolerant and Fast-growing cells treated with DMSO, 100 µM R-ketorolac, 25 µM NSC23766 and 6.25 µM 1A-116 for 24 hours. Data ( n = 3 biological replicates) analysed by two-way repeated measure ANOVA for each treatment versus vehicle. Error bars are SEM, or smaller than the symbol. F Schematic of tamoxifen resistant MMTV-PyMT:Rac1-FRET biosensor mouse with titanium window implanted over mammary tumour to monitor Rac1-FRET activity after treatment with NSC23766 (4 mg/kg; intraperitoneal injection) or R-ketorolac (1 mg/kg; oral gavage). G Representative image of Rac1-FRET activity in tamoxifen resistant (TamR) MMTV-PyMT tumours treated with a single injection 4 mg/kg NSC23766, and single cell based temporal quantitation of Rac1-FRET activity of tumour. Data ( n = 4 mice measured at 0, 2, 24 h; n = 2 at 6 h) analysed by one-way ANOVA with Dunnett’s multiple comparison test, error bars are SEM. H Representative image of Rac1-FRET activity in tamoxifen resistant (TamR) MMTV-PyMT tumours treated with 1 mg/kg R-ketorolac and imaged for up to 96 hours, and single cell based temporal quantitation of Rac1-FRET activity of tumour. Data ( n = 4 mice measured at 0, 6, 24, 48, 72, 96 h; n = 2 at 2 h) analysed by one-way ANOVA with Dunnett’s multiple comparison test, error bars are SEM. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were cyclin A (1:1000, #sc-239), cyclin D1 (1:500, #sc-20044), estrogen receptor-α (human) (1:500, #sc-543), β-actin (1:15,000, #sc-69879), and GAPDH (1:15,000, #sc-32233) from Santa Cruz Biotechnology; p21 (1:1000, #610234), and total Rb (1:500, #554136) from BD Biosciences; P-Rex1 (1:1000, #HPA001927) from Sigma-Aldrich; Rac1 (1:1000, #ARC03) from Cytoskeleton Inc; PAK2 (1:2000, #A4553) from AB Clonal; estrogen receptor-α (mouse) (1:1000, #ab32063) from Abcam; EGFR (1:1000, #H00001956-M02) from Abnova; pHER2 (Tyr1221/1222) (1:1000, #2243); pEGFR (Tyr1045) (1:1000, #2237), HER2 (1:1000, #2165) and pERK (Thr202/Tyr204) (1:1000, #9101) from Cell Signalling Technology; and pIR/IGF1R (Tyr1162/Tyr1163) (1:1000, #44-804 G) from Invitrogen.

Techniques: Diff-Quik, Staining, Activity Assay, Injection, Single Cell, Quantitation Assay, Comparison