rac1  (Cytoskeleton Inc)

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

Journal: Development (Cambridge, England)

Article Title: Mouse scalp development requires Rac1 and SRF for the maintenance of mechanoresponsive mesenchyme

doi: 10.1242/dev.204965

Figure Lengend Snippet: (A) Conditional knockout strategy for Pdgfra Cre Rac1 Flox/Flox . (B) Lateral and dorsal view of Rac1 -Wt and -KO P1 pups. The arrow indicates the scalp defect (n=8/8). (C) Genotypes of pups at P1, two-tailed Chi-square test of observed genotypes. 100% of Pdgfra Cre Rac1 Flox/Flox mice exhibited defects shown in Fig.1B . (D) Skeletal preparation of P1 skull with alizarin red and alcian blue staining (n=5). Dotted lines outline the individual bones of the calvarium. (E) Quantification of cranial bones (n=5). Data normalized to the length of the skull. Data represented as mean ± SD, Unpaired t-test with Welch’s correction. (F) Micro CT surface rendering of skulls at P1 showing loss of calvaria bone volume.

Article Snippet: The proteins were transferred to nitrocellulose membrane, washed with 1X TBST (1x TBS + 0.1%Tween 20), blocked with 5% BSA, and then subjected to detection with primary antibodies (anti-mouse Rac1 CST (Cell Signaling Technology) #8631, anti-rabbit SRF CST#5147, anti-mouse LaminA/C CST#4777, anti-rabbit β-Tubulin CST#2146S, anti-rabbit α-SMA CST#19245, anti-rabbit MKL1/MRTF-A CST#14760, anti-rabbit MKL2/MRTF-B CST#14613 and anti-rabbit GAPDH CST#5174, all at 1:2000 in 5% BSA) at 4°C O/N.

Techniques: Knock-Out, Two Tailed Test, Staining, Micro-CT

Journal: Development (Cambridge, England)

Article Title: Mouse scalp development requires Rac1 and SRF for the maintenance of mechanoresponsive mesenchyme

doi: 10.1242/dev.204965

Figure Lengend Snippet: (A) Western blot with cytoplasmic (c) and nuclear (n) fractions (n=3). (B) Western blot for whole cell lysate showing reduction in SRF and its target gene αSMA (n=3). (C) qPCR data for Rac1 , Srf and Acta2 levels (n=6), Data represented as mean ± SD, Unpaired t-test with Welch’s correction. (D) Collagen lattice contraction, expressed as a percentage of the lattice area at 24 hours, with representative images at 0 and 24 hr after lattice detachment (n=5 biological replicates per genotype with 2 technical replicates). Data are plotted as mean ± SD, Unpaired t-test with Welch’s correction. (E-F) Immunostaining of SRF and DAPI for E12.5 and 13.5 Rac1 -Wt and -KO (n=4–5). Scale=50μm. Quantification of Tom + Srf + cells per section in the apical mesenchyme for E12.5 and 13.5 Rac1 -Wt and -KO. Data represented as mean ± SD, Unpaired t-test with Welch’s correction.

Article Snippet: The proteins were transferred to nitrocellulose membrane, washed with 1X TBST (1x TBS + 0.1%Tween 20), blocked with 5% BSA, and then subjected to detection with primary antibodies (anti-mouse Rac1 CST (Cell Signaling Technology) #8631, anti-rabbit SRF CST#5147, anti-mouse LaminA/C CST#4777, anti-rabbit β-Tubulin CST#2146S, anti-rabbit α-SMA CST#19245, anti-rabbit MKL1/MRTF-A CST#14760, anti-rabbit MKL2/MRTF-B CST#14613 and anti-rabbit GAPDH CST#5174, all at 1:2000 in 5% BSA) at 4°C O/N.

Techniques: Expressing, Western Blot, Immunostaining

Journal: Development (Cambridge, England)

Article Title: Mouse scalp development requires Rac1 and SRF for the maintenance of mechanoresponsive mesenchyme

doi: 10.1242/dev.204965

Figure Lengend Snippet: (A) Lateral view of Rac1 -Wt and -KO embryos from E11.5 to E18.5. The orange arrows indicate blebbing/oedema, hematoma (dotted arrow), and scalp defect (E11.5 – n=17 Wt and 2 KO; E12.5 – n=44 Wt and 12 KO; E13.5 – n=71 Wt and 20 KO; E14.5 – n=40 Wt and 16 KO; E18.5 - n=71 Wt and 16 KO). (B) Graphical representation of coronal plane of section for E14.5 and E18.5 mouse embryos shown in C-D. (C) H&E staining of coronal sections of Rac1 -Wt and -KO E14.5 embryos. Dotted box=insets, dotted arrow=hypoplasia of basolateral mesenchyme, arrow=blebbing/oedema of intermediate and apical mesenchyme. The insets are represented as 1=apical, 2=intermediate, and 3=basolateral region of the head (n=3). Apical oedematous area is quantified in Supplemental Figure 1E . Scale=50μm (D) Trichrome staining of coronal sections of Rac1 -Wt and -KO E18.5 head. Arrows in Rac1 -KO indicate the apical limits of the intact calvaria, with the brain protruding through the gap and covered by a thin layer of the rudimentary dermis. Scale=50μm. The dotted box = Inset. The bottom panel represents the inset 1 = basolateral, 2 = apical, d = dermis, and s = skull (n=3–4). Scale=50μm (E) Quantification of cranial gap and dermal thickness of E18.5 Rac1 -Wt and -KO represented in Fig. 2D . Data represented as mean ± SD, Unpaired, one-tailed t-test with Mann-Whitney test.

Article Snippet: The proteins were transferred to nitrocellulose membrane, washed with 1X TBST (1x TBS + 0.1%Tween 20), blocked with 5% BSA, and then subjected to detection with primary antibodies (anti-mouse Rac1 CST (Cell Signaling Technology) #8631, anti-rabbit SRF CST#5147, anti-mouse LaminA/C CST#4777, anti-rabbit β-Tubulin CST#2146S, anti-rabbit α-SMA CST#19245, anti-rabbit MKL1/MRTF-A CST#14760, anti-rabbit MKL2/MRTF-B CST#14613 and anti-rabbit GAPDH CST#5174, all at 1:2000 in 5% BSA) at 4°C O/N.

Techniques: Staining, One-tailed Test, MANN-WHITNEY

Journal: Development (Cambridge, England)

Article Title: Mouse scalp development requires Rac1 and SRF for the maintenance of mechanoresponsive mesenchyme

doi: 10.1242/dev.204965

Figure Lengend Snippet: (A-C) Immunostaining of S100a6, αSMA, and DAPI for E12.5-E14.5 Rac1 -Wt and -KO (n=3). b=brain. Scale=50μm (D) Quantification of αSMA area at the apex of head mesenchyme (n=3). Data represented as mean ± SD, Unpaired t-test with Welch’s correction (E) Graphical representation of dorsal head development. From E12.5-E13.5, the Tomato + head mesenchyme expands from a simple layer of αSMA + S100a6 + cells into a multilayered tissue with αSMA + S100a6 + dermal cells overlying αSMA neg S100a6 + meningeal cells. In the mutant, αSMA is low, and layers do not expand.

Article Snippet: The proteins were transferred to nitrocellulose membrane, washed with 1X TBST (1x TBS + 0.1%Tween 20), blocked with 5% BSA, and then subjected to detection with primary antibodies (anti-mouse Rac1 CST (Cell Signaling Technology) #8631, anti-rabbit SRF CST#5147, anti-mouse LaminA/C CST#4777, anti-rabbit β-Tubulin CST#2146S, anti-rabbit α-SMA CST#19245, anti-rabbit MKL1/MRTF-A CST#14760, anti-rabbit MKL2/MRTF-B CST#14613 and anti-rabbit GAPDH CST#5174, all at 1:2000 in 5% BSA) at 4°C O/N.

Techniques: Immunostaining, Mutagenesis

Journal: Development (Cambridge, England)

Article Title: Mouse scalp development requires Rac1 and SRF for the maintenance of mechanoresponsive mesenchyme

doi: 10.1242/dev.204965

Figure Lengend Snippet: (A-C) Staining of Edu for E12.5-E14.5 Rac1 -Wt and -KO (n=3) basolateral and apical mesenchyme. Scale = 50μm (D) Quantification of Tom + Edu + cells in basolateral and apical mesenchyme for E12.5-E14.5 (n=3), Data represented as mean ± SD, Unpaired t-test with Welch’s correction. (E) In vitro proliferation (Crystal violet assay) for Rac1 -Wt and -KO dermal fibroblasts (n=2 biological replicates per genotype with 3 technical replicates), Data represented as mean ± SD. (F) Immunostaining of phalloidin and αSMA for Rac1 -Wt and -KO dermal fibroblasts (n=3) Scale=50μm

Article Snippet: The proteins were transferred to nitrocellulose membrane, washed with 1X TBST (1x TBS + 0.1%Tween 20), blocked with 5% BSA, and then subjected to detection with primary antibodies (anti-mouse Rac1 CST (Cell Signaling Technology) #8631, anti-rabbit SRF CST#5147, anti-mouse LaminA/C CST#4777, anti-rabbit β-Tubulin CST#2146S, anti-rabbit α-SMA CST#19245, anti-rabbit MKL1/MRTF-A CST#14760, anti-rabbit MKL2/MRTF-B CST#14613 and anti-rabbit GAPDH CST#5174, all at 1:2000 in 5% BSA) at 4°C O/N.

Techniques: Staining, In Vitro, Crystal Violet Assay, Immunostaining

Journal: Development (Cambridge, England)

Article Title: Mouse scalp development requires Rac1 and SRF for the maintenance of mechanoresponsive mesenchyme

doi: 10.1242/dev.204965

Figure Lengend Snippet: (A) Srf -Wt and -KO E18.5 (Lateral view) pups. The arrow indicates the apical head defect (n=13/13). (B) Genotypes of pups at E18.5, Chi-square analysis of genotypes with two-tailed P value, 100% of Pdgfra Cre ;Srf Flox/Flox mice exhibit apical head defects. (C) Skeletal prep of the E18.5 skull with alizarin red and alcian blue staining (n=7). (D) Quantification of calvaria bones (n=7). Data normalized to the length of the skull. Data represented as mean ± SD, Unpaired t-test with Welch’s correction. (E) Lateral view of Srf -Wt and -KO embryos from E12.5 to E18.5. The orange arrows indicate blebbing/oedema, brain protrusion, and apical head defect (E12.5 - n=57 Wt and 11 KO; E13.5 – n=108 Wt and 9 KO; E14.5 – n=69 Wt and 9 KO; E18.5 – n=159 Wt and 13 KO). (F) Graphical representation of coronal plane of section for E12.5 and E14.5 mouse embryos shown in G and Suppl. Fig. 6B . (G) H&E staining of coronal sections of Srf -Wt and -KO E12.5 embryos. Dotted box=insets, arrow=hypoplasia and edema of basolateral through intermediate and apical mesenchyme. The insets are represented as 1=apical, 2=intermediate, and 3=basolateral region of the head (n=3). Scale=50μm

Article Snippet: The proteins were transferred to nitrocellulose membrane, washed with 1X TBST (1x TBS + 0.1%Tween 20), blocked with 5% BSA, and then subjected to detection with primary antibodies (anti-mouse Rac1 CST (Cell Signaling Technology) #8631, anti-rabbit SRF CST#5147, anti-mouse LaminA/C CST#4777, anti-rabbit β-Tubulin CST#2146S, anti-rabbit α-SMA CST#19245, anti-rabbit MKL1/MRTF-A CST#14760, anti-rabbit MKL2/MRTF-B CST#14613 and anti-rabbit GAPDH CST#5174, all at 1:2000 in 5% BSA) at 4°C O/N.

Techniques: Two Tailed Test, Staining

Journal: Development (Cambridge, England)

Article Title: Mouse scalp development requires Rac1 and SRF for the maintenance of mechanoresponsive mesenchyme

doi: 10.1242/dev.204965

Figure Lengend Snippet: (A-C) Immunostaining of S100a6, αSMA, and DAPI for E12.5-E14.5 Srf -Wt and -KO (n=2–6). b=brain. Scale=50μm (D) Comparison of the αSMA area at the apical mesenchyme for both Rac1 -KO and Srf -KO. Rac1-KO data are the same as Figure 3D . Data represented as mean ± SD, Unpaired t-test with Welch’s correction.

Article Snippet: The proteins were transferred to nitrocellulose membrane, washed with 1X TBST (1x TBS + 0.1%Tween 20), blocked with 5% BSA, and then subjected to detection with primary antibodies (anti-mouse Rac1 CST (Cell Signaling Technology) #8631, anti-rabbit SRF CST#5147, anti-mouse LaminA/C CST#4777, anti-rabbit β-Tubulin CST#2146S, anti-rabbit α-SMA CST#19245, anti-rabbit MKL1/MRTF-A CST#14760, anti-rabbit MKL2/MRTF-B CST#14613 and anti-rabbit GAPDH CST#5174, all at 1:2000 in 5% BSA) at 4°C O/N.

Techniques: Immunostaining, Comparison