dominant negative rhoa  (Addgene inc)

Journal: The EMBO Journal

Article Title: Macrophages form dendrite-like pseudopods to enhance bacterial ingestion

doi: 10.1038/s44318-025-00515-z

Figure Lengend Snippet: ( A ) Schematic diagram showing the screening and sorting of none-DLPs (P−) and more-DLPs (P + ) iBMDM macrophages. ( B ) Illustration of upregulated genes in P− and P+ iBMDMs with LPS treatment. ( C ) Biological process enrichment analysis of upregulated genes in P+ but not in P− iBMDMs with LPS treatment. The top ten enriched terms are shown in the bubble chart. ( D ) Heatmap showing the transcriptional levels of surface markers of macrophages with LPS stimulation in P+ and P− iBMDMs by RNA sequencing. ( E ) Cellular component enrichment analysis of upregulated genes in P+ but not in P− iBMDMs. The top 10 enriched items are shown in the bubble chart. In ( C ) and ( E ), the colored bubbles displayed from red to blue indicating the descending order of P . adjust value. The sizes of the bubbles are displayed from small to large in ascending order of gene counts. The x and y axis represent the gene ratio and the GO terms, respectively. ( F ) Heatmap showing the transcriptional levels of RhoA GEFs and GAPs by RNA sequencing. ( G ) ARHGEF3 transcriptional levels were measured by RT-qPCR in P− and P+ iBMDM macrophages. ( H ) Western blot analysis of ARHGEF3 protein levels upon LPS stimulation in P− and P+ iBMDM macrophages. ( I ) Western blot analysis of ARHGEF3 and the active levels of RhoA in THP-1 macrophages treated with LPS for indicated times. ( J ) Quantification of the relative luciferase activity upon LPS or LPS + TAK242 treatment. ( K ) Western blot analysis of ARHGEF3 and the active levels of RhoA upon LPS, TAK-242 or combined LPS and TAK-242 treatment. ( L ) Upper panel showing the diagram of ARHGEF3 knockdown procedure in THP-1 macrophages. Pie charts in lower panels illustrating the percentage of three shapes with RNAi treatment and salmonella infection for 6 h. Ctrl (no transfection), n = 758; siNC, n = 384 cells; siARHGEF3, n = 590 cells. ( M ) Diagram of the RhoA-ROCK signaling pathway. ( N ) Upper panel showing the diagram of RhoA CA/DN plasmid transfection procedure in THP-1 macrophages. Pie charts in lower panels illustrating the percentage of three shapes with transfection and salmonella infection for 6 h. EGFP (Control), n = 236; RhoA CA, n = 244 cells; RhoA DN, n = 277 cells. ( O ) Upper panel showing the diagram of Y-27632 treatment procedure. Pie charts in lower panels illustrating the percentage of three shapes with or without Y-27632 treatment and with Salmonella infection for 6 h. Veh., n = 228 cells; Y-27632 treatment, n = 186 cells. ( P ) Co-immunoprecipitation (Co-IP) verified the interaction between mTLR4 and ARHGEF3 in iBMDM cells stably expressed mTLR4-HA. The protein complex was enriched using Protein A/G agarose beads. ( Q ) Representative images of THP-1 macrophages with DLPs stimulated by LPS without or with TAK242 pretreatment and stained with ARHGEF3 and TLR4 antibodies. White dash lines depict the outline of the cell. Cropped views depict DLPs regions. Yellow arrows depict the DLPs. Bars, 20 µm (in cell images) and 5 µm (in the magnified images). ( R ) Quantification of the mean intensity of ARHGEF3 in cell body (except nucleus) and the DLPs of THP-1 macrophages treated with LPS. Data are presented as mean ± s.d. from three independent experiments. Dots in quantifications presented individual cells. P values from left to right ( G ): P = 0.28437, P = 0.00013. P = 0.00035 ( J ). P values from left to right ( L ): P = 0.01918, P = 0.00050. P = 0.00057 ( N ), P = 7.67308E-05 ( O ). P = 3.63749E-20 ( R ). ns P > 0.05; *** P < 0.001; **** P < 0.0001: unpaired two-tailed Student’s t test ( G , J , O , R ), one-way ANOVA with Sidak’s analysis ( L , N ), two-way ANOVA with Sidak’s analysis ( F ), Benjamini–Hochberg (BH) analysis ( C , E ). .

Article Snippet: THP-1 macrophages were transiently transfected with constitutively active RhoA (CA, L63) (#15900, Addgene), dominant negative RhoA (DN, N19) (#15901, Addgene) or EGFP (negative control) by using LipofectamineTM LTX (#15338100, Invitrogen), and then bright-field live-cell imaging was conducted.

Techniques: RNA Sequencing, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Knockdown, Infection, Transfection, Plasmid Preparation, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Stable Transfection, Staining, Two Tailed Test

Journal: Life science alliance

Article Title: AR activates YAP/TAZ differentially in prostate cancer.

doi: 10.26508/lsa.202201620

Figure Lengend Snippet: Figure 3. AR-YAP/TAZ activation is regulated by RhoA-SRF. (A) Western blot analysis of LNCaP lysates from three different populations of cells, expressing short hairpin (sh) shCon LNCaP, targeting RhoA mRNA (shRhoA #1), and shRhoA #1 LNCaP exogenously expressing AR as illustrated. All cells were cultured in FBS. (B) qRT-PCR analysis of YAP, TAZ, CYR61, and CTGF mRNA from shRhoA LNCaP cells compared with shCon LNCaP cells. (C) qRT-PCR analysis of YAP, TAZ, CYR61, and CTGF mRNA from shRhoA LNCaP cells exogenously expressing AR, labelled shRhoA/AR++, compared with shCon LNCaP cells. (D) Confocal images of LNCaP cells expressing empty vector and LNCaP cells expressing dominant negative RhoA N19 stimulated with

Article Snippet: For the purpose of IF experiments, RhoA KD cells and shCon cells treated were transfected with the dominant negative RhoA N19 plasmid Addgene (#15901) (Nobes & Hall, 1999) or SRF(HA) plasmid, pCGN-SRF, Addgene (#11977) (Johansen & Prywes, 1993) using Lipofectamine LTX transfection reagent (Invitrogen) following the manufacturer’s protocol.

Techniques: Activation Assay, Western Blot, Expressing, Cell Culture, Quantitative RT-PCR, Plasmid Preparation, Dominant Negative Mutation

Journal: PLoS ONE

Article Title: RhoA promotes epidermal stem cell proliferation via PKN1-cyclin D1 signaling

doi: 10.1371/journal.pone.0172613

Figure Lengend Snippet: (A) Transfection of RhoA (+/+) (G14V), RhoA (-/-) (T19N) or pGFP control plasmid (1.5 μg/ml each plasmid) into huESCs using Lipofectamine transfection reagent. (B) After transfection, cell proliferation was quantitated using a CCK-8 assay according to the manufacturer’s instructions. (C) DNA synthesis was quantified based on [ 3 H]-thymidine uptake in disintegrations per minute (DPM). * P<0.05 versus control, **P<0.01 versus control. Data are shown as the mean ± SD of duplicate experiments on samples from three different donors (unpaired, two-tailed Student's t-test).

Article Snippet: Vectors encoding dominant-negative RhoA (−/−) (RhoA T19N: threonine at residue 19 replaced with asparagine) or dominant positive RhoA +/+ (RhoA G14V: glycine at residue 14 replaced with valine) and the pGFP control plasmid were purchased from Fisher Scientific (Waltham, MA, USA) [ ].

Techniques: Transfection, Control, Plasmid Preparation, CCK-8 Assay, DNA Synthesis, Two Tailed Test

Journal: PLoS ONE

Article Title: RhoA promotes epidermal stem cell proliferation via PKN1-cyclin D1 signaling

doi: 10.1371/journal.pone.0172613

Figure Lengend Snippet: HuESCs were transfected for 48 h with RhoA (+/+) (G14V), RhoA (-/-) (T19N) or pGFP control plasmid (1.5 μg/ml each plasmid) using Lipofectamine transfection reagent. (A) Cell lysates were subjected to Western blot assays with anti-Thr 774 phosphorylated (p-)PKN1, anti-total PKN1 and anti-GAPDH antibodies. (B) Thr 774 p-PKN1/total-PKN1 was determined after densitometric analysis. *P<0.05 versus control, **P<0.01 versus control. The data are presented as the mean ± SD (error bars) of at least three separate experiments (unpaired, two-tailed Student's t-test).

Article Snippet: Vectors encoding dominant-negative RhoA (−/−) (RhoA T19N: threonine at residue 19 replaced with asparagine) or dominant positive RhoA +/+ (RhoA G14V: glycine at residue 14 replaced with valine) and the pGFP control plasmid were purchased from Fisher Scientific (Waltham, MA, USA) [ ].

Techniques: Transfection, Control, Plasmid Preparation, Western Blot, Two Tailed Test