Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: Overall survival and the expression levels of Rho/Rac family genes in DLBCL. Kaplan–Meier curve of overall survival for individual member ( A ) RAC1, ( B ) RAC2, ( C ) RND1, ( D ) CDC42, ( E ) RHOQ, ( F ) RHOF; HR, hazard ratio.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques: Expressing

Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: Correlation between the expression of Rho/Rac family members’ and BTK. ( A ) Co-expression heatmap of Rho/Rac family genes and BTK; ( B ) Scatter diagram demonstrated the correlation between BTK and RAC1 , ( C ) CDC42, ( D ) RHOF and ( E ) RHOQ. * p <0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques: Expressing

Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: RAC1 expression levels in 18 different types of tumor tissues and paired adjacent normal tissues. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: non-significance. BLCA, Bladder Urothelial Carcinoma; BRCA, Breast invasive carcinoma; CHOL, Cholangio carcinoma; COAD, Colon adenocarcinoma; ESCA, Esophageal carcinoma; HNSC, Head and Neck squamous cell carcinoma; KICH, Kidney Chromophobe; KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma; PAAD, Pancreatic adenocarcinoma; PRAD, Prostate adenocarcinoma; READ, Rectum adenocarcinoma; STAD, Stomach adenocarcinoma; THCA, Thyroid carcinoma; UCEC, Uterine Corpus Endometrial Carcinoma.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques: Expressing

Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: Correlation between RAC1 expression and DLBCL clinical characteristics.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques: Expressing

Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: Logistic analyses of RAC1 expression and clinical characteristics of DLBCL.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques: Expressing

Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: RAC1 expression in patients with DLBCL according to different clinical stages.( A ) In the TCGA-DLBC dataset, RAC1 was upregulated in stage I/II as compared with that in stage III/IV. ( B ) RAC1 protein IHC staining in lymphadenitis and ( C ) DLBCL stage II, ( D ) stage III, ( E ) stage IV. Magnification 20 × 20, * p < 0.05. The staining of 3,3′-diaminobenzidine (DAB) was employed for RAC1 expression (yellow and brown) compared to negative sample (blue). Scale bar (bottom, right) = 100 μm.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: Cox regression analyses of clinical features associated with DLBCL overall survival.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Rac1 Is Crucial for Hair Follicle Integrity but Is Not Essential for Maintenance of the Epidermis

doi: 10.1128/mcb.00075-06

Figure Lengend Snippet: FIG. 7. Analysis of signaling pathway activation, protein expression, and activity of GTPases in Rac1-deficient epidermis. (A) Western blot analysis of c-Myc in epidermal lysates from 3-day-old, 15-day-old, and 8-month-old control (ctrl) and mutant (KO) mice showed no significant changes in expression of the protein in Rac1-deficient keratinocytes. Tubulin or GAPDH (*) content was used as a loading control. (B) Western blot analysis of signaling pathway activation in the epidermis of 3-day-old and 8-month-old control and mutant mice showed no significant changes in activation of NF-B, JNK, Akt, or p38 in the absence of Rac1. Levels of IB- were checked as an indirect test of NF-B activation. GAPDH content and total levels of JNK, Akt, and p38 were used as a loading control (p, phosphorylated forms). (C) Western blot analysis of GTPase activity performed with epidermal lysates from 3-day-old and 8-month-old control and mutant mice showed no significant differences in activation of Cdc42 or RhoA. The active form of Rac2 was not detected in lysates from 8-month-old mouse epidermis. Tubulin or GAPDH (*) content was used as a loading control. (D) No Rac3 protein was detected in epidermal lysates from 8-month-old control or mutant mice. Brain lysate was analyzed as a positive control, and tubulin content was used as a loading control. (E) Real-time PCR analysis of RhoG expression in 6-month-old mouse epidermis was performed with two different sets of primers. No significant changes in RhoG mRNA levels where detected in Rac1-null tissue in comparison to the control. GAPDH expression levels were used for standardization of samples.

Article Snippet: The following primary antibodies were incubated with membranes under the conditions and at the concentrations suggested by the manufacturers: Rac1 (clone 102), Cdc42 (clone 44; both from Transduction Laboratories), Rac1 (clone 23A8; Upstate), Rac3 (kind gift from Ivan de Curtis, San Rafaele Scientific Institute, Milan, Italy) (2), RhoA (119), c-Myc (N-262), I B- (C-21), Rac2 (all from Santa Cruz), phospo-Akt (Ser473), Akt, phospo-p38, p38 mitogenactivated protein kinase (all from Cell Signaling), phospho-JNK (Biosource, Cell Signaling), JNK (Biosource), tubulin (YL1/2; kindly provided by Jürgen Wehland, GBF, Braunschweig, Germany), and glyceraldehyde-3-phosphate dehydrogenase(GAPDH;Chemicon).

Techniques: Activation Assay, Expressing, Activity Assay, Western Blot, Control, Mutagenesis, Positive Control, Real-time Polymerase Chain Reaction, Comparison

Journal: RSC advances

Article Title: Bio-fabrication of pigment-capped silver nanoparticles encountering antibiotic-resistant strains and their cytotoxic effect towards human epidermoid larynx carcinoma (HEp-2) cells.

doi: 10.1039/c9ra01072f

Figure Lengend Snippet: Fig. 11 Apoptotic mechanism of HEp-2 cells treated with pcAgNPs. DNA fragmentation of HEp-2 cells exposed to pcAgNPs (a). RT-PCR analysis of p53 expression, leading to apoptotic cell death (b and c); pcAgNP activation of p53, leading to increased catalase expression. HEp-2 cells treated with 15 mg mL1 pcAgNPs for 24 h, showing p53, p21 and catalase expressions assessed using Western blotting analysis. b-actin served as a loading control. Numbers were calculated by quantitative densitometric analysis and indicate the ratio of specific proteins versus b-actin. (d) Data are representative of at least three independent experiments.

Article Snippet: In the western blotting analysis, the following primary antibodies were used: rabbit polyclonal anti-p21 (1 : 100) (cat. no. TA322940, OriGene, USA), mouse monoclonal anti-p53 (1 : 500) (cat. no. TA327920, OriGene, USA), andmouse monoclonal anticatalase (1 : 100) (cat. no. TA502496, OriGene, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Activation Assay, Western Blot, Control

Journal: PLoS ONE

Article Title: Rac2 Controls Tumor Growth, Metastasis and M1-M2 Macrophage Differentiation In Vivo

doi: 10.1371/journal.pone.0095893

Figure Lengend Snippet: (A & B) WT and Rac2-/- mice (n = 8-10) were subcutaneously implanted with 1×10 5 LLC, B16F10 and 2×10 6 NB9464D cells on the dorsal flank. Tumor growth was monitored regularly, until tumors were harvested on day 21. Mean tumor volume (A) and mass (B) for each group (n = 8) is plotted. Graphs present mean ± SEM of 8 mice in each group. Statistical significance is assessed by two sample t -test where *denotes P <0.05, ** denotes P <0.01 and *** denotes P <0.001. Experiment was repeated 7–8 times with similar results. (C) Left panel shows representative immunofluorescent staining of tumor vasculature by CD31 (green) and counterstain by DAPI (blue) on frozen tumor sections of LLC and B16 tumors implanted subcutaneously in WT and Rac2-/- mice. Right panel shows reduced microvascular density (MVD) in tumors isolated from Rac2-/- animals as compared to WT animals. MVD was determined by counting the number of microvessels per high-power field (HPF) in the section with an antibody reactive to CD31. Microvessels were counted blindly in 5–10 randomly chosen fields and data is representative of three independent experiments with 4–5 mice. * P <0.05 vs. WT. (D) H & E stained images (magnification 4X and 20X) showing invasive interface between the skin and muscularis layer of subcutaneous implanted LLC tumor into WT and Rac2-/- animals. The invasive interface is shown by arrows. Same results were obtained with 7–8 mice in each group and experiment is repeated 7–8 times.

Article Snippet: Rac2 antibody is from Novus Biologicals.

Techniques: Staining, Isolation

Journal: PLoS ONE

Article Title: Rac2 Controls Tumor Growth, Metastasis and M1-M2 Macrophage Differentiation In Vivo

doi: 10.1371/journal.pone.0095893

Figure Lengend Snippet: (A) Experimental metastasis of B16 melanoma cells in WT and Rac2-/- mice (n = 5). B16 F10 melanoma cells (5×10 5 cells) were injected through the tail vein, and after 15 days, the lungs were removed and the photographs were taken shown as upper panel in the figure. Lower panel shows mean number of tumor nodules visible on the surface of the lungs in WT and Rac2-/- mice. Surface tumor nodules in lungs were counted under dissecting microscope. Values are mean ± SEM ( n = 5 or 6; P <0.001; pair wise two-sided Student's t test). A marked suppression in the number of metastatic nodules was observed in Rac2-/- mice.(B) H&E-stained lung tissues demonstrating large macroscopic nodules (black arrows) were greatly increased in number and size in the WT vs. Rac2-/- mice, 15 d after B16 tumor cell injection. (C) Upper panel shows tumor mass of pancreatic tumors implanted orthotopically in WT and Rac2-/- mice. Panc02 (1×10 6 ) cells were injected in the pancreas of WT and Rac2-/- mice (n = 10). Tumors were removed 30 days after tumor implantation. Values are mean ± SEM ( n = 10; P<0.05; pair wise two-sided Student's t test). Bottom panel shows representative images of pancreatic tumors isolated from pancreas of WT and Rac2-/- mice. (D) Left panel shows macroscopic view of Panc02 metastatic mesenteric lymph nodes from WT and Rac2-/- mice. Right panel shows number of metastatic mesenteric lymph nodes/mesentery. Values are mean ± SEM ( n = 5 or 6; P<0.001; pair wise two-sided Student's t test). The data are representative of three independent experiments performed.

Article Snippet: Rac2 antibody is from Novus Biologicals.

Techniques: Injection, Microscopy, Staining, Tumor Implantation, Isolation

Journal: PLoS ONE

Article Title: Rac2 Controls Tumor Growth, Metastasis and M1-M2 Macrophage Differentiation In Vivo

doi: 10.1371/journal.pone.0095893

Figure Lengend Snippet: (A) Left panel shows BMDMs from WT, and Rac2-/- mice were tested in haptotaxis assay for capacity to migrate on different matrix proteins or fragments of fibronectin, vitronectin (via α v β 3 /α v β 5 ); H296, via α 4 β 1 ; CH271, via α 5 β 1 and collagen via α 2 β 1 /α 2 β 2 . Comparison of WT to Rac2-/- BMDMs shows significant difference on H296 peptide ( P <0.001), CH271 peptide ( P <0.05), VN protein ( P <0.01). Data represent mean ± SEM, representative of 4 independent experiments performed (n = 3). Right Upper panel shows Rac2 pull down assay indicating the extent of Rac2 activation in WT BMDMs under conditions of adhesion to: NS, no stimulation; Vitronectin (α v β 3 /α v β 5 ); H296, fibronectin fragment for α 4 β 1 ; CH271, fibronectin fragment for α 5 β 1 ; and Collagen (α 2 β 1 /α 2 β 2 ). Conversion of GDP-Rac2/Rac1 to GTP-Rac2/Rac1 was determined by using GST fusion protein representing the GTP-Rac-binding CRIB domain of the PAK-1 kinase. Cell lysate used in this comparison contained equal amounts of protein per lane. Total Rac1 and Rac2 protein was loaded for control. Right Lower panel shows Rac2 pull down assay indicating the extent of Rac2 activation in WT and α4Y991A knock in BMDMs under conditions of adhesion to H296, fibronectin fragment for α 4 β 1. Experiments were repeated 2-3 times with similar results. (B) LLC cells were inoculated subcutaneously in WT and α4Y991A mice (n = 6–8) and tumor growth was recorded as described in . Values represent mean ± SEM ( n = 6–8 mice per group; P<0.001) (C) Quantitative PCR analysis of mRNA for M1, M2 specific genes in the macrophages sorted from LLC tumors grown in WT and α4Y991A mice (n = 3–4). LLC tumors implanted in WT and α4Y991A mice were used for FACS sorting of macrophages on the basis of F4/80 and CD11b staining as described in . RNA was isolated from these macrophages and was used for real-time PCR analysis of the indicated genes described in . Values are mean ± SEM ( n = 3–4). Statistical significance is assessed by two sample t -test where *denotes P <0.05, ** denotes P <0.01 and *** denotes P <0.001.). (D) Left panel shows representative photograph of pulmonary metastatic foci produced 15 days after intravenous injection of B16F10 cells in WT and α4Y991A mice (n = 6–8). Right panel shows mean number of tumor nodules visible on the surface of the lungs in WT and α4Y991A mice. Values are mean ± SEM (n = 6; P<0.001; pair wise two-sided Student's t test). (E), Quantitative PCR analysis of mRNA for IL 1, uPA, TNFα, MMP9, Mgl1, MMR, YM1 and TGF-β in BMDMs isolated from WT and α4Y991A knock in mice and cultured in MCSF in vitro . Data are representative of three independent experiments, shown are mean ± SEM, *P<0.05, **P<0.01 and ***P<0.001 vs. WT, t test.

Article Snippet: Rac2 antibody is from Novus Biologicals.

Techniques: Comparison, Pull Down Assay, Activation Assay, Binding Assay, Control, Knock-In, Real-time Polymerase Chain Reaction, Staining, Isolation, Produced, Injection, Cell Culture, In Vitro

Journal: PLoS ONE

Article Title: Rac2 Controls Tumor Growth, Metastasis and M1-M2 Macrophage Differentiation In Vivo

doi: 10.1371/journal.pone.0095893

Figure Lengend Snippet: (A) Identification of F4/80 + macrophages by immunofluorescence microscopy in the frozen sections of LLC and B16 tumors stained with antibodies against F4/80 and imaged by fluorescence microscopy. The average no. of macrophages per HPF for 3 different experiments were 42±8, (WT) 38±6 (Rac2-/-) for LLC tumors and 50±5, (WT) 45±10, (Rac2-/-) for B16 tumors. Macrophages were counted blindly by 3 individuals in 5-10 randomly chosen fields and data is representative of three independent experiments with 4 mice. (B) Figure represents FACS data showing the quantification of CD11b and F480 + macrophages infiltrated in LLC tumors implanted in WT and Rac2-/- mice. Experiment was repeated 4-5 times with 3-4 mice in each group and similar results were obtained. (C) Quantitative PCR analysis of mRNA for M1, M2 specific genes in the macrophages sorted from LLC tumors grown in WT and Rac2-/- mice (n = 3–4) as described in . Values are mean ± SEM. Statistical significance is assessed by two sample t -test where *denotes P <0.05, ** denotes P <0.01 and *** denotes P <0.001. (D) Arginase activity was measured in macrophages sorted from LLC tumors injected in WT and Rac2-/- mice as described in . (E) Nitrite production in macrophages sorted from LLC tumors injected in WT and Rac2-/- and stimulated with 10 ng/ml LPS for 24 h. Supernatants were collected, and nitrite concentration was measured as described in . Results are mean ± SEM (n = 3–4 mice) for 3 independent experiments performed in triplicate (P<0.05 for arginase activity and P <0.001 for nitrite assay; student's t test).

Article Snippet: Rac2 antibody is from Novus Biologicals.

Techniques: Immunofluorescence, Microscopy, Staining, Fluorescence, Real-time Polymerase Chain Reaction, Activity Assay, Injection, Concentration Assay, Nitration

Journal: PLoS ONE

Article Title: Rac2 Controls Tumor Growth, Metastasis and M1-M2 Macrophage Differentiation In Vivo

doi: 10.1371/journal.pone.0095893

Figure Lengend Snippet: (A) Quantitative PCR analysis of mRNA for IL 1, uPA, TNFα, MMP9, Mgl1, MMR, YM1 and TGF-β in BMDMs isolated from WT and Rac2-/- mice and cultured in MCSF in vitro . Values are mean ± SEM (n = 3-4 mice). Statistical significance is assessed by two sample t -test where *denotes P <0.05, ** denotes P <0.01 and *** denotes P <0.001. (B) & (C), Heat map generated from microarray analysis of BMDMs isolated from WT and Rac2-/- mice (n = 5 in each group) as described in Materials and Methods. Colors illustrate fold changes, Red: up-regulation; green: down- regulation; black: no change. The bar code on the bottom represents the color scale of the log 2 values. The differential expression of genes related to cell cycle, angiogenesis and invasion are shown in B and M1-M2 polarization are shown in (C). (D) & (E), Heatmap representation of metabolites across BMDMs from WT (n = 5) and Rac2-/- (n = 5) mice. Shades of yellow represent elevation of a metabolite and shades of blue represent decrease of a metabolite relative to the median metabolite levels (see color scale). Colors illustrate fold changes, Yellow: up-regulation; blue: down-regulation; black: no change. Data shows higher expression of metabolites related to carbohydrate (D) and lipid metabolism (E) in Rac2-/- BMDMs.

Article Snippet: Rac2 antibody is from Novus Biologicals.

Techniques: Real-time Polymerase Chain Reaction, Isolation, Cell Culture, In Vitro, Generated, Microarray, Quantitative Proteomics, Expressing

Journal: PLoS ONE

Article Title: Rac2 Controls Tumor Growth, Metastasis and M1-M2 Macrophage Differentiation In Vivo

doi: 10.1371/journal.pone.0095893

Figure Lengend Snippet: (A) Upper panel shows that MCSF signaling differentially activates Rac2 while GMCSF signaling does not result in significant Rac2 activation. BMDMs or GBMDMs cultured in MCSF or GMCSF for 7 days were serum starved for 4 hrs and stimulated with 50 ng/ml of MCSF or GMCSF for 15 min followed by Rac2 GTP pull down assays as described in . Lower panel shows the differential activation of Rac2 when Mθs are costimulated through the MCSFR and α 4 β 1 integrin. WT BMDMs cultured in MCSF for 7 days were serum starved for 4 hrs, trypsinized and were allowed to engage with α 4 β 1 ligand (H296) or α 2 β 1 ligand (collagen), followed by MCSF stimulation (50 ng/ml) for 15 min and Rac2 GTP pull down assay as described before. (B & C) Tumor volume (B) and mass (C) of LLC tumors grown in Rac2-/- mice treated with or without 1 million WT BMDMs or Rac2-/- BMDMs or WT GBMDMs. After 5 days of LLC tumor inoculation, Rac2-/- mice were treated either with 1 million WT BMDMs or GBMDMs or Rac2-/- BMDMs or every third day, until tumors were removed on day 21. Values are mean ± SEM ( n = 6-8). Statistical significance is assessed by two sample t -test where *denotes P <0.05, ** denotes P <0.01 and *** denotes P <0.001. Experiment was repeated three times with similar results. (D) Reversal of B16 metastasis by injection of WT BMDMs and not by WT GBMDMs in Rac2-/- mice. Figure shows representative photograph of pulmonary metastatic foci produced 15 days after intravenous injection of B16F10 cells in Rac2-/- mice and treated with 1 million WT BMDMs or WT GBMDMs. One dose of 1 million BMDMs or GBMDMs were given to Rac2-/- mice two days before inoculating 5×10 5 B16F10 cells intravenously followed by treatment with 1 million BMDM or GBMDM every third day, lungs were harvested on day 15. Data are representative of three independent experiments with 6–8 mice in each group. (E) Reversal of metastatic defect in Rac2-/- mice by local injections of 1 million WT BMDMs in the right lobe of lungs and 1 million WT GBMDMs or Rac2-/- BMDMs in the left lobe of Rac2-/- mice, followed by tail vein injections of B16 luciferase cells (5×10 5 ) after 2 days. The luciferase signal was monitored every third day on IVIS by injecting luciferin, until lungs were harvested on day 15 (n = 5). Data are representative of two independent experiments with 5–6 mice in each group.

Article Snippet: Rac2 antibody is from Novus Biologicals.

Techniques: Activation Assay, Cell Culture, Pull Down Assay, Injection, Produced, Luciferase

Journal: PLoS ONE

Article Title: Rac2 Controls Tumor Growth, Metastasis and M1-M2 Macrophage Differentiation In Vivo

doi: 10.1371/journal.pone.0095893

Figure Lengend Snippet: (A) Graphic representation of novel integrin-Rac2 signaling axis in macrophages required for tumor growth, invasion, metastasis and the polarization of macrophages into M2 phenotype. (B) Interactome map developed from multiple-omic data which predicts how Rac2 regulates macrophage M2 differentiation. Nodes indicate genes either preselected for hierarchical analysis (colored borderer) or prioritized directly by the network propagation algorithm. Edges indicate protein-protein interactions. Node color indicates the protein or gene expression change in the Rac2-/- vs. WT condition.

Article Snippet: Rac2 antibody is from Novus Biologicals.

Techniques: Protein-Protein interactions, Gene Expression

Journal: Nature Communications

Article Title: Gadd45g insufficiency drives the pathogenesis of myeloproliferative neoplasms

doi: 10.1038/s41467-024-47297-2

Figure Lengend Snippet: a c-kit + BM cells from diseased Gadd45g +/ − and Ctrl mice were lysed, precipitated with anti-GADD45g antibody, and detected by Western blot with anti-RAC2 and -GADD45g antibodies. b Representative immunofluorescence micrographs showing colocalization of GADD45g with RAC2 in c-kit + BM cells of Ctrl mice. Panels represent nucleus (blue), GADD45g (green), RAC2 (yellow), and merged images, respectively. Arrows in merged image indicate colocalization of GADD45g with RAC2. Bar represents 10 μm. c Representative immunofluorescence micrographs showing cellular distribution of GADD45g, RAC2 and RAC1 in cord blood CD34 + cells from healthy human donors. Panels represent nucleus (blue), GADD45g (green), RAC2 (orange), RAC1 (pink), and merged images, respectively. Arrows in merged image indicate colocalization of GADD45g with RAC2. Bar represents 5 μm. d Western blot analysis of RAC2-GTP and total RAC2 protein levels in c-kit + BM cells from diseased Gadd45g +/− and Ctrl mice. e HEL and SET-2 cells transfected with GADD45g-specific shRNA or shCtrl were lysed, precipitated with anti-GADD45g antibody, and detected by Western blot with anti-RAC2 and -GADD45g antibodies. f Western blot analysis of RAC2-GTP and total RAC2 protein levels in HEL and SET-2 cells transfected with GADD45g- specific shRNA or shCtrl. g Western blot analysis of p-PAK1 and total PAK1 protein levels in c-kit + BM cells from diseased Gadd45g +/ − and Ctrl mice. h Western blot analysis of p-PAK1 and total PAK1 protein levels in HEL and SET-2 cells transfected with GADD45g- specific shRNA or shCtrl. i HEL and SET-2 cells were transfected with GADD45g- specific shRNA or shCtrl for 48 h, followed by transfection with RAC2-specific shRNA (shRAC2) or scrambled control (Scr) for another 48 h. The protein levels of p-PAK1, total PAK1, p-PI3K, total PI3K, pAKT-Ser473 and total AKT were examined by Western blot. j HEL and SET-2 cells were transfected with GADD45g- specific shRNA or shCtrl for 48 h, followed by treatment with vehicle or IPA-3 (10 μM for 48 h). The protein levels of p-PI3K, total PI3K, pAKT-Ser473 and total AKT were examined by Western blot. For ( a–j ): At least three independent experiments with similar results were performed.

Article Snippet: GADD45g (sc393261, Santa Cruz), RAC2 antibody (abx001053, Abbexa, Cambridge, UK) and RAC1 antibody (ab97732, Abcam) were used at 1:100 and incubated overnight at 4 °C.

Techniques: Western Blot, Immunofluorescence, Transfection, shRNA

Journal: Nature Communications

Article Title: Gadd45g insufficiency drives the pathogenesis of myeloproliferative neoplasms

doi: 10.1038/s41467-024-47297-2

Figure Lengend Snippet: a , b HEL and SET-2 cells were transfected with GADD45g- specific shRNA or shCtrl for 48 h, followed by transfection with RAC2-specific shRNA (shRAC2) or scrambled control (Scr) for another 48 h. Effects of RAC2 knockdown on colony formation ( a ) and apoptosis ( b ) of these cells. ( c ) HEL and SET-2 cells were transfected with GADD45g- specific shRNA or shCtrl for 48 h. Cells were then plated in methylcellulose containing IPA-3 (10 μM). Colonies were counted and analyzed on day 14. d HEL and SET-2 cells were transfected with GADD45g- specific shRNA or shCtrl for 48 h, followed by treatment with vehicle or IPA-3 (10 μM) for 48 h, and the percentage of apoptosis cells was examined. e Effects of EHT 1864 treatment (5 μM for 10–14 days) on colony formation of primary BM CD34 + cells from GADD45g high and GADD45g low patients with MPN ( n = 3 per group). f Effects of IPA-3 treatment (10 μM for 10–14 days) on colony formation of primary BM CD34 + cells from GADD45g high and GADD45g low patients with MPN ( n = 3 per group). For ( a – d ) : Figures shown are representative of three independent experiments with similar results. Data are shown as mean ± SD ( n = 3 technical replicates). Comparisons were evaluated by two-tailed Student’s t test, and multiple groups were analyzed with one-way ANOVA.

Article Snippet: GADD45g (sc393261, Santa Cruz), RAC2 antibody (abx001053, Abbexa, Cambridge, UK) and RAC1 antibody (ab97732, Abcam) were used at 1:100 and incubated overnight at 4 °C.

Techniques: Transfection, shRNA, Two Tailed Test