Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: The list of antibodies used for Western blotting.

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: The sequences of primers used for real-time PCR.

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: (A) Decreasing mRNA expression levels of RASAL1 upon the time of high Pi treatment after 24, 48 and 72 hours. Results were normalized to reference gene GAPDH (expression is presented as means ± s.d., n = 3 independent experiments, *P<0.05, **P<0.01, ***P<0.001). (B) Western blot confirming the decreased protein expression of RASAL1 in Pi treated cells. (C) MeDIP result showing the methylated promoter of RASAL1 along the treatment time, correlated with the reduced expression of RASAL1 in both mRNA and protein level. (D) Ras activity was measured by ELISA assay, untreated cells served as controls. Pi- treated cells showed the hyper-activation of total Ras.

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Expressing, Western Blot, Methylated DNA Immunoprecipitation, Methylation, Activity Assay, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: Cells were treated with Pi 3mM for 72 hours. (A) MeDIP result showing the demethylated promoter of RASAL1 by using PFA, correlated with restored mRNA expression of RASAL1. (B) qPCR analysis showed the restored RASAL1 mRNA expression levels by using phosphate transporter inhibitor (PFA). Results were normalized to reference gene GAPDH (expression is presented as means ± s.d., n = 3 independent experiments, **P<0.01). (C) ELISA assay indicating the normalization of total Ras with combination of PFA or RAS inhibitor farnesylthiosalicylic acid (FTS). (D) bright-field images showing the morphology change of HCAEC cells cultured under normal control condition, high Pi conditions, and Pi combined with FTS or PFA. Scale bars 25 μm. (E) Western blot analysis showing the expression of endothelial cell marker CD31 and fibroblast cell marker S100A4 in HCAEC cells exposed to normal control condition, high Pi conditions, and Pi combined with FTS or PFA (F) qRT-PCR data showing the mRNA expression levels of EndMT transcriptional factors (SNAIL, SLUG, and TWIST) and FSP1 in HCAEC cells under four conditions indicated above. Results were normalized to reference gene GAPDH (expression is presented as means ± s.d., n = 3 independent experiments, ***P<0.001).

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Methylated DNA Immunoprecipitation, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Western Blot, Marker, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: (A) Simplified schematic showing the human RASAL1 promoter along with exons (black boxes), translational start site (black arrow), locations of RASAL1 chip primer for amplicon1 and amplicon2, and amplicon2 primers serve as negative control chip primer. (B-C) The binding properties of HDAC2 and DNMT1 to the RASAL1 promoter region were analyzed by chromatin immunoprecipitation (ChIP) assay and detected by qRT-PCR in Pi treated (B) or in control cells (C). IgG purified from the same species serve as negative control for ChIP (expression are presented as means ± s.d., n = 3 independent experiments, **P<0.01, ***P<0.001, n.s. no significance).

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Negative Control, Binding Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Control, Purification, Expressing

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: In the physiological conditions, the CpG islands located in RASAL1 promoter are unmethylated (top panel) as indicated by open circles, and RASAL1 is transcriptional active. Under pathological conditions, initially when endothelial cells are exposed to stimulus, such as TGFβ1 or high concentration of Pi resulting in RASAL1 silencing through condensed chromatin structure at RASAL1 promoter mediated by HDAC2. While the RASAL1 promoter remains unmethylated (middle panel) and RASAL1 is transiently silenced. When the cells are continuously exposed to the stimulus, the CpG islands located in RASAL1 promoter are methylated (indicated by filled circles) by DNMT1 recruited through the interaction with HDAC2. Therefore, RASAL1 is permanently silenced due to promoter hypermethylation (lower panel).

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Concentration Assay, Methylation

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance.

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: Fig. 1 Residual tumours, but not adjacent normal tissues, are enriched with RASAL2. A UMAP plots of epithelial cells from primary TNBC tumours showing correlation between CytoTRACE score (1—least differentiation, 0 – most differentiation) and RASAL2 expression. B Violin plots of RASAL2 expression and residual tumour signature expression in the four clusters of epithelial cells identified in the TNBC tumours in (A). Cluster 1 expressions are significantly higher compared to all other clusters. Data are represented as mean ± SEM. P value by one-way ANOVA. C Heatmap of RASAL2 expression in pre- and post-treatment breast cancer patients [19]. Fold change (FC) was determined relative to pre-treatment expression level. P value by paired T-test. D Dot plots of RASAL2 expression in pre- versus post-treatment TNBC/BRCA-mutant breast cancer patients [20]. Probes that recognise RASAL2 variant 2 (ILMN_1813701 and ILMN_1673455) show significant increase following treatment. Data are represented as mean ± SEM. P value by two-tailed T-test. E Immunohistochemistry of fixed post-treatment TNBC patient breast specimens. RASAL2 (brown stain) was enriched in the tumour compartment versus adjacent normal epithelia. Data are represented as mean ± SEM. P value by two-tailed T-test. Scale bar, 20 µm. F Immunoblotting of fresh post-treatment TNBC patient specimens. RASAL2 was enriched in the tumour (T) versus adjacent normal (N) tissues in TNBC patients. LE long exposure.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Expressing, Mutagenesis, Variant Assay, Two Tailed Test, Immunohistochemistry, Staining, Western Blot

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance.

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: Fig. 2 RASAL2 promotes pan-resistance to cytotoxic agents beyond platinum. A Correlation between RASAL2 expression and sensitivity to indicated chemotherapy. The area under percent-viability curves (AUC) was computed as a metric of drug sensitivity, as derived from the Cancer Therapeutics Response Portal. Pearson r and P value are reported. B Cell viability assay. Vector control and RASAL2-overexpressing MDA-MB-468 cells were treated as indicated. Data are represented as mean ± SEM, n = 3 biological replicates. P value by paired T-test. C Spheroid assay. Viability of vector control and RASAL2-overexpressing TNBC spheroids was measured following treatment with vehicle DMSO, doxorubicin (DOXO) or gemcitabine (GEM). Representative images of HCC1806 spheroids are shown. Data are represented as mean ± SEM, n = 3 biological replicates. P value by paired T-test. Scale bar, 250 µm. Cell viability assay. Vector control and RASAL2- overexpressing (D) or -knockdown (E) HCC1937 cells were treated as indicated. Data are represented as mean ± SEM, n = 3 biological replicates. P value by paired T-test. F Correlation between RASAL2 expression and in vivo tumour response to doxorubicin. Mice were treated with vehicle control or 2 mg/kg doxorubicin [22]. Each dot represents an independent TNBC PDX model. Tumour growth inhibition was defined as [1 −(mean volume of treated tumours)/(mean volume of control tumours)] × 100%. Pearson r and P value are reported. G Change in tumour volume following doxorubicin in two TNBC PDX models. Mice were treated as described in (F), n = 8–9 per group. TM00099 tumours had the lowest RASAL2 expression, whereas TM01278 had the highest RASAL2 expression. P value by two-way ANOVA test.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Expressing, Derivative Assay, Viability Assay, Plasmid Preparation, Control, Knockdown, In Vivo, Inhibition

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance.

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: Fig. 4 Transcription factor CREB1 drives RASAL2 and BCL2 expression. A Venn diagram showing the overlapped predicted transcription factors binding on the promoters of RASAL2 and BCL2 using computational tools, JASPAR and LASAGNA. B Analyses of candidate transcription factors in breast cancer patient cohorts. Heatmap shows the fold changes of RASAL2, BCL2 and candidate gene transcription factors in patient-matched breast tumour specimens post- versus pre-treatment (top, [19]). Correlation between RASAL2/BCL2 expression and candidate transcription factors in TNBC patients in the TCGA-BRCA cohort (bottom). P value by two-sided Pearson correlation analysis. C Consensus binding motifs of transcription factor CREB1. D Decrease in the relative mRNA expression of RASAL2 and BCL2 following siRNA- mediated knockdown of CREB1 in MDA-MB-468 cells. Data are represented as mean ± SEM, n = 3 biological replicates. P value by two-tailed T-test. E Immunoblotting of TNBC cells transfected with siCREB1 or control siRNA. CREB1, RASAL2 and BCL2 were decreased in expression in cells treated with siCREB1 compared to control. F ChIP-qPCR confirmation of CREB1 binding to predicted sites on RASAL2 and BCL2 promoters. TSS denotes transcription start site. Data are represented as mean ± SEM, n = 3 biological replicates. P value by two-tailed T-test. G Decrease in the relative luciferase units in siCREB1 MDA-MB-468 cells compared to siControl. pRL-CMV Renilla luciferase plasmid was co-transfected for normalisation. Data are represented as mean ± SEM, n = 3 biological replicates. P value by two-way ANOVA. H Decrease in the relative luciferase units in MDA-MB-468 cells with truncated RASAL2 promoter without CREB1-binding sequence compared to those with wild-type (WT) RASAL2 promoter. pRL-CMV Renilla luciferase plasmid was co-transfected for normalisation. Data are represented as mean ± SEM, n = 3 biological replicates. P value by one-way ANOVA test.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Expressing, Binding Assay, Knockdown, Two Tailed Test, Western Blot, Transfection, Control, ChIP-qPCR, Luciferase, Plasmid Preparation, Sequencing

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance.

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: Fig. 5 Mitochondria is a common homing site for BCL2 and RASAL2. A Immunofluorescence of BCL2 and RASAL2 in primary TNBC patient tumour. Scale bar, 20 µm. B Immunofluorescence of BCL2 and RASAL2 in TNBC cells. Bottom graph shows the line scan quantification of BCL2 (red) and RASAL2 (green). Scale bar, 30 µm. C Confocal imaging of BCL2 and RASAL2 in TNBC cells. Panels on the left show exemplary co-localisation of signals within the boxed region of the cell. Scale bar, 10 µm. D AlphaFold prediction of the interaction between BCL2 and the N-terminus of RASAL2. pLDDT score (0–100) is a confidence score, and pTM score (0–1) is a metric for the structural congruency between two folded protein structures, with higher scores corresponding to higher confidence. PAE plot of the top ranked model is shown on the right [33, 34]. E Co-immunoprecipitation of BCL2 and RASAL2 in MDA-MB-468 cells. F Immunoblotting of cytoplasmic versus mitochondrial fractions of mammary cell lines. BCL2 was not detected in 4T1 murine cells as the antibody used was reactive only to human. AKT and TOM20 serve as cytoplasmic and mitochondrial markers, respectively. G Confocal imaging of RASAL2 and MitoTracker in TNBC cells. Scale bar, 5 µm.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Imaging, Immunoprecipitation, Western Blot

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance.

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: Fig. 6 BCL2 upregulation attenuate mitochondrial depolarisation by attenuating BAX oligomerisation. A Live-cell imaging. RASAL2 depletion increases the rate of GFP-BAX accumulation (green) in HCC1937 cells following exposure to 20 µM staurosporine. Number denotes time in seconds. Scale bar, 10 µm. B Quantification of change in BAX intensity. Fluorescence intensity of individual BAX foci was tracked over time and quantified, n = 4 foci per condition. Data are represented as mean ± SEM. P value by two-tailed T-test. C Schematic for live mitochondrial outer membrane permeabilisation (MOMP) assay. D MOMP assays revealing attenuated cytochrome c release in RASAL2-overexpressing TNBC cells. TOM20 serves as mitochondrial marker. E JC-1 mitochondrial membrane potential assay. TNBC cells were treated with vehicle DMSO or 5 µM doxorubicin (DOXO), and subsequently stained with JC-1 reagent. JC-1 aggregates (indicating high mitochondrial membrane potential) were observed as red, while JC-1 monomers (indicating low mitochondrial membrane potential) were green. Representative images of vector control and RASAL2-overexpressing TNBC cells are shown. Scale bar, 100 µm. F Quantification of the ratio of integrated intensity of red to green fluorescence in (E). Data are represented as mean ± SEM, n = 5 random fields of view per condition. P value by two-tailed T-test.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Live Cell Imaging, Fluorescence, Two Tailed Test, Membrane, Marker, Staining, Plasmid Preparation, Control

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance.

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: Fig. 7 Mechanism of apoptotic regulation by the CREB1-RASAL2-BCL2 axis. RASAL2 and BCL2 share common transcription factor motifs in their promoter regions. Transcription factor CREB1 binds to these promoter regions, and drives the expression of RASAL2 and BCL2. This upregulation is supported by CREB1-interactor YAP, a transcription co-factor that is regulated by RASAL2, thus forming a positive loop in the CREB1-RASAL2-BCL2 axis. Both RASAL2 and BCL2 colocalise at the mitochondria. Their presence confers mitochondrial resilience by mitigating mitochondrial outer membrane depolarisation, which occurs, for example, during BAX/tBID-triggered apoptosis. Consequently, in high RASAL2/BCL2 chemoresistant tumour cells, there is reduced cytochrome c release upon apoptosis induction and thereby attenuation of cell death.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Expressing, Membrane

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Lipid Receptor GPR31 (G-Protein–Coupled Receptor 31) Regulates Platelet Reactivity and Thrombosis Without Affecting Hemostasis

doi: 10.1161/atvbaha.120.315154

Figure Lengend Snippet: Figure 6. GPR31 (G-protein–coupled receptor 31) regulates PAR (protease-activated receptor)-4–mediated platelet activation resulting in orchestrated control of Rap1-RASA3–mediated αIIbβ3 activation. A, Fura-2–labeled transiently transfected GPR31/Chinese hamster ovary (CHO) cells were stimulated with 10 and 20 μM 12(S)-HETE, and Ca2+ flux was measured over time. B, Fura-2–labeled human platelets were stimulated with 10 μM 12(S)-HETE, and Ca2+ flux was measured over time. C, GPR310 (G-protein–coupled receptor 310) blocks PAR4-mediated calcium flux on human platelets. Platelets were preincubated with 3 μM GPR310 or vehicle before stimulation with 160 μM AYPGKF. D, Immunoblot showing 3 nmol/L thrombin activation of pAKT or pERK phosphorylation after 15 min in human platelets pretreated with 1 μM GPR310 as indicated. β-actin is used as loading control. E, Gel-purified platelets were treated with GPR310 (3 μM), AZD1283 (10 μM), or dual inhibition before thrombin stimulation (3 nmol/L) for 15 min, and Rap1 activation was measured. The bottom panels represent total Rap1 as loading control. F, Human platelets were treated with GPR310 (1 μM), AZD1283 (10 μM), dual inhibition, or LY294002 (10 μM), before thrombin stimulation with 3 nmol/L thrombin for 5 min. After membrane preparation, samples were immunoblotted with RASA3 ab. β-actin is used as loading control. G, Schematic of the PAR4, GPR31, and P2Y12 signaling in human platelet. Molecular weight markers (kDa). 12-LOX indicates 12-lipoxygenase.

Article Snippet: For RASA3, platelet lysates were ultracentrifuged at 100K for 2 hours at 4 °C and membrane protein quantified using the Bradford assay, equally loaded and resolved by SDS-PAGE and Western blot and Rasa3 levels detected with Rasa3 Ab (Proteintech; No. 27835-1-AP).

Techniques: Activation Assay, Control, Labeling, Transfection, Western Blot, Phospho-proteomics, Purification, Inhibition, Membrane, Molecular Weight

Journal: PLOS One

Article Title: IQGAP1 participates in endothelial cell apoptosis and regulates atherosclerosis by targeting YAP

doi: 10.1371/journal.pone.0328345

Figure Lengend Snippet: (A) The Annexin V–FITC/propidium iodide (PI) assay results indicate that Si-IQGAP1 can slightly decrease the apoptosis rate of normal cells, whereas knocking down IQGAP1 in PA-induced cells (PA + Si-IQGAP1) can significantly reduce the apoptosis rate. (B) The EdU assay reveals that Si-IQGAP1 can slightly increase EC proliferation. PA significantly inhibits HUVEC proliferation, while knocking down IQGAP1 can reverse the PA-induced proliferation stagnation. (C,D,E,F,G) Western blot and densitometric analysis of each protein relative to β-actin demonstrates that Si-IQGAP1 can partially decrease cleaved caspase-3 and BAX expression while enhancing BCL-2 expression. PA can significantly induce the upregulation of apoptotic proteins, but knocking down IQGAP1 can prevent this change, thereby protecting HUVECs from apoptosis. (H) Overexpression of IQGAP1 (pcDNA-IQGAP1) in normal cells can induce apoptosis and a higher apoptosis rate while treated with PA. (I) After transfection with pcDNA-IQGAP1, cell proliferation decreased significantly. (J,K,L,M,N) HUVECs treated with pcDNA-IQGAP1 shows an upregulation of cleaved caspase-3 and BAX expression as well as downregulation of BCL-2 expression. HUVECs, human umbilical vein endothelial cells; EC, endothelial cells; PA, palmitic acid; IQGAP1, IQ motif containing GTPase activating protein 1; Bcl‐2, B‐cell lymphoma 2; Bax, Bcl‐2 associated X; ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the cultured HUVECs were partitioned into groups, as follows: Untreated , normal control without any treated; si-NC , transfected with negative control siRNA (Jima); Si-IQGAP1, transfected with small interfering RNA of IQGAP1; PA , 400 μg/ml PA (HY-N0830; Med Chem Express) and negative control siRNA, PA + Si-IQGAP1 , 400 μg/ml PA and transfected with small interfering RNA of IQGAP1; pcDNA-NC , transfected with pcDNA3.1-3xFlag-negative control; pcDNA-IQGAP1 , transfected with pcDNA3.1-3xFlag-IQGAP1 plasmid; Si-IQGAP1 + Si-NC , transfected with small interfering RNA of IQGAP1 and negative control siRNA of YAP; and Si-IQGAP1 + Si-YAP , transfected with small interfering RNA of IQGAP1 and small interfering RNA of YAP.

Techniques: EdU Assay, Western Blot, Expressing, Over Expression, Transfection

Journal: PLOS One

Article Title: IQGAP1 participates in endothelial cell apoptosis and regulates atherosclerosis by targeting YAP

doi: 10.1371/journal.pone.0328345

Figure Lengend Snippet: (A) Semi-quantitative immunofluorescence analysis indicates that YAP expression increases upon IQGAP1 knockdown. (B) Western blot analysis shows that Si-IQGAP1 increased YAP expression and inhibits the phosphorylation of the S127 site of YAP. (C) Active intracellular YAP expression were decreased in Si-IQGAP1 group. (D) Semi-quantitative immunofluorescence analysis indicates that overexpression of IQGAP1 can decrease the expression of YAP. (E, F) Overexpression of IQGAP1 can reduce the proportion of YAP/ YAP-S127 and increase the phosphorylation of YAP at S127 site. ns P > 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Subsequently, the cultured HUVECs were partitioned into groups, as follows: Untreated , normal control without any treated; si-NC , transfected with negative control siRNA (Jima); Si-IQGAP1, transfected with small interfering RNA of IQGAP1; PA , 400 μg/ml PA (HY-N0830; Med Chem Express) and negative control siRNA, PA + Si-IQGAP1 , 400 μg/ml PA and transfected with small interfering RNA of IQGAP1; pcDNA-NC , transfected with pcDNA3.1-3xFlag-negative control; pcDNA-IQGAP1 , transfected with pcDNA3.1-3xFlag-IQGAP1 plasmid; Si-IQGAP1 + Si-NC , transfected with small interfering RNA of IQGAP1 and negative control siRNA of YAP; and Si-IQGAP1 + Si-YAP , transfected with small interfering RNA of IQGAP1 and small interfering RNA of YAP.

Techniques: Immunofluorescence, Expressing, Knockdown, Western Blot, Phospho-proteomics, Over Expression

Journal: PLOS One

Article Title: IQGAP1 participates in endothelial cell apoptosis and regulates atherosclerosis by targeting YAP

doi: 10.1371/journal.pone.0328345

Figure Lengend Snippet: (A) Flow cytometry results indicate that inhibiting YAP expression using Si-YAP increased the HUVEC apoptosis. (B,C,D,E,F,G)) Western blot analysis reveals that when both Si-IQGAP1 and Si-YAP are co-transfected, Si-YAP increased the expression of cleaved caspase-3 and BAX as well as the decrease in BCL-2. (H) The EdU analysis demonstrates that Si-YAP can decline the cell proliferation. ns P > 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Subsequently, the cultured HUVECs were partitioned into groups, as follows: Untreated , normal control without any treated; si-NC , transfected with negative control siRNA (Jima); Si-IQGAP1, transfected with small interfering RNA of IQGAP1; PA , 400 μg/ml PA (HY-N0830; Med Chem Express) and negative control siRNA, PA + Si-IQGAP1 , 400 μg/ml PA and transfected with small interfering RNA of IQGAP1; pcDNA-NC , transfected with pcDNA3.1-3xFlag-negative control; pcDNA-IQGAP1 , transfected with pcDNA3.1-3xFlag-IQGAP1 plasmid; Si-IQGAP1 + Si-NC , transfected with small interfering RNA of IQGAP1 and negative control siRNA of YAP; and Si-IQGAP1 + Si-YAP , transfected with small interfering RNA of IQGAP1 and small interfering RNA of YAP.

Techniques: Flow Cytometry, Expressing, Western Blot, Transfection

Journal: PLOS One

Article Title: IQGAP1 participates in endothelial cell apoptosis and regulates atherosclerosis by targeting YAP

doi: 10.1371/journal.pone.0328345

Figure Lengend Snippet: (A) H&E and (B, C) Oil Red O staining shows AS in the AAV-NC and AAV-Si-IQGAP1 groups following 12 weeks of HFD. However, the atherosclerotic plaque size and area in AAV-Si-IQGAP1 mice are smaller than those in the AAV-NC group. (D) The TUNEL assay demonstrates that the number of apoptotic cells are significantly decreased in the AAV-Si-IQGAP1 group. (E,F) Western blot and densitometric analyses of each protein relative to β‐actin reveal decreased expression of IQGAP1, and pro-apoptotic proteins (cleaved caspase-3 and BAX), while YAP and the anti-apoptotic protein BCL-2 are increased in the aortic wall of the AAV-Si-IQGAP1 groups. (G) IQGAP1 knockdown also leads to an increase of YAP in mouse aortic. HFD, high-fat diet; AS, atherosclerosis; IQGAP1, IQ motif containing GTPase activating protein 1; Bcl‐2, B‐cell lymphoma 2; Bax, Bcl‐2 associated X. **P < 0.01, ***P < 0.001.

Article Snippet: Subsequently, the cultured HUVECs were partitioned into groups, as follows: Untreated , normal control without any treated; si-NC , transfected with negative control siRNA (Jima); Si-IQGAP1, transfected with small interfering RNA of IQGAP1; PA , 400 μg/ml PA (HY-N0830; Med Chem Express) and negative control siRNA, PA + Si-IQGAP1 , 400 μg/ml PA and transfected with small interfering RNA of IQGAP1; pcDNA-NC , transfected with pcDNA3.1-3xFlag-negative control; pcDNA-IQGAP1 , transfected with pcDNA3.1-3xFlag-IQGAP1 plasmid; Si-IQGAP1 + Si-NC , transfected with small interfering RNA of IQGAP1 and negative control siRNA of YAP; and Si-IQGAP1 + Si-YAP , transfected with small interfering RNA of IQGAP1 and small interfering RNA of YAP.

Techniques: Staining, TUNEL Assay, Western Blot, Expressing, Knockdown

Journal: Frontiers in Immunology

Article Title: The Rac-GEF Tiam1 controls integrin-dependent neutrophil responses

doi: 10.3389/fimmu.2023.1223653

Figure Lengend Snippet: Tiam1 controls the expression of small GTPase and cytoskeletal regulators, and the association of Rasa3 with Rac in neutrophils adhering to ICAM1. Wild type and Tiam1 –/– neutrophils were allowed to adhere to 50 mm glass coverslips coated with ICAM1 during priming with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 50 min at 37°C, 5% CO 2 . DFP was added for 10 min before neutrophils were stimulated with 1.5 µM fMLP for 1 min and then lysed. The cleared supernatants (total lysates) were incubated with immobilised Rac1 G15A to isolate proteins that interact with nucleotide-free Rac. (A) Targeted mass spectrometry was performed for 21 proteins found to bind Rac1 G15A (left). 14 proteins could be compared between wild type (Wt) and Tiam1 –/– samples, 12 proteins in at least 2 out of 3 independent experiments (right). Wt/ Tiam1 –/– ratios are mean ± SEM or range, as appropriate, of 2-3 independent experiments; coloured dots depict the different experiments. Statistics are one-way ANOVA with Dunnett’s multiple comparisons test on log-transformed ratios. (B) Total lysates from the experiments in (A) were western blotted with the indicated antibodies. Representative western blots and coomassie loading control are shown. Blots of selected proteins were quantified by densitometry (right). Wt/ Tiam1 –/– ratios are expressed as mean ± SEM of 3 independent experiments; statistics are two-way ANOVA with Sidak’s multiple comparisons test on raw band intensities. Bottom panel: The Wt/ Tiam1 –/– Rac1 G15A binding ratio is expressed as a function of the Wt/ Tiam1 –/– expression level for the indicated proteins.

Article Snippet: Total lysates were western blotted using the following antibodies: 14-3-3 (pan, Cell Signaling Technology, 8312, 1:1000), α-Pix (Cell Signaling Technology, 4573, 1:1000), Git2 (Invitrogen, PA5-78301, 1:1000), Graf1 (Proteintech, 55139-1-AP, 1:1000), Psd4 (Abcam, ab154008, 1:1000), Rap1gds1/SmgGDS (Novus Biologicals, NBP1-87027, 1:1000), Rasa3 (Proteintech, 27835, 1:500), Talin-1 (Cell Signaling Technology, 4021, 1:500), Tiam1 (Bethyl Laboratories, A300-099A, 1:1000) and Tiam2 ( ) (1:750).

Techniques: Expressing, Incubation, Mass Spectrometry, Transformation Assay, Western Blot, Control, Binding Assay