itga3  (Proteintech)

Journal: Pharmaceuticals

Article Title: Oligomeric Proanthocyanidins Reverse Lenvatinib Resistance in Hepatocellular Carcinoma Through ITGA3-Mediated Pathway

doi: 10.3390/ph18091361

Figure Lengend Snippet: Gene Profiling Analysis Reveals Genes Involved in Lenvatinib Resistance. ( A ) Workflow showing the identification of differentially expressed genes from the GSE186191 dataset comparing parental and Lenvatinib-resistant Huh-7 and Hep3B cells. A total of 225 commonly altered genes were identified and analyzed using the DAVID Bioinformatics Database. ( B ) Pathway enrichment analysis of the 225 shared genes identified the top 10 KEGG pathways, with the ECM-receptor interaction pathway being the most enriched, including ITGA3 and ITGB8, which were also commonly found in the Focal Adhesion and PI3K/AKT signaling pathways. ( C ) qRT-PCR analysis of ITGA3 and ITGB8 expression in parental and rHuh-7 and rPLC cells. Both resistant cell lines exhibited significantly higher expression of these genes than their parental cell lines. ( D ) ITGA3 expression in 83 paired clinical samples (tumor tissue vs. adjacent non-tumor tissue). ( E ) Kaplan–Meier survival curve showing worse prognosis for patients with high ITGA3 expression ( p < 0.001; HR = 2.73, 95% CI: 1.56–4.78). Data represent mean ± SD (* p < 0.05 vs. control).

Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies: GAPDH (1:5000, Proteintech, Cat# 10494-1-AP), PARP (1:1000, CST, Cat# 9532S), Cleaved-PARP (1:1000, CST, Cat# 5625S), Caspase-3 (1:1000, CST, Cat# 9662S), Cleaved-Caspase-3 (1:1000, CST, Cat# 9661S), Bax (1:1000, CST, Cat# 41162), CD44 (1:1000, CST, Cat# 3570S), CD133 (1:1000, Merck Millipore, Cat# MAB4399), OCT4 (1:1000, CST, Cat# 75463S), ITGA3 (1:1000, Proteintech, Cat# 66070-1-Ig), EGFR (1:1000, CST, Cat# 4267T), Phospho-EGFR (Ser473, 1:1000, CST, Cat# 3777S), AKT (1:1000, CST, Cat# 9272), and Phospho-AKT (Ser473, 1:1000, CST, Cat# 4060S).

Techniques: Protein-Protein interactions, Quantitative RT-PCR, Expressing, Control

Journal: Pharmaceuticals

Article Title: Oligomeric Proanthocyanidins Reverse Lenvatinib Resistance in Hepatocellular Carcinoma Through ITGA3-Mediated Pathway

doi: 10.3390/ph18091361

Figure Lengend Snippet: siRNA-mediated knockdown of ITGA3 suppresses tumorigenic phenotypes and induces apoptosis in HCC cell lines. ( A ) Proliferation assay after ITGA3 siRNA transfection. ( B ) Annexin V apoptosis assay in rHuh-7 and rPLC after ITGA3 knockdown. ITGA3 knockdown significantly increased apoptosis rates compared to control siRNA (siNC). ( C ) Western blot analysis showing decreased ITGA3 expression and inhibition of the EGFR–AKT signaling pathway following ITGA3 knockdown in rHuh-7 and rPLC. GAPDH was used as an internal control. ( D ) Representative images of anoikis assays in rHuh-7 and rPLC after ITGA3 knockdown. Calcein AM was used to stain viable cells (green), and EthD-1 was used to stain dead cells (red). Data represent mean ± SD (* p < 0.05 vs. control). (See also for functional assays demonstrating suppression of colony formation, migration, invasion, and stemness-related gene expression following ITGA3 knockdown).

Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies: GAPDH (1:5000, Proteintech, Cat# 10494-1-AP), PARP (1:1000, CST, Cat# 9532S), Cleaved-PARP (1:1000, CST, Cat# 5625S), Caspase-3 (1:1000, CST, Cat# 9662S), Cleaved-Caspase-3 (1:1000, CST, Cat# 9661S), Bax (1:1000, CST, Cat# 41162), CD44 (1:1000, CST, Cat# 3570S), CD133 (1:1000, Merck Millipore, Cat# MAB4399), OCT4 (1:1000, CST, Cat# 75463S), ITGA3 (1:1000, Proteintech, Cat# 66070-1-Ig), EGFR (1:1000, CST, Cat# 4267T), Phospho-EGFR (Ser473, 1:1000, CST, Cat# 3777S), AKT (1:1000, CST, Cat# 9272), and Phospho-AKT (Ser473, 1:1000, CST, Cat# 4060S).

Techniques: Knockdown, Proliferation Assay, Transfection, Apoptosis Assay, Control, Western Blot, Expressing, Inhibition, Staining, Functional Assay, Migration, Gene Expression

Journal: Pharmaceuticals

Article Title: Oligomeric Proanthocyanidins Reverse Lenvatinib Resistance in Hepatocellular Carcinoma Through ITGA3-Mediated Pathway

doi: 10.3390/ph18091361

Figure Lengend Snippet: OPCs restore anoikis sensitivity and overcome Lenvatinib resistance by targeting ITGA3 and inhibiting EGFR–AKT signaling. ( A ) qRT-PCR analysis of ITGA3 expression in rHuh-7 and rPLC cell lines treated with Lenv, OPCs, or the combination of both. OPCs treatment significantly downregulated ITGA3 expression at the mRNA level. ( B ) Western blot analysis showing the effect of treatments on ITGA3 expression and EGFR–AKT signaling pathway activation in rHuh-7 and rPLC. OPCs and the combination treatment suppressed ITGA3 and the phosphorylation of EGFR, AKT. (C) Representative images from anoikis assays in rHuh-7 and rPLC treated with control, Lenvatinib, OPCs, or the combination. Calcein AM was used to stain viable cells (green), and EthD-1 was used to stain dead cells (red). Combination therapy restored anoikis sensitivity in both cell lines. Data represent mean ± SD (* p < 0.05 vs. control).

Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies: GAPDH (1:5000, Proteintech, Cat# 10494-1-AP), PARP (1:1000, CST, Cat# 9532S), Cleaved-PARP (1:1000, CST, Cat# 5625S), Caspase-3 (1:1000, CST, Cat# 9662S), Cleaved-Caspase-3 (1:1000, CST, Cat# 9661S), Bax (1:1000, CST, Cat# 41162), CD44 (1:1000, CST, Cat# 3570S), CD133 (1:1000, Merck Millipore, Cat# MAB4399), OCT4 (1:1000, CST, Cat# 75463S), ITGA3 (1:1000, Proteintech, Cat# 66070-1-Ig), EGFR (1:1000, CST, Cat# 4267T), Phospho-EGFR (Ser473, 1:1000, CST, Cat# 3777S), AKT (1:1000, CST, Cat# 9272), and Phospho-AKT (Ser473, 1:1000, CST, Cat# 4060S).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Activation Assay, Phospho-proteomics, Control, Staining

Journal: Cancer Science

Article Title: SOX10 Regulates Melanoma Metastasis Through the IRF1 ‐ ITGA3 / EphA2 ‐ FAK Pathway

doi: 10.1111/cas.70173

Figure Lengend Snippet: SOX10 negatively regulates ITGA3 and EphA2 expression. (A) Violin plots of ITGA3 or EphA2 expression levels in subpopulations are shown using the same single‐cell RNA‐seq data with Figure . The subpopulations with higher invasive scores was marked by red line. (B) A t‐SNE analysis of single‐cell RNA‐seq data from GSE134432 . Violin plots of SOX10, ITGA3, or EphA2 expression levels in each cluster are shown. (C) Human melanoma cells were transfected with siCNTL or siSOX10 (#9 or #10) and whole‐cell lysates 3 days after siRNA transfection were subjected to Western blotting. (D) A2058 cells were infected with shRNA for control (shSCR) or SOX10 (shSOX10). After puromycin selection, whole‐cell lysates were subjected to Western blotting. (E) A2058 cells were transfected with siCNTL, siSOX10 (#9 or #10), or siMITF (#1 or #2). Three days after siRNA transfection, whole‐cell lysates were subjected to Western blotting. Other conditions were similar to those in Figure .

Article Snippet: The primary antibodies used were MITF (C‐5) from Dr. Fisher, D.E. (MGH), AXL (#8661), EphA2 (#6997), Phospho‐EphA2 (Ser‐897; #6347), IRF1 (#8478), FAK (#3285), and Phospho‐FAK (Tyr‐397; #8556) from Cell Signaling Technology (Beverly, MA, USA), and ITGA3 (sc‐374242), ITGB1 (sc‐374429), SOX10 (sc‐514302), and β‐actin (sc‐47778) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, RNA Sequencing, Transfection, Western Blot, Infection, shRNA, Control, Selection

Journal: Cancer Science

Article Title: SOX10 Regulates Melanoma Metastasis Through the IRF1 ‐ ITGA3 / EphA2 ‐ FAK Pathway

doi: 10.1111/cas.70173

Figure Lengend Snippet: SOX10 regulates melanoma adhesion and motility through ITGA3 and EphA2, respectively. (A) A2058/Luc cells were transfected with siCNTL or siSOX10 (#9 or #10). Three days after transfection, cells (1.25 × 10 3 cells/well) were added to non‐ (−) or laminin‐332‐coated wells (LN‐332). The intensity of luminescence was measured by an in vivo imaging system. Data are the means ± SD with each data point. * p < 0.01 versus siCNTL‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test. (B, C) A2058/Luc or A2058 cells were transfected with siCNTL, siSOX10 #10, siITGA3 (#1 or #2), or siEphA2 (#1 or #2) for 3 days. A2058/Luc cells were subjected to an adhesion assay (left) and A2058 cells to a random motility assay using the time‐lapse imaging system (center), while whole‐cell lysates were subjected to Western blotting (right). Data are the means ± SD with each data point. * p < 0.01 versus siSOX10‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test. (D) Human melanoma cells were transfected with siCNTL or siSOX10 (#9 or #10). Three days after transfection, cells were subjected to the transwell‐chamber migration assay. After hematoxylin and eosin staining, the number of migrated cells was counted under a microscope. Data are the means ± SD with each data point. * p < 0.01 versus siCNTL‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test in each cell line. (E) A2058 cells were transfected with siCNTL, siSOX10#10, siITGA3 (#1), or siEphA2 (#2) for 3 days. * p < 0.01 versus siSOX10‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test. Other conditions were similar to those in (D).

Article Snippet: The primary antibodies used were MITF (C‐5) from Dr. Fisher, D.E. (MGH), AXL (#8661), EphA2 (#6997), Phospho‐EphA2 (Ser‐897; #6347), IRF1 (#8478), FAK (#3285), and Phospho‐FAK (Tyr‐397; #8556) from Cell Signaling Technology (Beverly, MA, USA), and ITGA3 (sc‐374242), ITGB1 (sc‐374429), SOX10 (sc‐514302), and β‐actin (sc‐47778) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, In Vivo Imaging, Cell Adhesion Assay, Motility Assay, Imaging, Western Blot, Migration, Staining, Microscopy

Journal: Cancer Science

Article Title: SOX10 Regulates Melanoma Metastasis Through the IRF1 ‐ ITGA3 / EphA2 ‐ FAK Pathway

doi: 10.1111/cas.70173

Figure Lengend Snippet: SOX10 regulates EphA2 and ITGA3 through the transcription factor IRF1. (A) Genes that positively correlated with EphA2 or ITGA3 are shown in melanoma from the CCLE dataset. The negative correlation of SOX10 with EphA2 and ITGA3 is shown in the upper panel. (B–F) A2058 cells were transfected with siCNTL, siSOX10 #10, or siIRF1 (#1 or #2). Three days after transfection, whole‐cell lysates were subjected to Western blotting (B). Three days after transfection, transfected cells were subjected to an adhesion assay (C), random motility assay (D), transwell‐migration assay (E), or experimental lung metastasis assay (F). Data show the mean ± SD (C–E) or ± SEM (F) with each data point. * p < 0.01 versus siSOX10‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test.

Article Snippet: The primary antibodies used were MITF (C‐5) from Dr. Fisher, D.E. (MGH), AXL (#8661), EphA2 (#6997), Phospho‐EphA2 (Ser‐897; #6347), IRF1 (#8478), FAK (#3285), and Phospho‐FAK (Tyr‐397; #8556) from Cell Signaling Technology (Beverly, MA, USA), and ITGA3 (sc‐374242), ITGB1 (sc‐374429), SOX10 (sc‐514302), and β‐actin (sc‐47778) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Western Blot, Cell Adhesion Assay, Motility Assay, Transwell Migration Assay

Journal: Cancer Science

Article Title: SOX10 Regulates Melanoma Metastasis Through the IRF1 ‐ ITGA3 / EphA2 ‐ FAK Pathway

doi: 10.1111/cas.70173

Figure Lengend Snippet: SOX10 regulates melanoma metastasis to laminin‐332 through the ITGA3/EphA2‐FAK axis. (A) A gene set enrichment analysis was performed by KEGG gene sets using bulk RNA‐seq data after the knockdown of SOX10 from Figure . The enrichment plot of KEGG_Focal_Adhesion is shown. (B, C) A2058 cells were transfected with siCNTL, siSOX10 #10, siIRF1 (#1 or #2), siEphA2 #2, or siITGA3 #1. Three days after transfection, whole‐cell lysates were subjected to Western blotting. (D) A2058 cells were transfected with siCNTL or siSOX10 #10. Three days after transfection, transfected cells were treated with defactinib (0.1 or 1 μM) for 2 h and then subjected to a transwell‐migration assay. Defactinib was added to both the upper and lower chambers during the transwell‐migration assay (left panel). After 2 h of exposure to defactinib, whole‐cell lysates were subjected to Western blotting (right panel). Data show the mean ± SD with each data point. * p < 0.01 versus siSOX10‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test. (E) A2058/Luc cells were transfected with siCNTL or siSOX10 #10. Three days after transfection, transfected cells were treated with defactinib (1 μM) for 8 h and then subjected to the experimental lung metastasis assay. Data are shown as the mean ± SEM ( n = 5) with each data point. * p < 0.01 versus siSOX10‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test.

Article Snippet: The primary antibodies used were MITF (C‐5) from Dr. Fisher, D.E. (MGH), AXL (#8661), EphA2 (#6997), Phospho‐EphA2 (Ser‐897; #6347), IRF1 (#8478), FAK (#3285), and Phospho‐FAK (Tyr‐397; #8556) from Cell Signaling Technology (Beverly, MA, USA), and ITGA3 (sc‐374242), ITGB1 (sc‐374429), SOX10 (sc‐514302), and β‐actin (sc‐47778) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: RNA Sequencing, Knockdown, Transfection, Western Blot, Transwell Migration Assay