Journal: bioRxiv

Article Title: INHBA promotes the progression of gastric cancer by activating MAPK signaling pathway via targeting ITGA6

doi: 10.1101/2025.04.05.647266

Figure Lengend Snippet: (A) Differential expression genes (DEGs) heat map identified by RNA-seq. (B) The number of DEGs identified by RNA-seq. (C) Overlap analysis of RNA-seq and STAD-DEGs-up identified genes. (D) Expression ITGA6 in GC paired tissue cohort from the GEPIA2 database. (E) Subcellular localization of ITGA6 from GeneCards. (F, G) the interaction between INHBA and ITGA6 was verified by Co-IF (F) and Co-IP (G) experiments. Scale bar, 10μm. (H) qRT-PCR was used to detect ITGA6 mRNA expression after INHBA knockdown in NUGC-3 cells. (I) WB was used to detect ITGA6 protein expression after INHBA knockdown in NUGC-3 cells. (J) qRT-PCR was used to detect ITGA6 mRNA expression after INHBA overexpression in AGS cells. (K) WB was used to detect ITGA6 protein expression after INHBA overexpression in AGS cells. Data are presented as means ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001

Article Snippet: In this study, the following antibodies were used: GAPDH antibody (diluted 1:500; Goodhere-Bio; Catalog number: AB-P-R001), INHBA antibody (diluted 1:500; Proteintech, Wuhan, Hubei, P.R.C; Catalog number: 17524-1-AP), ITGA6 antibody (diluted 1:500; Proteintech, Catalog number: 27189-1-AP), MEK1/2 antibody (diluted 1:1000; Proteintech; Catalog number: 11049-1-AP), phospho-MEK1 antibody (diluted 1:1000; Proteintech; Catalog number: 28930-1-AP), ERK1/2 antibody (diluted 1:1000; Proteintech; Catalog number: 66192-1-Ig). phospho-ERK1/2 antibody (diluted 1:100; Proteintech; Catalog number: 28733-1-AP).

Techniques: Expressing, RNA Sequencing, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Knockdown, Over Expression

Journal: bioRxiv

Article Title: INHBA promotes the progression of gastric cancer by activating MAPK signaling pathway via targeting ITGA6

doi: 10.1101/2025.04.05.647266

Figure Lengend Snippet: (A) The basal expression of ITGA6 mRNA in GC cell lines and GES-1 was detected by qRT-PCR. (B) The basal expression of ITGA6 protein in cell lines was detected by WB. (C) The expression of ITGA6 protein in 30 pairs of GC paired tissues was detected by IHC. (magnification ×400). (D-E) Four siRNA-ITGA6 sequences were transfected simultaneously into AGS cells. (F-G) The relative expression level of INHBA mRNA and protein were detected by qRT-PCR and WB after instantaneous co-transfection of INHBA siRNA and ITGA6 overexpression plasmid in NUGC-3. (H-I) The relative expression level of INHBA mRNA and protein were detected by qRT-PCR and WB after instantaneous co-transfection of ITGA6 siRNA and INHBA overexpression plasmid in AGS. Data are presented as means ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001

Article Snippet: In this study, the following antibodies were used: GAPDH antibody (diluted 1:500; Goodhere-Bio; Catalog number: AB-P-R001), INHBA antibody (diluted 1:500; Proteintech, Wuhan, Hubei, P.R.C; Catalog number: 17524-1-AP), ITGA6 antibody (diluted 1:500; Proteintech, Catalog number: 27189-1-AP), MEK1/2 antibody (diluted 1:1000; Proteintech; Catalog number: 11049-1-AP), phospho-MEK1 antibody (diluted 1:1000; Proteintech; Catalog number: 28930-1-AP), ERK1/2 antibody (diluted 1:1000; Proteintech; Catalog number: 66192-1-Ig). phospho-ERK1/2 antibody (diluted 1:100; Proteintech; Catalog number: 28733-1-AP).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Cotransfection, Over Expression, Plasmid Preparation

Journal: bioRxiv

Article Title: INHBA promotes the progression of gastric cancer by activating MAPK signaling pathway via targeting ITGA6

doi: 10.1101/2025.04.05.647266

Figure Lengend Snippet: (A) KEGG enrichment result of the differentially expressed gene sets from the GEPIA2 database. (B) The expression levels of MAPK signaling pathway related proteins were detected by WB after instantaneous co-transfection of INHBA siRNA and ITGA6 overexpression plasmid in NUGC-3. (C, D, E, F, G) The effects of ITGA6 on INHBA in NUGC-3 cells were detected by CCK-8 assay (C), colony formationassay (D), wound healing assay (E), Scale bar, 200 μm, Transwell migration assay (F) and Transwell invasion assay (G). Scale bar, 100 μm. (H, I, J, K, L) The effects of ITGA6 on INHBA in AGS cells were detected by CCK-8 assay (H), colony ormation assay (I), wound healing assay J), Scale bar, 200 μm, Transwell migration assay (K) and Transwell invasion assay (L). Scale bar, 100 μm.. Data are presented as means ± SD. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001

Article Snippet: In this study, the following antibodies were used: GAPDH antibody (diluted 1:500; Goodhere-Bio; Catalog number: AB-P-R001), INHBA antibody (diluted 1:500; Proteintech, Wuhan, Hubei, P.R.C; Catalog number: 17524-1-AP), ITGA6 antibody (diluted 1:500; Proteintech, Catalog number: 27189-1-AP), MEK1/2 antibody (diluted 1:1000; Proteintech; Catalog number: 11049-1-AP), phospho-MEK1 antibody (diluted 1:1000; Proteintech; Catalog number: 28930-1-AP), ERK1/2 antibody (diluted 1:1000; Proteintech; Catalog number: 66192-1-Ig). phospho-ERK1/2 antibody (diluted 1:100; Proteintech; Catalog number: 28733-1-AP).

Techniques: Expressing, Cotransfection, Over Expression, Plasmid Preparation, CCK-8 Assay, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: NPJ Precision Oncology

Article Title: INHBA(+) cancer-associated fibroblasts generate an immunosuppressive tumor microenvironment in ovarian cancer

doi: 10.1038/s41698-024-00523-y

Figure Lengend Snippet: a Western blot of PD-L1, phosphorylated SMAD2 (pSMAD2), SMAD2, pSMAD3, and SMAD3 in 781T CAFs were stably transfected with either INHBA cDNA/control cDNA, or transiently transfected with INHBA siRNAs/control siRNAs, or treated with recombinant Activin A. GAPDH was used as a loading control. INHBA mRNA levels measured by qRT-PCR in cell cultures grown in parallel with the cell cultures used for Western blotting are plotted in Supplementary Fig. . b Western blot detection of PD-L1, pSMAD2, SMAD2 in 781T CAFs were treated with negative controls, or Activin A and control siRNAs, or Activin A and SMAD2 siRNAs, followed by INHBA mRNA levels measured by qRT-PCR in cell cultures grown in parallel with the cell cultures used for Western blotting are plotted in Supplementary Fig. . c Measurements of GLuc and SEAP activities in the media using a GloMax microplate reader in 781T CAFs treated as described in b . Normalized promoter activity was calculated as the ratio of GLuc and SEAP activities (paired t test). d Representative flow cytometry graphs of live CD4(+) T cells showing the frequency of CD25(+) FOXP3(+) Tregs after human pan T cells were activated with CD3/CD28 antibodies and co-cultured in direct contact (3 independent experiments) or indirect contact (Transwell membrane; 3 independent experiments) with 781T CAFs treated for 24 hours with negative controls, or INHBA siRNAs and control cDNA, or INHBA siRNAs and PD-L1 cDNA. e Quantification of Tregs in co-cultures of activated human pan-T cells and 781T CAFs in direct and indirect contact (one-way ANOVA test).

Article Snippet: A vector expressing human INHBA cDNA (#RC203226, OriGene) and control cDNA (#PS100001, OriGene) were introduced into CAFs using the lipofectamine 3000 reagent (#L3000015, Thermo Fisher Scientific) according to the transfection protocol.

Techniques: Western Blot, Stable Transfection, Transfection, Control, Recombinant, Quantitative RT-PCR, Activity Assay, Flow Cytometry, Cell Culture, Membrane