anti tgfβ1 2 3  (R&D Systems)

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: GO and KEGG pathway enrichment analyses of DEGs in MCF-7 cells after GGCT knockdown. (A) GO enrichment analysis for biological processes (up-regulated and down-regulated genes), and KEGG pathway analysis of DEGs, using samples collected 3 days after knockdown. The top 10 biological process terms ranked according to p-Values are shown. (B) Hypothetical schema of cell cycle regulation following GGCT knockdown in MCF-7 cells, constructed based on DEGs enriched in the KEGG pathway ‘hsa04110: Cell cycle’ (http://www.kegg.jp). (C) Relative mRNA expression patterns of GGCT, TGF-β2, CDKN1A (p21 Cip1 ), and CDKN2B (p15 INK4b ) were measured with qRT-PCR. n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 using two-tailed Student’s t-test for pairwise comparisons. CDKN1A, Cyclin dependent kinase inhibitor 1A; CDKN2B, cyclin dependent kinase inhibitor 2B; DEGs, differentially expressed genes; GGCT, γ-glutamylcyclotransferase; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; qRT-PCR, quantitative reverse-transcription-polymerase chain reaction; TGF-β2, transforming growth factor-β2.

Article Snippet: Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies; p15 INK4b (1:500; cat. no. sc-271791; Santa Cruz Biotechnology, Dallas, TX, USA); p21 WAF1/CIP1 (1:500; cat. no. 556430; BD Biosciences, San Jose, CA, USA); α-tubulin (1:1,000; cat. no. T9026; Sigma–Aldrich Co. LCC, Tokyo, Japan); GGCT (1:500; cat. no. HPA020735; Sigma–Aldrich Co. LCC); TGF-β2 (1:1,000; cat no. 19999-1-AP; Proteintech, Rosemont, IL, USA), phospho-SMAD2 (1:500; cat. no. 3108; Cell Signaling Technology, Danvers, MA, USA); phospho-SMAD3 (1:500; cat. no. 9520; Cell Signaling Technology); SMAD2/3 (1:500; cat. no. 3102; Cell Signaling Technology).

Techniques: Knockdown, Construct, Expressing, Quantitative RT-PCR, Two Tailed Test, Reverse Transcription, Polymerase Chain Reaction

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: TGF-β2/SMAD signaling axis regulates p15 INK4b and p21 Cip1 expression in GGCT-depleted MCF-7 cells. (A) mRNA expression levels of GGCT and TGF-β2 were analyzed by qRT-PCR 3 days after transfection with GGCT- and/or TGF-β2-siRNA. (B) Western blot analysis of GGCT and TGF-β2 expression 4 days after transfection with GGCT- and/or TGF-β2-siRNA. (C) Western blotting analysis of p15 INK4b , p21 Cip1 , phospho-SMAD2 (pSMAD2), SMAD2, phospho-SMAD3 (pSMAD3), SMAD3, GGCT, and α-tubulin in MCF-7 at 4 days after transfection with GGCT-siRNA and/or TGF-β2-siRNA, or non-target control siRNA. (D) Western blotting analysis of p15 INK4b , p21 Cip1 , pSMAD3, SMAD3, GGCT, and α-tubulin in MCF-7 cells at 4 days after transfection with GGCT-siRNA and/ or SMAD3-siRNA, or non-target control siRNA. (E) The number of viable cells in the trypan blue dye exclusion test and (F) representative images of MCF- 7 cells at 4 days after transfection with the indicated siRNAs. Scale bar: 200 μm; n=3 per group; *p<0.05, **p<0.01, and ***p<0.001 vs. control, †p<0.05; ††p<0.01, and †††p<0.001 vs. GGCT, one-way ANOVA followed by Tukey’s post hoc test. ANOVA, Analysis of variance; GGCT, γ-glutamylcyclotransferase; pSMAD, phosphorylated SMAD; qRT-PCR, quantitative reverse-transcription-polymerase chain reaction; siRNA, small-interfering RNA.

Article Snippet: Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies; p15 INK4b (1:500; cat. no. sc-271791; Santa Cruz Biotechnology, Dallas, TX, USA); p21 WAF1/CIP1 (1:500; cat. no. 556430; BD Biosciences, San Jose, CA, USA); α-tubulin (1:1,000; cat. no. T9026; Sigma–Aldrich Co. LCC, Tokyo, Japan); GGCT (1:500; cat. no. HPA020735; Sigma–Aldrich Co. LCC); TGF-β2 (1:1,000; cat no. 19999-1-AP; Proteintech, Rosemont, IL, USA), phospho-SMAD2 (1:500; cat. no. 3108; Cell Signaling Technology, Danvers, MA, USA); phospho-SMAD3 (1:500; cat. no. 9520; Cell Signaling Technology); SMAD2/3 (1:500; cat. no. 3102; Cell Signaling Technology).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Control, Reverse Transcription, Polymerase Chain Reaction, Small Interfering RNA

Journal: Cancer Genomics & Proteomics

Article Title: γ-Glutamylcyclotransferase Depletion Induces p15INK4b and p21Cip1-mediated Senescence via TGF-β2/SMAD3 Pathway Activation in Breast Cancer Cells

doi: 10.21873/cgp.20571

Figure Lengend Snippet: p15 INK4b , p21 Cip1 , and their upstream regulator TGF-β2 are involved in the induction of cellular senescence following GGCT-KD in MCF-7 cells. (A) Representative images of SA-β-Gal staining 4 days after transfection with the indicated siRNAs, including simultaneous knockdown of GGCT, p15, and p21. Scale bar: 50 μm. (B) The proportion of SA-β-Gal-positive cells in MCF-7 cells are shown. (C) Representative images of SA-β-Gal staining at 4 days after transfection with the indicated siRNAs, including simultaneous knockdown of GGCT and TGF-β2. Scale bar: 50 μm. (D) The proportion of SA-β-Gal-positive cells in MCF-7 cells are shown. n=3 per group; *p<0.05, **p<0.01, ***p<0.001, using one-way ANOVA followed by Tukey’s post hoc test. ANOVA, Analysis of variance; GGCT, γ-glutamylcyclotransferase; KD, knockdown; SA-β-Gal, senescence-associated β-galactosidase; siRNA, smallinterfering RNA; TGF-β2, transforming growth factor-β2.

Article Snippet: Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies; p15 INK4b (1:500; cat. no. sc-271791; Santa Cruz Biotechnology, Dallas, TX, USA); p21 WAF1/CIP1 (1:500; cat. no. 556430; BD Biosciences, San Jose, CA, USA); α-tubulin (1:1,000; cat. no. T9026; Sigma–Aldrich Co. LCC, Tokyo, Japan); GGCT (1:500; cat. no. HPA020735; Sigma–Aldrich Co. LCC); TGF-β2 (1:1,000; cat no. 19999-1-AP; Proteintech, Rosemont, IL, USA), phospho-SMAD2 (1:500; cat. no. 3108; Cell Signaling Technology, Danvers, MA, USA); phospho-SMAD3 (1:500; cat. no. 9520; Cell Signaling Technology); SMAD2/3 (1:500; cat. no. 3102; Cell Signaling Technology).

Techniques: Staining, Transfection, Knockdown

Journal: Scientific Reports

Article Title: Cholangiocarcinoma derived exosomes attenuate the anti-tumor functions of NK cells

doi: 10.1038/s41598-026-36706-9

Figure Lengend Snippet: Effect of RBE-Exos on the adhesion of NK-92 cells. ( A ) Growth status of NK-92 cells. NK cells treated with RBE-Exos lost the normal cluster growing. ( B ) CD11a protein levels. ( C ) CD18 protein levels. ( D ) CD54 protein levels. Western blot showed that the protein levels of CD11a, CD18 and, CD54 were obviously decreased in NK-92 cells treated with RBE-Exos. ( E ) Relative transcriptional levels of CD11a , CD18 , CD54 and, CD2 in NK-92 cells. qRT-PCR displayed that RBE-Exos downregulated the transcriptional levels of CD11a , CD18 , CD54 , but not CD2 . Data were presented as mean ± SEM for three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The primary antibodies were as follows: rabbit anti-human CD63, BCL-2, BCL-XL, CD11a, β-actin (Abcam, USA), mouse anti-human HSP70 (Santa-Cruz, USA), rabbit anti-human CD18 (Proteintech, China), mouse anti-human CD54 (Proteintech, China).

Techniques: Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Cholangiocarcinoma derived exosomes attenuate the anti-tumor functions of NK cells

doi: 10.1038/s41598-026-36706-9

Figure Lengend Snippet: Cytotoxicity and expression of adhesion molecules in primary NK cells. ( A ) Cytotoxicity of primary NK cells from CCA patients and healthy people against target cells. LDH detection displayed the cytotoxicity of NK cells from CCA patients was significantly lower than that of NK cells healthy people. Data are presented as the mean ± SEM from n = 3 independent experiments. ( B ) Protein levels of CD18 in primary NK cells from CCA patients and healthy people. Data are presented as the mean ± SEM from n = 3 independent experiments. ( C ) Protein levels of CD54 in primary NK cells from CCA patients and healthy people. Data are presented as the mean ± SEM from n = 3 independent experiments. ** P < 0.01.

Article Snippet: The primary antibodies were as follows: rabbit anti-human CD63, BCL-2, BCL-XL, CD11a, β-actin (Abcam, USA), mouse anti-human HSP70 (Santa-Cruz, USA), rabbit anti-human CD18 (Proteintech, China), mouse anti-human CD54 (Proteintech, China).

Techniques: Expressing