Journal: Oncology Reports

Article Title: Knockdown of CCT2 inhibits the malignant progression of hepatocellular carcinoma cells by impairing STAT3 activation

doi: 10.3892/or.2026.9086

Figure Lengend Snippet: Knockdown of CCT2 inhibits STAT3 signaling activation in hepatocellular carcinoma cells. The protein levels of STAT3, p-STAT3, MCL1, MMP2 and SOX2 in (A) Huh-7 and (B) HCCLM3 cells were measured by western blotting. The protein levels of (C) p-STAT3, (D) MCL1, (E) MMP2 and (F) SOX2 in subcutaneous tumor tissue were detected by immunohistochemical staining. *P<0.05, **P<0.01 vs. sh-NC. CCT2, chaperonin containing TCP1 subunit 2; sh, short hairpin; NC, negative control; p, phosphorylated; MCL1, myeloid cell leukemia sequence 1; SOX2, SRY-box transcription factor 2.

Article Snippet: The primary antibodies were as follows: CCT2 (cat. no. 24896-1-AP), β-actin (cat. no. 66009-1-Ig), MMP2 (cat. no. 10373-2-AP), myeloid cell leukemia sequence 1 (MCL1; cat. no. 16225-1-AP) and SRY-box transcription factor 2 (SOX2; cat. no. 11064-1-AP; all Proteintech Group, Inc.) and STAT3 (cat. no. 4904) and phosphorylated (p-)STAT3 (Tyr705; cat. no. 4113; both Cell Signaling Technology, Inc.) The membranes were washed three times in TBST (0.1% Tween-20) for 5 min each at room temperature.

Techniques: Knockdown, Activation Assay, Western Blot, Immunohistochemical staining, Staining, Negative Control, Sequencing

Journal: Experimental and Therapeutic Medicine

Article Title: IL-32γ promotes integrin αvβ6 expression through the activation of NF-κB in HSCs

doi: 10.3892/etm.2017.4956

Figure Lengend Snippet: Effect of different concentrations (0, 5, 10 and 20 ng/ml) of IL-32γ on LX-2 activation phenotypes. (A) Growth curves of LX-2 demonstrated that upregulation of IL-32γ promoted proliferation of LX-2. (B) Reverse transcription-quantitative polymerase chain reaction assessing mRNA levels of α-SMA, (C) collagen I, (D) TIMP1, (E) MMP2 and (F) MMP9, representing the activation level of LX-2. (G) Western blot analysis was used to measure collagen I, MMP9, MMP2, α-SMA, TIMP1 and GAPDH expression in whole-cell extracts. Data are presented as the mean ± standard deviation of three experiments. *P<0.01 and **P<0.05 vs. 0 ng/ml IL-32γ. IL-32γ, interleukin-32γ; α-SMA, α-smooth muscle actin; TIMP1, tissue inhibitor of metalloproteinase 1; MMP, matrix metalloproteinases; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Primary antibodies were as follows: Anti-integrin αVβ6 (cat. no. ab97588; Abcam, Cambridge, MA, USA), anti-GAPDH (cat. no. KGAA002-2; Nanjing KeyGen Biotech Co., Ltd.), anti-α-SMA (cat. no. G6669; Sigma-Aldrich; Merck KGaA), collagen type I antibody (cat. no. 600-402-103; Rockland, Limerick, PA, USA), TIMP1 (cat. no. 8946), MMP2 (cat. no. 87809), MMP9 (cat. no. 13667), NF-κB: p65 (cat. no. 8242), p50 (cat. no. 3035) (all Cell Signaling Technology, Inc., Danvers, MA, USA) and IL-32γ (cat. no. 513501; Biolegend, Inc.).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Standard Deviation, Quantitative RT-PCR

Journal: Genome Medicine

Article Title: Small-molecule MMP2/MMP9 inhibitor SB-3CT modulates tumor immune surveillance by regulating PD-L1

doi: 10.1186/s13073-020-00780-z

Figure Lengend Snippet: Downregulation of MMP2/9 by SB-3CT treatment reduced PD-L1 expression. a Spearman’s correlation of group4 score and PD-L1 mRNA expression across 33 cancer types. b – g Evaluation of PD-L1 expression derived from SK-MEL-28 melanoma cell lines and A549 lung cancer cell lines treated with DMSO, SB-3CT (25 μM), IFNγ (200 ng/mL), and IFNγ/SB-3CT in combination for 24 h. b , c PD-L1 expression by RT-PCR in b SK-MEL-28 melanoma cell lines and c A549 lung cancer cell lines. d , e Western blot (left panel: representative images, right panel: quantification) of PD-L1 protein levels in d SK-MEL-28 and e A549. f , g Flow cytometry of PD-L1 + membrane level in f SK-MEL-28 and g A549. h – m PD-L1 expression of SK-MEL-28 melanoma cell line transfected with shMMP2 ( h – j ) and shMMP9 ( k – m ) or the scrambled negative control shRNA (shNC). Western blot ( h , k ) quantification of MMP2, MMP9, and PD-L1 protein expression ( i , l ), and RT-PCR analysis of MMP2, MMP9, and PD-L1 mRNA expression ( j , m )

Article Snippet: Human anti-PD-L1-Rb (ab213524) and mouse anti-PD-L1-Rb (ab213480) were purchased from Abcam; anti-CD8α-Rb (GB11068 and GB11068-1) was purchased from Servicebio; anti-PD-1-Rb (84651) was purchased from Cell Signaling Technology; the antibodies specific for human anti-MMP2-Rb (10375-2-AP), anti-MMP9-Rb (10373-2-AP), anti-MMP9-Rb (10373-2-AP), anti-GAPDH-M (60004-1-Ig), and anti-PD-L1-M (66248) were purchased from Proteintech; SB-3CT was purchased from Selleck(S7430); and in vivo mAb anti-PD-1-M (BE0146), anti-CTLA-4-M (BE0164), and IgG isotype control (BE0086, BE0089) were purchased from Bioxcell.

Techniques: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry, Transfection, Negative Control, shRNA

Journal: Genome Medicine

Article Title: Small-molecule MMP2/MMP9 inhibitor SB-3CT modulates tumor immune surveillance by regulating PD-L1

doi: 10.1186/s13073-020-00780-z

Figure Lengend Snippet: Regulation of PD-L1 expression through MMP2/9 has an anti-tumor effect. a – f Analysis of PD-L1 expression of SK-MEL-28 melanoma cell line with overexpression (oe) MMP2 ( a – c ), oeMMP9 ( d – f ). Western blot ( a , d ), quantification ( b , e ), and RT-PCR analysis ( c , f ) of MMP2, MMP9, and PD-L1 protein or mRNA expression. g – l PD-L1 expression of shMMP2 ( g – i ) and shMMP9 ( j – l ) SK-MEL-28 melanoma cell line treated with SB-3CT. Western blot ( g , j ); quantification of MMP2, MMP9, and PD-L1 protein ( h , k ); and mRNA expression ( i , l ). m Western blot showed the protein expression of PD-L1 for SK-MEL-28 melanoma cells with overexpression of PD-L1, treated with or without SB-3CT. n Z -scale normalization expression of differentially expressed genes (fold change > 1.5 and two-sided Student’s t test p < 0.05) between A375 melanoma cell lines treated with IFNγ/SB-3CT in combination and IFNγ. o Enriched signaling pathways for genes downregulated in A375 melanoma cell lines treated with SB-3CT and IFNγ in combination (Fisher’s exact test p < 0.05). All experiments were repeated three times independently. Results are mean ± s.d. NS, p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001, as determined by one-way ANOVA and Dunnett’s multiple comparison test

Article Snippet: Human anti-PD-L1-Rb (ab213524) and mouse anti-PD-L1-Rb (ab213480) were purchased from Abcam; anti-CD8α-Rb (GB11068 and GB11068-1) was purchased from Servicebio; anti-PD-1-Rb (84651) was purchased from Cell Signaling Technology; the antibodies specific for human anti-MMP2-Rb (10375-2-AP), anti-MMP9-Rb (10373-2-AP), anti-MMP9-Rb (10373-2-AP), anti-GAPDH-M (60004-1-Ig), and anti-PD-L1-M (66248) were purchased from Proteintech; SB-3CT was purchased from Selleck(S7430); and in vivo mAb anti-PD-1-M (BE0146), anti-CTLA-4-M (BE0164), and IgG isotype control (BE0086, BE0089) were purchased from Bioxcell.

Techniques: Expressing, Over Expression, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: American Journal of Translational Research

Article Title: Overexpression of TEM8 promotes ovarian cancer progression via Rac1/Cdc42/JNK and MEK/ERK/STAT3 signaling pathways

doi:

Figure Lengend Snippet: TEM8 promoted invasion and migration in ovarian cancer cells. A-D. Transwell assay showed that TEM8 overexpression promoted invasion of ovarian cancer cells, however, TEM8 knockdown reversed this effect (n = 9; ×200, scale bar = 100 μm). E-H. Scratch assay showed that TEM8 overexpression promoted migration of ovarian cancer cells, however, TEM8 knockdown reversed this effect (n = 9; ×100, scale bar = 100 μm). I, J. Representative images and quantitation of the western blot showed that the protein expression of MMP2, MMP9, and VEGFA in the TEM8 overexpression/knockdown groups (n = 3). GAPDH was used as an internal control. Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Proteins were separated using 10% SDS-PAGE, transferred to PVDF membranes and blocked using 5% BSA at room temperature for 2 h. Primary antibodies were incubated at 4°C overnight (dilution ratio of 1:1000): rabbit anti-TEM8 (Abcam, Cambridge, UK), rabbit anti-GATA2 (Abcam, Cambridge, UK), rabbit anti-Rac1 (Abcam, Cambridge, UK), rabbit anti-Cdc42 (Wanleibio, Shenyang, China), rabbit anti-p-Rac1/cdc42 (Cell Signaling Technology, California, USA), rabbit anti-JNK (Cell Signaling Technology, California, USA), mouse anti-p-JNK (Cell Signaling Technology, California, USA), rabbit anti-STAT3 (Wanleibio, Shenyang, China), rabbit anti-MEK (Santa Cruz, USA), rabbit anti-p-MEK (Cell Signaling Technology, California, USA), rabbit anti-ERK (Cell Signaling Technology, California, USA), rabbit anti-p-ERK (Cell Signaling Technology, California, USA), rabbit anti-p-STAT3 (Ser727) (Wanleibio, Shenyang, China), anti-Ki-67 (Wanleibio, Shenyang, China), anti-cyclin D1 (Cell Signaling Technology, California, USA), rabbit anti-MMP2 (Proteintech, Wuhan, China), anti-rabbit MMP9 (Proteintech, Wuhan, China), mouse anti-Bcl2 (Proteintech, Wuhan, China), rabbit anti-Bax (Proteintech, Wuhan, China), rabbit anti-VEGFA (Abcam, Cambridge, UK), mouse anti-GAPDH (1:2000, ZSGB-BIO Technology Co., Ltd., Beijing, China).

Techniques: Migration, Transwell Assay, Over Expression, Wound Healing Assay, Quantitation Assay, Western Blot, Expressing