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: Oncotarget

Article Title: Filamin C, a dysregulated protein in cancer revealed by label-free quantitative proteomic analyses of human gastric cancer cells

doi:

Figure Lengend Snippet: (A) DU145 cells with filamin C silencing were fluorescence-labeled and analyzed in a zebrafish cancer metastasis model. The selected pictures were shown from 0 to 3 days post injection. The areas indicated with arrows were enlarged for visualizing disseminated cancer cells. (B) Filamin C was silenced in DU145 cells and the proenzyme (Pro) and activated form of MMP2 were analyzed. (C) Migration of DU145 cells with or without filamin C silencing were analyzed. The third group with filamin C silencing was further treated with the MMP2 inhibitor ARP100. The statistical analysis results were shown in Figure and the Western blot results were shown in Figure . FLNC, filamin C.

Article Snippet: Antibody for human MMP2 was purchased from Proteintech (Wuhan, China).

Techniques: Fluorescence, Labeling, Injection, Migration, Western Blot

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