Journal: International Journal of Oncology

Article Title: Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels

doi: 10.3892/ijo.2022.5310

Figure Lengend Snippet: An inactive catalytic domain mutant 786-O C431S exhibits a similar phenotype as the control cells. (A) Cells with a PARK2 C431S mutation were generated by transducing cells with the pLVX-EF1a-PARKC431S-IRES-mCherry plasmid. (B) PARK2 mRNA expression levels were examined by RT-qPCR. TBP and β-actin were used as housekeeping genes. Data are presented as the mean + SEM of triplicate experiments. Kruskal-Wallis test was performed followed by Dunn's post hoc test; * P<0.05. (C) RT-qPCR analysis revealed no difference in CKS2 mRNA expression levels between the different transduced ccRCC cell lines. Data are presented as the mean ± SEM. (D) Transwell Boyden chamber migration assays were conducted with mutant cell lines 786-O C431S and RCC-MH C431S . Magnification, ×10. Data were compared by Kruskal-Wallis followed by Dunn's post hoc test; n=3; * P<0.05, *** P<0.001, **** P<0.0001. (E) CKS2 mRNA expression levels were assessed after siRNA knockdown. The experiment was conducted in triplicate. TBP and β-actin served as housekeeping genes. Data are presented at the mean ± SEM. (F) Migratory capacity was assessed after siRNA-mediated knockdown of CKS2 and (G) significance was assessed by Kruskal-Wallis followed by Dunn's post hoc test; n=2; ** P<0.01, *** P<0.001. Magnification, ×10. ccRCC, clear cell renal cell carcinoma; CKS2, CDC28 protein kinase regulatory subunit 2; EF1a, elongation factor 1α; EV, empty vector; IBR, in-between RING fingers domain; IRES, internal ribosome entry site; ns, not significant; PARK2, parkin; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA; TBP, TATA-box binding protein; Ubl, ubiquitin-like domain; WT, wild-type.

Article Snippet: 786-O and 293T cell lines were ordered from American Type Culture Collection, and RCC-MH cells were purchased from Cell Lines Service GmbH.

Techniques: Mutagenesis, Control, Generated, Plasmid Preparation, Expressing, Quantitative RT-PCR, Migration, Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA, Binding Assay, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: PBRM1 directs PBAF to pericentromeres and protects centromere integrity

doi: 10.1038/s41467-025-57277-9

Figure Lengend Snippet: a Clonogenic survival of RPE1 parental and PBRM1 knockouts treated with increasing doses of the CDK1 inhibitor RO-3306 relative to DMSO-only treated cells. n = 6, mean ± SEM, data were analysed using a 2way ANOVA with Dunnett’s test, ** p = 0.0053, *** p = 0.0005. b Representative image from clonogenic survival assay in ( a ). c Experimental outline for quantifying nuclear defects induced following CDK1 inhibition by RO-3306. d % of cells with mild (left) or severe (right, also see panel f ) nuclear defects after 24 h of treatment with DMSO (-) or RO-3306 (+). n = 4, mean ± SEM, data were analysed by 2way ANOVA with Dunnett’s test, **** p < 0.0001. 600-1500 nuclei were analysed per condition. e Representative images from d , showing nuclear morphology after 24 h of treatment with DMSO (-) or RO-3306 (+). Scale bar corresponds to 40 µm. f Quantification of types of severe nuclear morphology defects in parental or PBRM1 knockout cells treated as per the schematic in c . n = 4, mean ± SEM. g Clonogenic survival of RPE1 parental and PBRM1 knockouts following CCNB1 siRNA (si1 or si2) treatment, normalised to survival after treatment with a scramble (scr) siRNA. Points correspond to independent biological replicates, n = 3, mean ± SEM, data were analysed by 2way ANOVA with Dunnett’s test, *** p = 0.001 and *** p = 0.002 for KO1 and KO2, respectively. h Representative image from clonogenic survival assay in g . i Clonogenic survival of a panel of renal cell carcinoma (RCC) cell lines, which are PBRM1-proficient (blue) or -deficient due to loss-of-function mutations (red). Survival was measured after CCNB1 depletion with two independent CCNB1 siRNAs, normalised to survival after treatment with a scramble siRNA. Points correspond to independent biological replicates, n = 4 (RCC-FG2, Caki-1, RCC-4), n = 5 (786-O), or n = 8 (769-P, Caki-2). Boxes contain the 25 th to 75 th percentiles with line at median, and whiskers extend to 10th and 90th percentiles. j Western blotting for PBRM1 in RCC cell lines used in ( i ). α-tubul i n is used as a loading control. k Representative images in PBRM1-proficient (786-O) and -deficient (RCC-FG2) cell line from the survival assay in ( i ). Source data are provided as a source data file.

Article Snippet: 786-O (catalogue number CRL-1932), 769-P (catalogue number CRL-1933), and RCC-FG2 (Cell Lines Service, CLS GmbH, catalogue number 300249) cells were cultured in RPMI 1640 medium (Sigma) supplemented with 10% FBS and 1% P/S.

Techniques: Clonogenic Cell Survival Assay, Inhibition, Knock-Out, Western Blot, Control

Journal: EBioMedicine

Article Title: The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

doi: 10.1016/j.ebiom.2017.07.018

Figure Lengend Snippet: FLRT3, a physiological ligand of LPHN1, induces galectin-9 and Tim-3 secretion. (A) THP-1 cells were exposed for 16 h to 10 nM extracellular domain of human recombinant FLRT3 followed by measurement of released Tim-3 and galectin-9 by ELISA. (B) THP-1 cells were exposed to mouse bone marrow (mBM) extracts for 16 h with or without 1 h pre-treatment with 5 μg/ml anti-FLRT3 antibody. The presence of FLRT3 in mBM extracts was confirmed by Western blot analysis. Secreted Tim-3 and galectin-9 were measured by ELISA. (C, left) RCC-FG1 cells express FLRT3 as confirmed by Western blotting. (C, right) RCC-FG1 cells were co-cultured with THP-1 cells at a ratio of 1 THP-1:2 RCC-FG1 with or without 1 h pre-treatment with 5 μg/ml FLRT3 neutralizing antibody. Secreted galectin-9 and Tim-3 were measured by ELISA. Images are from one experiment representative of three which gave similar results. Quantitative data depict mean values ± SEM of three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control. Symbols “a” or “b” are used instead of “*” to indicate differences vs. cells treated with mBM extracts or co-cultured with RCC-FG1 cells, respectively.

Article Snippet: Renal clear cell carcinoma RCC-FG1 cells were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture, Control

Journal: Molecular Genetics & Genomic Medicine

Article Title: miR‐33a inhibits cell growth in renal cancer by downregulation of MDM4 expression

doi: 10.1002/mgg3.833

Figure Lengend Snippet: Inhibitory effects of miR‐33a on cell proliferation and cell cycle in renal cell cancer (RCC) cell lines. (a) Expression of miR‐33a in Caki‐1 and 786‐O cells after transfection with miR‐33a mimics and miR‐33a inhibitor or NC. NC represents negative control of miRNA. Results were expressed as of three independent experiments, with at least three replicates in each independent experiment, ** p < 0.01 versus NC. (b) CCK‐8 assays indicated that the effects of miR‐33a on the growth of RCC cell lines. Results were expressed as of three independent experiments, with at least three replicates in each independent experiment, * p < 0.05 versus NC. (c, d) The results of flow cytometry showed that upregulation of miR‐33a significantly increased the percentages of cells in the G0/G1 phase, which showed that the upregulation of miR‐33a could suppress RCC cells proliferation. * p < 0.05 versus Control

Article Snippet: Normal primary renal tubular HK‐2 cell lines and RCC cell lines (Caki‐1, ACHN and 786‐O) were purchased from China Center For Type Culture Collection (Wuhan, China).

Techniques: Expressing, Transfection, Negative Control, CCK-8 Assay, Flow Cytometry, Control

Journal: Molecular Genetics & Genomic Medicine

Article Title: miR‐33a inhibits cell growth in renal cancer by downregulation of MDM4 expression

doi: 10.1002/mgg3.833

Figure Lengend Snippet: miR‐33a directly targets Mouse double minute 4 (MDM4). (a) The predicted miR‐33a binding site within MDM4 3′ UTR and miR‐33a mutated version by site mutagenesis; (b) Caki‐1 cells were transfected with reporter constructs containing either wild type (WT) MDM4 , or MDM 4 3′ UTR with mutation (MUT), along with miR‐33a mimics, or negative control, respectively. Relative luciferase activity was measured. (c) qPCR analysis of MDM4 expression in renal cell cancer (RCC) Caki‐1 cells after overexpression or knockdown of miR‐33a . (d, e) Western blotting analysis of MDM4 protein expression in RCC Caki‐1 cells after overexpression or knockdown of miR‐33a . (f) Western blot analysis revealed that transfection of MDM4 siRNA into Caki‐1 cells resulted in decreased MDM4 expression compared to the cells transfected with scrambled siRNA. These effects of siRNA were attenuated by anti‐ miR‐33a inhibitor transfection. NC represents normal control, * p < 0.05, ** p < 0.01 versus NC

Article Snippet: Normal primary renal tubular HK‐2 cell lines and RCC cell lines (Caki‐1, ACHN and 786‐O) were purchased from China Center For Type Culture Collection (Wuhan, China).

Techniques: Binding Assay, Mutagenesis, Transfection, Construct, Negative Control, Luciferase, Activity Assay, Expressing, Over Expression, Knockdown, Western Blot, Control

Journal: Molecular Genetics & Genomic Medicine

Article Title: miR‐33a inhibits cell growth in renal cancer by downregulation of MDM4 expression

doi: 10.1002/mgg3.833

Figure Lengend Snippet: The effect of mouse double minute 4 ( MDM4 ) on cell growth in renal cell cancer (RCC). (a, b, c) RT‐qPCR and western blot analysis of MDM4 expression in RCC cells. (d) Kaplan–Meier analysis of overall survival in 30 RCC patients with low median ( n = 15) and high median ( n = 15) expression levels of MDM4 . (e) CCK‐8 assays indicated that the effects of MDM4 on growth of RCC cell lines. Results were expressed as ± x - s of three independent experiments, with at least three replicates in each independent experiment, * p < 0.05 versus NC. (f) Western blot analysis of p53 expression after miR‐33a mimics and ‐inhibitor transfection in RCC cells

Article Snippet: Normal primary renal tubular HK‐2 cell lines and RCC cell lines (Caki‐1, ACHN and 786‐O) were purchased from China Center For Type Culture Collection (Wuhan, China).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, CCK-8 Assay, Transfection