Journal: Cell Death & Disease

Article Title: REGγ deficiency suppresses tumor progression via stabilizing CK1ε in renal cell carcinoma

doi: 10.1038/s41419-018-0646-2

Figure Lengend Snippet: a , b Expression of REGγ in RCC tissues (T) and normal kidney tissues (N) as detected by IHC ( a ) and WB ( b ). c Expression of REGγ in four RCC cell lines (A498, 786-O, ACHN, and caki-1) and the normal renal tubular epithelial cell line (HK-2) as detected by western blot. d Comparison of REGγ expression in different Fuhrman grades I–IV of RCC tissue samples via IHC staining (left panel: magnification ×200, scale bar = 50 μm; right panel: magnification ×400, scale bar = 20 μm). e REGγ (PSME3) expression (median of expression intensity) in different pathological types and grades of RCC derived from Oncomine database ( https://www.oncomine.org/ ). f Kaplan–Meier analysis of the correlation between REGγ expression and the survival time in RCC patients. Cases were classified into lower expression group and higher expression group as described in methods. g Prognosis of RCC (KIRC) patients with high or low expression of REGγ (PSME3) derived from OncoLnc database ( https://www.oncolnc.org/ )

Article Snippet: The stable REGγ knockdown RCC cell lines ACHN and A498 were generated by integration of retroviral shREGγ vectors specific for REGγ or a control gene (GFP) from OriGene (Rockville, MD), which was performed as previously described .

Techniques: Expressing, Western Blot, Immunohistochemistry, Derivative Assay

Journal: Cell Death & Disease

Article Title: REGγ deficiency suppresses tumor progression via stabilizing CK1ε in renal cell carcinoma

doi: 10.1038/s41419-018-0646-2

Figure Lengend Snippet: a PSME3 upregulation is closely correlated with renal cell carcinoma revealed by KEGG pathway analysis. b GSEA indicated that cell proliferation and anti-apoptosis were positively correlated with elevated PSME3 expression in RCC from database GSE89563 (GO_0008284 and GO_0006915). NES, normalized enrichment score. c Stable knockdown of REGγ (shREGγ) in RCC cell lines (ACHN and A498) confirmed by WB. d , e Effect of shREGγ on RCC cells growth as determined by MTT assay. f Representative images of colony formation of RCC cells after transfection of shREGγ versus shNC. g , h Representative images of EdU incorporation assay after transfection of shREGγ versus shNC. i , j Representative flow cytometry plots of cell cycle distribution from ACHN and A498 cells transfected with shREGγ and shNC. k , l Apoptosis rate from ACHN and A498 cells after transfected with shREGγ and shNC as detected by flow cytometry. Data are shown as mean ± SD. * P < 0.05

Article Snippet: The stable REGγ knockdown RCC cell lines ACHN and A498 were generated by integration of retroviral shREGγ vectors specific for REGγ or a control gene (GFP) from OriGene (Rockville, MD), which was performed as previously described .

Techniques: Expressing, MTT Assay, Transfection, Flow Cytometry

Journal: Cell Death & Disease

Article Title: REGγ deficiency suppresses tumor progression via stabilizing CK1ε in renal cell carcinoma

doi: 10.1038/s41419-018-0646-2

Figure Lengend Snippet: a , b Representative images and the relative quantification of wound-healing assay in RCC cells transfected with shREGγ and shNC. c , d Representative images and the relative quantification of transwell invasion assay in ACHN and A498 cells transfected with shREGγ and shNC. Data are shown as mean ± SD. * P < 0.05

Article Snippet: The stable REGγ knockdown RCC cell lines ACHN and A498 were generated by integration of retroviral shREGγ vectors specific for REGγ or a control gene (GFP) from OriGene (Rockville, MD), which was performed as previously described .

Techniques: Wound Healing Assay, Transfection, Transwell Invasion Assay

Journal: Molecular Oncology

Article Title: Obesity alters the fitness of peritumoral adipose tissue, exacerbating tumor invasiveness in renal cancer through the induction of ADAM12 and CYP1B1

doi: 10.1002/1878-0261.13782

Figure Lengend Snippet: Adipose peritumoral tissue affects the expression of inflammatory genes by renal cell carcinoma (RCC) cells. (A) Relative mRNA expression of the different tumor suppressor genes in the healthy and tumor kidney tissues of RCC patients ( n = 10), including VHL , PBRM1 , SETD2 , and BAP1 , was measured by real‐time PCR. Cyclophilin‐A was used as an internal control. Data are shown as mean ± SD from at least 3 separate experiments. P ‐values were determined by Student's t ‐test. (B) Schematic figure illustrating the protocol for differentiation of adipocytes from peritumoral adipose tissue (AT) of lean, overweight, and obese RCC patients. (C) Relative mRNA expression of the leptin and adiponectin of the differentiated adipocytes among the lean, overweight, and obese groups ( n = 6). Data are shown as mean ± SD from at least 3 separate experiments. P ‐values were determined by one‐way ANOVA. (D) Schematic figure of the conditioned medium (CM) experiment, showing RCC cells treated with conditioned media (CMs) of lean, overweight, and obese differentiated adipocytes. (E) Relative mRNA expression of the IL6 , CXCR4 , SDF1 , and BAFFR of the A498 cells treated with CMs collected from differentiated adipocytes derived from the lean, overweight, and obese groups ( n = 6). Data are shown as mean ± SD from at least 3 separate experiments. P ‐values were determined by one‐way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The human renal cancer cell line (A498) was grown in DMEM (Microgem); supplemented with 10% FBS, 1% Pen/Strep, and 1% L‐Glutamine, at 37 °C in 5% CO 2 .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Derivative Assay

Journal: Journal of Oncology

Article Title: Effects of the Targeted Regulation of CCRK by miR-335-5p on the Proliferation and Tumorigenicity of Human Renal Carcinoma Cells

doi: 10.1155/2022/2960050

Figure Lengend Snippet: CCRK promoted ccRCC cell proliferation in vitro . (a) and (b) Analysis of the CCRK expression and cell proliferation rates in A498 and ACHN cells transfected with scr-shRNA or CCRK-shRNA by western blotting and CCK-8 assay. (c) The CCRK plasmid (pcDNA3.1) was transfected by Lipofectamine 2000 in A498 cells with knockdown of CCRK, CCRK expression and cell proliferation were partly recovered. (d) Colony formation assay in A498 and ACHN cells.

Article Snippet: In brief, A498 cells (transfected with CCRK-shRNA or scr-shRNA) were transplanted into the subcutaneous tissue of 6-week-old male BALB/c nude mice (Shanghai Sippr BK Laboratory Animals Ltd., Shanghai, China) at a concentration of 2 × 10 6 cells/mL ( n = 4 mice per group).

Techniques: In Vitro, Expressing, Transfection, shRNA, Western Blot, CCK-8 Assay, Plasmid Preparation, Colony Assay

Journal: Journal of Oncology

Article Title: Effects of the Targeted Regulation of CCRK by miR-335-5p on the Proliferation and Tumorigenicity of Human Renal Carcinoma Cells

doi: 10.1155/2022/2960050

Figure Lengend Snippet: CCRK inhibited apoptosis of RCC cells in vitro. (a) and (b) Analysis of the cell proliferation index at 24 h, 48 h, and 72 h in A498 and ACHN cells transfected with CCRK-shRNA or src-shRNA by flow cytometry. (c) and (d) Analysis of the cell apoptosis rates in A498 and ACHN cells transfected with CCRK-shRNA or src-shRNA by flow cytometry.

Article Snippet: In brief, A498 cells (transfected with CCRK-shRNA or scr-shRNA) were transplanted into the subcutaneous tissue of 6-week-old male BALB/c nude mice (Shanghai Sippr BK Laboratory Animals Ltd., Shanghai, China) at a concentration of 2 × 10 6 cells/mL ( n = 4 mice per group).

Techniques: In Vitro, Transfection, shRNA, Flow Cytometry

Journal: Journal of Oncology

Article Title: Effects of the Targeted Regulation of CCRK by miR-335-5p on the Proliferation and Tumorigenicity of Human Renal Carcinoma Cells

doi: 10.1155/2022/2960050

Figure Lengend Snippet: Western blotting in vitro and tumour volume analysis in vivo . (a) Analysis of the expression of cell cycle marker (cyclin D1) and apoptosis markers (caspase-3, cleaved caspase-3, and Bax) in A498 and ACHN cells transfected with or without lentivirus CCRK-shRNA by western blotting. (b) Analysis of tumour volume in vivo after subcutaneous inoculation of nude mice in A498 cells transfected with or without lentivirus CCRK-shRNA ( n = 4 mice). (c) The IHC stain in tumour tissues of nude mice ( n = 3). (d) Analysis of optical density (OD) value of Ki-67 index in tumour tissues of nude mice ( n = 3).

Article Snippet: In brief, A498 cells (transfected with CCRK-shRNA or scr-shRNA) were transplanted into the subcutaneous tissue of 6-week-old male BALB/c nude mice (Shanghai Sippr BK Laboratory Animals Ltd., Shanghai, China) at a concentration of 2 × 10 6 cells/mL ( n = 4 mice per group).

Techniques: Western Blot, In Vitro, In Vivo, Expressing, Marker, Transfection, shRNA, Staining

Journal: Journal of Oncology

Article Title: Effects of the Targeted Regulation of CCRK by miR-335-5p on the Proliferation and Tumorigenicity of Human Renal Carcinoma Cells

doi: 10.1155/2022/2960050

Figure Lengend Snippet: miR-335-5p binding to 3'UTR of CCRK and its effects on ccRCC patients. (a) The volcano plot shows the upregulated (red dot) and downregulated (light-green dot) miRNAs in ccRCC patients and normal patients. (b) The Venn diagram showed the overlapping miRNAs (yellow, 5) between predicted CCRK-targeting miRNAs (blue, 422) by TargetScan 3.1 and downregulated miRNAs (orange, 15) in ccRCC tissues. (c) Comparison of the miR-335-5p expression levels between normal tissues and primary tumour tissues (ccRCC) based on TCGA database. (d) The relative expression of miR-335-5p in RCC cells (A498, 786-O, Caki-1, and ACHN). (e) Comparison of the relative expression of miR-335-5p among tumours of different pathological grades; (f) The miR-335-5p and CCRK 3′UTR had conservative binding sites. (g) The expression of CCRK protein whether adding miR-335-5p mimic into A498 and ACHN cells. (h) Analysis of the luciferase activity between miR-335-5p group and miR-NC group in A498 and ACHN cells transfected with wild-type (wt) or mutant (mu) CCRK 3′UTR. ∗P < 0.05; ∗∗P < 0.01; and ∗∗∗P < 0.001.

Article Snippet: In brief, A498 cells (transfected with CCRK-shRNA or scr-shRNA) were transplanted into the subcutaneous tissue of 6-week-old male BALB/c nude mice (Shanghai Sippr BK Laboratory Animals Ltd., Shanghai, China) at a concentration of 2 × 10 6 cells/mL ( n = 4 mice per group).

Techniques: Binding Assay, Expressing, Luciferase, Activity Assay, Transfection, Mutagenesis

Journal: Journal of Oncology

Article Title: Effects of the Targeted Regulation of CCRK by miR-335-5p on the Proliferation and Tumorigenicity of Human Renal Carcinoma Cells

doi: 10.1155/2022/2960050

Figure Lengend Snippet: expression of CCRK attenuated the effects of miR-335-5p in ccRCC cell lines. A498 cells and ACHN cells were transfected with miR-335-5p mimic or miR-NC and cotransfected with miR-335-5p mimic + LV-CCRK or miR-335-5p mimic + LV-NC. (a) and (b) Analysis of the cell proliferation rate of 4 groups in A498 cells and ACHN cells by the CCK-8 assay. (c), (d), (e), and (f) Analysis of the apoptosis rates and proliferation index of 4 groups in A498 cells and ACHN cells by flow cytometry. (g), (h), and (i) Analysis of the expression of CCRK, apoptosis markers (caspase-3, cleaved caspase-3, and Bax), and cell cycle marker (cyclin D1) of 4 groups by western blotting. The data are presented as the mean ± SEM. NC, negative control; LV, lentivirus. ∗ /# P < 0.05; ∗∗ /## P < 0.01; and ∗∗∗ /### P < 0.001. ∗ compared with miR NC, # compared with miR-335 mimic + LV-NC.

Article Snippet: In brief, A498 cells (transfected with CCRK-shRNA or scr-shRNA) were transplanted into the subcutaneous tissue of 6-week-old male BALB/c nude mice (Shanghai Sippr BK Laboratory Animals Ltd., Shanghai, China) at a concentration of 2 × 10 6 cells/mL ( n = 4 mice per group).

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