renal clear cell carcinoma cell line a498  (Procell Inc)

Journal: British Journal of Cancer

Article Title: Identification of low-dose multidrug combinations for sunitinib-naive and pre-treated renal cell carcinoma

doi: 10.1038/s41416-020-0890-y

Figure Lengend Snippet: Cell line-specific four-drug low-dose combinations and their efficacy in cell metabolic activity inhibition identified in the TGMO-based search.

Article Snippet: Human clear-cell renal cell carcinoma cell lines A498, Caki-1, and 786-O were purchased at ATCC.

Techniques: Activity Assay, Inhibition

Journal: British Journal of Cancer

Article Title: Identification of low-dose multidrug combinations for sunitinib-naive and pre-treated renal cell carcinoma

doi: 10.1038/s41416-020-0890-y

Figure Lengend Snippet: a General scheme of selected drugs and their targets within important cell signalling pathways. b The pipeline of the TGMO search to find optimised low-dose drug combinations. c Within three searches, over 200 drug combinations are experimentally tested; the results in cell metabolic activity inhibition are presented from the lowest to the highest efficacy. d Representation of the results of search 3 visualising the best drug candidates for the final drug combination of A498-ST. Regression coefficients of the models of efficacy (blue) and the therapeutic window (turquoise) are presented. Significance is represented with ** p < 0.01. e The activity of the final drug combination in cell metabolic activity (% Ctrl) and the monotherapies at corresponding doses. Error bars represent the standard deviation ( N = 2–3). Significances of ** p < 0.005 define the difference of the ODC treatment to the Ctrl and each corresponding monotherapy determined with one-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: Human clear-cell renal cell carcinoma cell lines A498, Caki-1, and 786-O were purchased at ATCC.

Techniques: Activity Assay, Inhibition, Standard Deviation, Comparison

Journal: British Journal of Cancer

Article Title: Identification of low-dose multidrug combinations for sunitinib-naive and pre-treated renal cell carcinoma

doi: 10.1038/s41416-020-0890-y

Figure Lengend Snippet: Efficacy of the ODC and corresponding monotherapies over a period of 70 days after chronic ( a ) or discontinued ( b ) treatment with 1 µM sunitinib. c Decreased efficacy of the ODC by reducing the dose to a final concentration of 75% or 50%, or by removing one drug (selumetinib). d Cell metabolic activity inhibition after prolonged incubation of A498-ST cells (72, 144 and 216 h) with the ODC and corresponding monotherapies, as well as sunitinib used as positive control. e Cross-validation of the optimised drug combination established for A498-ST in other RCC and non-cancerous cell lines. Error bars represent the standard deviation (cell metabolic activity measurements, N = 2–3). Significances of * p < 0.01 and ** p < 0.005 define the difference of the ODC treatment to the Ctrl and corresponding monotherapies determined with one-way or two-way ANOVA with Tukey’s post hoc test.

Article Snippet: Human clear-cell renal cell carcinoma cell lines A498, Caki-1, and 786-O were purchased at ATCC.

Techniques: Concentration Assay, Activity Assay, Inhibition, Incubation, Positive Control, Biomarker Discovery, Standard Deviation

Journal: British Journal of Cancer

Article Title: Identification of low-dose multidrug combinations for sunitinib-naive and pre-treated renal cell carcinoma

doi: 10.1038/s41416-020-0890-y

Figure Lengend Snippet: a Representative images of heterotypic 3D co-culture spheroids composed of cell tracker-stained A498-ST cells (green), NHDFα (blue) and ECRF24 cells (red), and the 3D spheroid development over the first 24 h. Scale bar represents 150 µm. b Spheroid cell metabolic activity in A498-ST 3D co-cultures after treatment with Ctrl, ODC, corresponding monotherapies or 1 µM sunitinib (positive control). The results were obtained after administering each treatment for 72 h at two treatment schedules, i.e. (i) day 2–5 after spheroid formation (left graph), or (ii) day 4–7 after spheroid formation (right graph). Error bars represent the standard deviation (metabolic activity measurements, N = 2–3). Significances of ** p < 0.005 define the difference of the ODC treatment to the Ctrl and each corresponding monotherapy determined with an ordinary one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Human clear-cell renal cell carcinoma cell lines A498, Caki-1, and 786-O were purchased at ATCC.

Techniques: Co-Culture Assay, Staining, Activity Assay, Positive Control, Standard Deviation

Journal: British Journal of Cancer

Article Title: Identification of low-dose multidrug combinations for sunitinib-naive and pre-treated renal cell carcinoma

doi: 10.1038/s41416-020-0890-y

Figure Lengend Snippet: a Representative images of A498 and A498-ST cells stained for f-actin (phalloidin, green) and nuclei (Dapi, blue). In total, 19.000 cells/well were seeded, incubated with the ODC or corresponding monotherapies and imaged after 24 h. Scale bar represents 20 µm. b Quantification of the presence and average length of connected and non-connected tunnelling nanotubes after treatment. c , d The size of the cell body (striped, above dashed line) and nucleus (full, below dashed line). e , f Quantified mitotic abnormalities plotted for Ctrl and ODC-treated A498 and A498-ST cells. Error bars represent the standard deviation ( N = 2, 6 images of two biological repeats). Significances of ** p < 0.005 define the difference of the ODC treatment to the Ctrl determined with paired t test.

Article Snippet: Human clear-cell renal cell carcinoma cell lines A498, Caki-1, and 786-O were purchased at ATCC.

Techniques: Staining, Incubation, Standard Deviation

Journal: BMC Biotechnology

Article Title: A TXNIP-driven bioluminescent reporter for high-throughput discovery of glycolytic inhibitors against renal cell carcinoma

doi: 10.1186/s12896-026-01111-7

Figure Lengend Snippet: 2-DG and its derivatives inhibit glycolysis and cell proliferation of A498 cells. ( A – C ) Proliferation of A498 cells measured by CCK-8 assay after treatment with 2-DG ( A ), 2-FG ( B ), and 2-DG-d ( C ). For 2-DG treatment, the concentration gradient was set as 0 mM (vehicle control, containing equal volume of DMSO), 5 mM, 10 mM, and 15 mM; while for 2-FG and 2-DG-d treatments, the concentration gradients were consistent: 0 mM (vehicle control), 1 mM, 2 mM, 5 mM, and 10 mM. Cell viability was assessed by CCK-8 assay at the indicated time points over a 4-day period. ( D - F ) Glucose uptake capacity of A498 cells after 2-DG ( D ), 2-FG ( E ), and 2-DG-d ( F ) treatment, measured using a glucose uptake assay kit. The concentration gradients of each compound were identical to those described in the aforementioned section for cell proliferation detection. ( G - I ) Lactate production levels in A498 cells following treatment with gradient concentrations of 2-DG ( G ), 2-FG ( H ), and 2-DG-d ( I ), detected by a lactate detection kit. The concentration gradients for each compound were consistent with those used in cell proliferation detection. Data are mean ± SEM ( n = 3); data in Figs. 1A-C were analyzed by two-way ANOVA with Dunnett’s multiple comparisons test; data in Figs. 1D-I were analyzed by one-way ANOVA with Dunnett’s multiple comparisons; * p < 0.05, *** p < 0.001, **** p < 0.0001

Article Snippet: The human renal clear cell carcinoma cell line A498 (Cat. No. CL-0254, RRID: CVCL_1056) was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: CCK-8 Assay, Concentration Assay, Control

Journal: BMC Biotechnology

Article Title: A TXNIP-driven bioluminescent reporter for high-throughput discovery of glycolytic inhibitors against renal cell carcinoma

doi: 10.1186/s12896-026-01111-7

Figure Lengend Snippet: 2-DG upregulates MLXIP and TXNIP expression in A498 cells. ( A ) Volcano plot of differentially expressed genes (DEGs). ( B ) Top 10 upregulated genes from RNA-seq analysis. ( C ) TXNIP mRNA detected by RT-qPCR in A498 cells after 48 h of treatment with 2-DG (0, 5, 10, and 15 mM). ( D ) Protein expression of TXNIP and MLXIP was detected by Western blotting in A498 cells treated with 2-DG at 0, 5, 10, and 15 mM for 48 h. The 0 mM group served as the vehicle control and contained an equal volume of DMSO. ( E ) MLXIP and TXNIP mRNA expression analyzed by RT-qPCR in A498 cells 48 h after transfection with MLXIP plasmid (0, 2, and 4 µg), where 0 µg MLXIP plasmid corresponds to the empty vector plasmid used as control. Data are mean ± SEM ( n = 3); Statistical significance was analyzed by one-way ANOVA with Dunnett’s multiple comparisons; **** p < 0.0001, *** p < 0.001

Article Snippet: The human renal clear cell carcinoma cell line A498 (Cat. No. CL-0254, RRID: CVCL_1056) was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, Control, Transfection, Plasmid Preparation

Journal: BMC Biotechnology

Article Title: A TXNIP-driven bioluminescent reporter for high-throughput discovery of glycolytic inhibitors against renal cell carcinoma

doi: 10.1186/s12896-026-01111-7

Figure Lengend Snippet: TXNIP inhibits glycolysis in A498 cells. ( A ) Volcano plot of TXNIP expression-related genes in KIRC generated using LinkedOmics analysis. ( B ) GO enrichment and KEGG pathway analysis of TXNIP expression-related genes in KIRC performed using DAVID. ( C ) Comparison of TXNIP expression levels between KIRC tumors and adjacent normal tissues, and ( D ) analysis of TXNIP expression across different tumor grades, both using UALCAN. ( E - G ) Glucose uptake ( E ) and lactate production ( F ) were measured in A498 cells transfected with increasing amounts (0, 0.5, 1, and 2 µg) of TXNIP plasmid, where 0 µg TXNIP plasmid corresponds to the empty vector plasmid used as control. TXNIP mRNA analyzed by RT-qPCR in A498 cells 48 h after transfection with TXNIP plasmid ( G ). Data are mean ± SEM ( n = 3); Statistical significance was analyzed by one-way ANOVA with Dunnett’s multiple comparisons; **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05

Article Snippet: The human renal clear cell carcinoma cell line A498 (Cat. No. CL-0254, RRID: CVCL_1056) was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing, Generated, Comparison, Transfection, Plasmid Preparation, Control, Quantitative RT-PCR

Journal: BMC Biotechnology

Article Title: A TXNIP-driven bioluminescent reporter for high-throughput discovery of glycolytic inhibitors against renal cell carcinoma

doi: 10.1186/s12896-026-01111-7

Figure Lengend Snippet: Characterization of a stable RCC reporter cell line with TXNIP promoter-driven luciferase expression. ( A ) Schematic diagrams of pGL4.19-TXNIP-Pro-Luc2 constructs. The TXNIP promoter fragment, spanning from − 1166 bp to + 312 bp relative to the transcription start site (TSS), was cloned into the pGL4.19-Luc2 vector to drive luciferase expression. ( B ) A498-TXNIP-Pro-Luc2 cells or A498-Luc2 cells were lysed for luciferase activity analysis. ( C ) A498-TXNIP-Pro-Luc2 and A498-Luc2 cells were imaged using the IVIS Lumina LT system to obtain flux measurements (left panel, images). Quantified flux data were averaged ( n = 3) and plotted (right panel, graph). ( D , E ) After transfection of the MLXIP plasmids into A498-TXNIP-Pro-Luc2 ( D ) or A498-Luc2 cells ( E ) for 48 h, imaging was performed (left panel, images). Quantified flux data were averaged ( n = 3) and plotted (right panel, graph). ( F ) After transfection of the MLXIP plasmids into A498-TXNIP-Pro-Luc2 or A498-Luc2 cells for 48 h, the cells were lysed for luciferase activity analysis. ( G , H ) A498-TXNIP-Pro-Luc2 cells ( G ) and A498-Luc2 cells ( H ) were serially diluted, placed into wells of a 96-well plate, and immediately imaged. Quantified flux data were averaged ( n = 3) and plotted. Data are mean ± SEM ( n = 3); Statistical significance for B and C was analyzed by one-way ANOVA; for D , E , and F , it was analyzed by one-way ANOVA with Dunnett’s multiple comparisons test; **** p < 0.0001

Article Snippet: The human renal clear cell carcinoma cell line A498 (Cat. No. CL-0254, RRID: CVCL_1056) was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Luciferase, Expressing, Construct, Clone Assay, Plasmid Preparation, Activity Assay, Transfection, Imaging

Journal: BMC Biotechnology

Article Title: A TXNIP-driven bioluminescent reporter for high-throughput discovery of glycolytic inhibitors against renal cell carcinoma

doi: 10.1186/s12896-026-01111-7

Figure Lengend Snippet: 2-DG and its derivatives activate TXNIP promoter-driven luciferase expression in A498 cells. ( A – C ) Luciferase activity driven by the TXNIP promoter (A498-TXNIP-Pro-Luc2 cells) after 48 h treatment with 2-DG ( A ), 2-FG ( B ), and 2-DG-d ( C ). For 2-DG treatment, the concentration gradient was set as 0 mM (vehicle control, containing equal volume of DMSO), 5 mM, 10 mM, and 15 mM; while for 2-FG and 2-DG-d treatments, the concentration gradients were consistent: 0 mM (vehicle control), 1 mM, 2 mM, 5 mM, and 10 mM. ( D – F ) Luciferase activity in control A498-Luc2 cells after 48 h treatment with 2-DG ( D ), 2-FG ( E ), and 2-DG-d ( F ). The concentration gradients for each compound were the same as those described for A498-TXNIP-Pro-Luc2 cells above. ( G – I ) After treating A498-TXNIP-Pro-Luc2 cells with 2-DG ( G ), 2-FG ( H ), and 2-DG-d ( I ), for 48 h, flux measurements were acquired using the IVIS Lumina LT system. ( J – L ) After treating A498-Luc2 cells with 2-DG ( J ), 2-FG ( K ), and 2-DG-d ( L ) for 48 h, flux measurements were acquired using the IVIS Lumina LT system. Top, cellular images; bottom, normalized fold induction of TXNIP-Pro-Luc2 or Luc2 treated with the indicated doses of drugs. Quantified flux data were averaged ( n = 3) and plotted. The dosage of each compound was consistent with that used in the previous luciferase activity assay. Data are mean ± SEM ( n = 3); Statistical significance was analyzed by one-way ANOVA with Dunnett’s multiple comparisons; **** p < 0.0001, ** p < 0.01, * p < 0.05

Article Snippet: The human renal clear cell carcinoma cell line A498 (Cat. No. CL-0254, RRID: CVCL_1056) was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Luciferase, Expressing, Activity Assay, Concentration Assay, Control

Journal: British Journal of Cancer

Article Title: CDK4/6 inhibitors dephosphorylate RNF26 to stabilize TSC1 and increase the sensitivity of ccRCC to mTOR inhibitors

doi: 10.1038/s41416-024-02750-3

Figure Lengend Snippet: 786-O and A498 cells were transfected with EV, HA-CDK4 (1 ng, +), and HA-CDK4 (5 ng, ++) for 24 h. Cells were harvested for western blotting ( a ) and RT-qPCR ( b ) assay. Data are presented as the mean ± SEM of three replicates. ns, not significant. c , d 786-O and A498 cells were transfected with indicated siRNAs for 48 h. Cells were harvested for western blotting ( c ) and RT-qPCR ( d ) assay. Data are presented as the mean ± SEM of three replicates. ns, not significant; *, P < 0.05. e , f 786-O and A498 cells were treated with vehicle, Palbociclib (1 μM, +), or (10 μM, ++) for 24 hours. Cells were harvested for western blotting ( e ) and RT-qPCR ( f ) assay. Data are presented as the mean ± SEM of three replicates. ns, not significant; **, P < 0.01. g–i 786-O cells were transfected with the indicated plasmids or siRNAs for 48 h, or treated with Palbociclib (5 μM) for 24 h. Cells were treated with CHX, and cells were collected for western blotting analysis at different time points. j 786-O cells were treated with indicated chemicals for 48 h and subjected to western blotting analysis. k 786-O cells were treated with indicated chemicals for 48 h and subjected to immunoprecipitation and western blotting analysis. l IHC staining of the tissue microarray of ccRCC (n = 38) with CDK4 or TSC1 antibodies.

Article Snippet: Renal clear cell carcinoma cell lines A498 (#CL-0254, Procell Life Science&Technology, Wuhan, China) and HUVEC (#CL-0122, Procell Life Science&Technology, Wuhan, China) were purchased from Procell Life Science&Technology and identified by short tandem repeat (STR) profiling.

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Immunohistochemistry, Microarray

Journal: British Journal of Cancer

Article Title: CDK4/6 inhibitors dephosphorylate RNF26 to stabilize TSC1 and increase the sensitivity of ccRCC to mTOR inhibitors

doi: 10.1038/s41416-024-02750-3

Figure Lengend Snippet: a 786-O cells were treated with or without Palbociclib (5 μM) for 24 h. Cells were harvested for immunoprecipitation and mass spectrometry analysis by using the IgG or TSC1 antibodies. b , c 786-O and A498 cells were harvested for immunoprecipitation by using the TSC1 or RNF26 antibodies. d 786-O cells were transfected with indicated plasmids for 48 h. Cells were harvested for immunofluorescence staining by using the RNF26 or TSC1 antibodies. e Proximity Ligation Assay (PLA) was performed in the 786-O cells by using the indicated antibodies. f A model depicting the domain of RNF26. g GST-pull down assay was performed by using the recombinant protein of RNF26. h 786-O cells were treated with indicated chemicals for 24 h. Cells were harvested for immunoprecipitation and western blotting assay. i, j 786-O cells were treated with indicated plasmids or siRNAs for 48 h. Cells were harvested for immunoprecipitation and western blotting assay. k 786-O cells were treated with indicated chemicals for 24 h. Cells were harvested for immunoprecipitation and western blotting assay. l 786-O cells were treated with indicated plasmids or chemicals for 48 h. Cells were harvested for immunoprecipitation and western blotting assay. m 786-O and A498 cells were harvested for immunoprecipitation by using the CDK4 or RNF26 antibodies. n GST-pull down assay was performed by using the recombinant protein of RNF26.

Article Snippet: Renal clear cell carcinoma cell lines A498 (#CL-0254, Procell Life Science&Technology, Wuhan, China) and HUVEC (#CL-0122, Procell Life Science&Technology, Wuhan, China) were purchased from Procell Life Science&Technology and identified by short tandem repeat (STR) profiling.

Techniques: Immunoprecipitation, Mass Spectrometry, Transfection, Immunofluorescence, Staining, Proximity Ligation Assay, Pull Down Assay, Recombinant, Western Blot

Journal: British Journal of Cancer

Article Title: CDK4/6 inhibitors dephosphorylate RNF26 to stabilize TSC1 and increase the sensitivity of ccRCC to mTOR inhibitors

doi: 10.1038/s41416-024-02750-3

Figure Lengend Snippet: a Analysis of the TCGA-KIRC dataset after sub-dividing the RNF26 into high or low expression group. b CancerSEA web tool ( http://biocc.hrbmu.edu.cn/CancerSEA/ ) was used to analyze the biological function of RNF26 in the RCC. c – j 786-O and A498 cells were infected with indicates shRNAs for 72 h. Cells were harvested for transwell, colony formation, tube formation, CCK-8 and nude mouse xenograft assay. Data are presented as the mean ± SEM of three replicates for in vitro assay and fix replicates for in vivo assay. ***, P < 0.001. k 786-O and A498 cells were transfected with indicates plasmids for 48 h. Cells were harvested for CCK-8, transwell and tube formation assay. Data are presented as the mean ± SEM of three replicates. ***, P < 0.001. l , m 786-O cells were transfected with indicated plasmids for 24 h. Cells were harvested for transwell and tube formation assay. Data are presented as the mean ± SEM of three replicates. ***, P < 0.001.

Article Snippet: Renal clear cell carcinoma cell lines A498 (#CL-0254, Procell Life Science&Technology, Wuhan, China) and HUVEC (#CL-0122, Procell Life Science&Technology, Wuhan, China) were purchased from Procell Life Science&Technology and identified by short tandem repeat (STR) profiling.

Techniques: Expressing, Infection, CCK-8 Assay, Xenograft Assay, In Vitro, In Vivo, Transfection, Tube Formation Assay

Journal: British Journal of Cancer

Article Title: CDK4/6 inhibitors dephosphorylate RNF26 to stabilize TSC1 and increase the sensitivity of ccRCC to mTOR inhibitors

doi: 10.1038/s41416-024-02750-3

Figure Lengend Snippet: a–c A498 cells were infected with indicated shRNAs for 72 h. Cells were harvested and subjected to RNA-seq analysis ( a ). KEGG and GSEA analysis were performed. d GSEA analysis of TCGA-KIRC was performed after sub-dividing the RNF26 into high or low expression group. e , f 786-O and A498 cells were infected with indicates shRNAs for 72 h. Cells were harvested for western blotting analysis ( e ) and ELISA assay ( f ). Data are presented as the mean ± SEM of three replicates. ***, P < 0.001. g , h 786-O and A498 cells were transfected with indicates plasmids for 48 h. Cells were harvested for western blotting analysis ( g ) and ELISA assay ( h ). Data are presented as the mean ± SEM of three replicates. ***, P < 0.001. i 786-O and A498 cells were transfected with indicated constructs for 72 h. Cells were harvested for western blotting analysis. SR, shRNA resistant. j , k 786-O cells were transfected with indicated constructs for 72 h. Cells were harvested and treated with a serial dose of Everolimus for 24 h to measure the IC50 values of Everolimus. l–n IHC staining of the tissue microarray of ccRCC (n = 38) with RNF26, p-S6K1 T389 or VEGFA antibodies.

Article Snippet: Renal clear cell carcinoma cell lines A498 (#CL-0254, Procell Life Science&Technology, Wuhan, China) and HUVEC (#CL-0122, Procell Life Science&Technology, Wuhan, China) were purchased from Procell Life Science&Technology and identified by short tandem repeat (STR) profiling.

Techniques: Infection, RNA Sequencing, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Construct, shRNA, Immunohistochemistry, Microarray

Journal: British Journal of Cancer

Article Title: CDK4/6 inhibitors dephosphorylate RNF26 to stabilize TSC1 and increase the sensitivity of ccRCC to mTOR inhibitors

doi: 10.1038/s41416-024-02750-3

Figure Lengend Snippet: a–d 786-O and A498 cells were transfected with indicated plasmids for 24 h. Then, these cells were treated with or without Everolimus and subjected to western blotting, transwell, CCK-8, and tube formation assay. Data are presented as the mean ± SEM of three replicates. Ns, not significant; **, P < 0.01; ***, P < 0.001. e–k 786-O and A498 cells were infected with indicated shRNAs for 72 h. Then, these cells were treated with or without Everolimus and subjected to western blotting, transwell, CCK-8, tube formation, and nude mouse xenografts assay. Data are presented as the mean ± SEM of three replicates for in vitro assay and six replicates for in vivo assay. Ns, not significant; *, P < 0.05; ***, P < 0.001.

Article Snippet: Renal clear cell carcinoma cell lines A498 (#CL-0254, Procell Life Science&Technology, Wuhan, China) and HUVEC (#CL-0122, Procell Life Science&Technology, Wuhan, China) were purchased from Procell Life Science&Technology and identified by short tandem repeat (STR) profiling.

Techniques: Transfection, Western Blot, CCK-8 Assay, Tube Formation Assay, Infection, In Vitro, In Vivo

Journal: British Journal of Cancer

Article Title: CDK4/6 inhibitors dephosphorylate RNF26 to stabilize TSC1 and increase the sensitivity of ccRCC to mTOR inhibitors

doi: 10.1038/s41416-024-02750-3

Figure Lengend Snippet: a , b 786-O and A498 cells were infected with indicated shRNAs for 72 h. Cells were harvested for western blotting ( a ) and RT-qPCR assay ( b ). Data are presented as the mean ± SEM of three replicates. Ns, not significant. c , d 786-O and A498 cells were transfected with indicated plasmids for 48 h. Cells were harvested for western blotting ( c ) and RT-qPCR assay ( d ). Data are presented as the mean ± SEM of three replicates. Ns, not significant. e , f A498 cells were transfected with indicated plasmids for 48 h. These cells were treated with or without MG132 or bafA1 and subjected to western blotting analysis. g A498 cells were transfected with indicated plasmids for 48 h and subjected to western blotting analysis. h , i 786-O cells were infected or transfected with the indicated shRNAs or plasmids for 72 h. Cells were treated with CHX, and cells were collected for western blotting analysis at different time points. j 786-O cells were infected with the indicated shRNAs for 72 h. Cells were harvested for immunoprecipitation and western blotting analysis. k , l IHC staining of the tissue microarray of ccRCC (n = 38) with RNF26 or TSC1 antibodies. m–o 786-O and A498 cells were transfected with indicated constructs for 72 h. Cells were harvested for western blotting analysis.

Article Snippet: Renal clear cell carcinoma cell lines A498 (#CL-0254, Procell Life Science&Technology, Wuhan, China) and HUVEC (#CL-0122, Procell Life Science&Technology, Wuhan, China) were purchased from Procell Life Science&Technology and identified by short tandem repeat (STR) profiling.

Techniques: Infection, Western Blot, Quantitative RT-PCR, Transfection, Immunoprecipitation, Immunohistochemistry, Microarray, Construct

Journal: BIO-PROTOCOL

Article Title: BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry

doi: 10.21769/bioprotoc.1912

Figure Lengend Snippet: Figure 1. Quantification and visualization of intracellular neutral LDs A. A498 cells were grown overnight in the presence of BSA (0.2%) or 30 μM oleic acid with BSA. The cells were analyzed according to the section titled “BODIPY staining for flow cytometry”. For each sample, mean FL-1 area was normalized to the mean FL-1 area for the BSA treated samples. Note that oleic acid increased FL-1 area. P-value was determined by an unpaired student’s t-test. ***P <0.001. B. Representative histograms of cells described in (A). C. A498 cells grown on coverslips were treated overnight with BSA (0.2%) or 30 μM oleic acid with BSA. The cells were analyzed according to the section titled “BODIPY staining for microscopy”. Scale bar = 5 μm.

Article Snippet: Cell line of interest *For correspondence: celeste2@mail.med.upenn.edu. uthor M anuscript A uthor M anuscript A uthor M anuscript Note: For this protocol, we utilize the A498 clear cell renal cell carcinoma cell line (ATCC, catalog number: A-498), but this method can be readily performed with other cell lines.

Techniques: Staining, Flow Cytometry, Microscopy