Journal: International Journal of Biological Sciences

Article Title: Suppression of ASNS expression by VHL-mediated ubiquitination hinders the progression of renal cell carcinoma through enhancing JUP expression and inhibiting PI3K-AKT and MAPK pathways

doi: 10.7150/ijbs.129332

Figure Lengend Snippet: L-Asparagine enhanced the growth and metastasis of RCC in vitro and in vivo . Cell plate cloning experiments assessing HK2, Caki-1 and 786-O cell lines treated with varying concentrations of L-Asparagine (L-Asn). (B) Cell plate cloning experiments evaluating HK2, Caki-1, and 786-O cell lines treated with L-ASNase (0.5 U/mL). (C and D) Experiments measuring cell migration and invasion in HK2, Caki-1, and 786-O cell lines treated with either L-Asn (0.5mM) or L-ASNase (0.5 U/mL). (E) In vivo imaging of mouse orthotopic renal tumors fed with either L-Asn-rich (3g L-Asn/kg feed) or deficient diets (Each group consists of 5 mice). (F) In vitro imaging of mouse lungs subjected to L-Asn-rich (3g/kg) or -deficient diets (Each group consists of 5 mice). Lung metastasis rate: CON: 5/5; L-Asn -: 4/5; L-Asn +: 5/5. (G) ELISA assay quantifying the levels of L-Asparagine synthase (ASNS) and L-ASNase in mouse kidney tumors. (H) ELISA assay quantifying the levels of ASNS and L-ASNase in plasma. (I) The relationship between the expression of ASNS in the plasma of these 63 VHL -mutant RCC patients and the disease-free survival (DFS) of patients. (J) The expression of ASNS in these 19 pairs of VHL -mutant RCC and their AN tissues was detected through immunohistochemical staining. (K) Immunohistochemical staining to detect ASNS protein in ccRCC tissues. (L) Western blotting analysis of ASNS protein in RCC tissues. (M) Western blotting analysis of ASNS protein in various RCC cell lines (A498, OSRC2, Caki-1, 769-P, 786-O, and RCC4).

Article Snippet: Six RCC cell lines A498, OSRC2, Caki-1 ( VHL wild-type, metastatic RCC cell line), 769-P, 786-O ( VHL mutant), RCC4 ( VHL mutant) and two control cell lines HK2 (Epithelial cells of human renal cortex proximal tubules), HEK-293 (Human embryonic kidney cells) were purchased from the ATCC (American Type Culture Collection) cell bank.

Techniques: In Vitro, In Vivo, Cloning, Migration, In Vivo Imaging, Imaging, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Expressing, Mutagenesis, Immunohistochemical staining, Staining, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Suppression of ASNS expression by VHL-mediated ubiquitination hinders the progression of renal cell carcinoma through enhancing JUP expression and inhibiting PI3K-AKT and MAPK pathways

doi: 10.7150/ijbs.129332

Figure Lengend Snippet: VHL interacted with ASNS protein and modulated its expression. (A) Western blotting analysis showing changes in ASNS protein expression in HK2, 786-O, RCC4, and Caki-1 cell lines after treatment with different concentrations of L-Asn. (B) Comparison of ASNS protein expression in HK2, 786-O, RCC4, and Caki-1 cell lines post L-Asn treatment. (C) ELISA analysis of L-Asn expression levels in 786-O and RCC4 cell lines following VHL overexpression. (D and E) Western blotting analysis of ASNS expression changes in 786-O and RCC4 cell lines after VHL overexpression. (F) Western blotting analysis of ASNS expression changes in HEK-293 and Caki-1 cell lines after VHL knockdown. (G) Western blotting analysis of ASNS expression levels in Caki-1 cells after VHL knockout. (H) Confirmation of the regulatory effects of L-Asn and VHL on ASNS expression in 786-O, RCC4, and Caki-1 cell lines.

Article Snippet: Six RCC cell lines A498, OSRC2, Caki-1 ( VHL wild-type, metastatic RCC cell line), 769-P, 786-O ( VHL mutant), RCC4 ( VHL mutant) and two control cell lines HK2 (Epithelial cells of human renal cortex proximal tubules), HEK-293 (Human embryonic kidney cells) were purchased from the ATCC (American Type Culture Collection) cell bank.

Techniques: Expressing, Western Blot, Comparison, Enzyme-linked Immunosorbent Assay, Over Expression, Knockdown, Knock-Out

Journal: International Journal of Biological Sciences

Article Title: Suppression of ASNS expression by VHL-mediated ubiquitination hinders the progression of renal cell carcinoma through enhancing JUP expression and inhibiting PI3K-AKT and MAPK pathways

doi: 10.7150/ijbs.129332

Figure Lengend Snippet: VHL facilitated the ubiquitination of ASNS and reduced its protein level. Western blotting analysis of protein stability in HER-293, 786-O, RCC4, and Caki-1 cell lines. (B) Western blotting analysis showing the impact of VHL overexpression on ASNS expression changes in 786-O and RCC4 cell lines following MG132 treatment. (C) Western blotting analysis showing the impact of VHL knockout on ASNS expression changes in HEK-293 and Caki-1 cell lines following MG132 treatment. (D) Confirmation of the interaction between VHL and ASNS proteins in 786-O, RCC4, HEK-293, and Caki-1 cell lines through immunoprecipitation (IP) experiments. (E) Validation of the role of VHL in regulating ASNS protein ubiquitination in 786-O, RCC4, HEK-293, and Caki-1 cell lines using ubiquitination IP experiments. (F) The effect of VHL knockout on ASNS protein ubiquitination using ubiquitination IP experiments in HEK-293 and Caki-1 cell lines.

Article Snippet: Six RCC cell lines A498, OSRC2, Caki-1 ( VHL wild-type, metastatic RCC cell line), 769-P, 786-O ( VHL mutant), RCC4 ( VHL mutant) and two control cell lines HK2 (Epithelial cells of human renal cortex proximal tubules), HEK-293 (Human embryonic kidney cells) were purchased from the ATCC (American Type Culture Collection) cell bank.

Techniques: Ubiquitin Proteomics, Western Blot, Over Expression, Expressing, Knock-Out, Immunoprecipitation, Biomarker Discovery

Journal: International Journal of Biological Sciences

Article Title: Suppression of ASNS expression by VHL-mediated ubiquitination hinders the progression of renal cell carcinoma through enhancing JUP expression and inhibiting PI3K-AKT and MAPK pathways

doi: 10.7150/ijbs.129332

Figure Lengend Snippet: VHL ubiquitinated the 510 lysine residue of ASNS protein to modulate its expression. Identification of distinct ubiquitination modification sites on ASNS protein in VHL-overexpressing 786-O cells compared to control cells through ubiquitination proteomic analysis. (B) Prediction of the binding site between VHL and ASNS proteins using the online docking tool HDOCK. (C) Assessment of the binding of VHL to five truncated ASNS proteins via IP experiments in HEK-293 and Caki-1 cell lines. (D) Assessment of the binding of VHL to five truncated ASNS proteins via IP experiments in VHL overexpressed 786-O cells. (E) Examination of the interaction between VHL and ASNS mutants in HEK-293 and Caki-1 cell lines. (F) Examination of the interaction between VHL and ASNS mutants in VHL overexpressed 786-O cells. (G) Western blotting analysis of the effects of VHL overexpression and MG132 treatments on ASNS protein levels in ASNS mutant RCC4 and 786-O cell lines. (H) Western blotting analysis of the effects of VHL knockout and MG132 treatments on ASNS protein levels in ASNS mutant Caki-1 cells. (I) Evaluation of changes in VHL expression in HEK-293 and Caki-1 cell lines following ASNS overexpression. (J) Assessment of VHL expression changes in HEK-293 and Caki-1 cell lines after ASNS knockdown.

Article Snippet: Six RCC cell lines A498, OSRC2, Caki-1 ( VHL wild-type, metastatic RCC cell line), 769-P, 786-O ( VHL mutant), RCC4 ( VHL mutant) and two control cell lines HK2 (Epithelial cells of human renal cortex proximal tubules), HEK-293 (Human embryonic kidney cells) were purchased from the ATCC (American Type Culture Collection) cell bank.

Techniques: Residue, Expressing, Ubiquitin Proteomics, Modification, Control, Binding Assay, Western Blot, Over Expression, Mutagenesis, Knock-Out, Knockdown

Journal: International Journal of Biological Sciences

Article Title: Suppression of ASNS expression by VHL-mediated ubiquitination hinders the progression of renal cell carcinoma through enhancing JUP expression and inhibiting PI3K-AKT and MAPK pathways

doi: 10.7150/ijbs.129332

Figure Lengend Snippet: ASNS interacted with JUP protein and affected its expression. Molecular volcano plot illustrating differential expression in ASNS-overexpressing Caki-1 cells compared to control Caki-1 cells based on TMT proteomic analysis. (B) GO pathway enrichment analysis for differentially expressed molecules. (C) Silver staining results showing ASNS overexpression and its control in IP samples from Caki-1 cells. (D) Combined analysis of TMT proteomics and mass spectrometry results from silver-stained differential protein bands. (E) Correlation analysis of JUP expression with ASNS and VHL expression using TCGA-KIRC data. (F) Correlation analysis of JUP expression with ASNS and VHL expression using the data from VHL -mutant ccRCC tissues in TCGA-KIRC. (G) Confirmation of the interaction between ASNS and JUP proteins in Caki-1 and 786-O cell lines through IP experiments. (H and I) Analysis of JUP expression characteristics in ccRCC and its association with various pathological stages, grades, and distant metastasis using TCGA-KIRC data. (J) Comparison of overall survival (OS) and DFS between patients with high and low JUP expression. (K) Validation of JUP expression in clinical samples from our center using immunohistochemical staining. (L) Western blotting analysis of JUP protein in ccRCC tissues. (M) Western blotting analysis of ASNS protein in various RCC cell lines (A498, OSRC2, Caki-1, 769-P, 786-O, and RCC4).

Article Snippet: Six RCC cell lines A498, OSRC2, Caki-1 ( VHL wild-type, metastatic RCC cell line), 769-P, 786-O ( VHL mutant), RCC4 ( VHL mutant) and two control cell lines HK2 (Epithelial cells of human renal cortex proximal tubules), HEK-293 (Human embryonic kidney cells) were purchased from the ATCC (American Type Culture Collection) cell bank.

Techniques: Expressing, Quantitative Proteomics, Control, Silver Staining, Over Expression, Mass Spectrometry, Staining, Mutagenesis, Comparison, Biomarker Discovery, Immunohistochemical staining, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Suppression of ASNS expression by VHL-mediated ubiquitination hinders the progression of renal cell carcinoma through enhancing JUP expression and inhibiting PI3K-AKT and MAPK pathways

doi: 10.7150/ijbs.129332

Figure Lengend Snippet: ASNS modulated the PI3K-AKT and MAPK pathways by interacting with JUP. Immunofluorescence staining revealed the sublocalization of ASNS and JUP protein expression in RCC tissues. (B) Western blotting analysis reveals changes in JUP protein expression following ASNS overexpression in Caki-1 cells. (C) Western blotting analysis shows alterations in JUP expression after ASNS knockdown in 786-O and RCC4 cell lines. (D) Western blotting analysis indicates changes in JUP expression after ASNS overexpression in 786-O and RCC4 cell lines. (E) A volcano plot illustrates the analysis of differentially expressed genes in JUP-overexpressing 786-O cells compared to control 786-O cells using RNA-seq. (F) The expression heat-map of these 49 differential genes. (G) KEGG pathway enrichment analysis for these differentially expressed genes. (H) Graph showing the number of differentially expressed genes included in the KEGG enrichment pathways. (I) Western blot analysis detects changes in protein expression levels of pERK1/2, pPI3K, pAKT, Cleaved-caspase 3, Bcl-2, BAX, E-cadherin, N-cadherin, and SNAIL following JUP overexpression in Caki-1 and 786-O cell lines. (J) Western blotting analysis shows changes in protein expression levels of pERK1/2, pPI3K, pAKT, Cleaved-Caspase 3, Bcl-2, BAX, E-cadherin, N-cadherin, and SNAIL after ASNS overexpression in Caki-1 and 786-O cell lines.

Article Snippet: Six RCC cell lines A498, OSRC2, Caki-1 ( VHL wild-type, metastatic RCC cell line), 769-P, 786-O ( VHL mutant), RCC4 ( VHL mutant) and two control cell lines HK2 (Epithelial cells of human renal cortex proximal tubules), HEK-293 (Human embryonic kidney cells) were purchased from the ATCC (American Type Culture Collection) cell bank.

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Over Expression, Knockdown, Control, RNA Sequencing

Journal: International Journal of Biological Sciences

Article Title: Suppression of ASNS expression by VHL-mediated ubiquitination hinders the progression of renal cell carcinoma through enhancing JUP expression and inhibiting PI3K-AKT and MAPK pathways

doi: 10.7150/ijbs.129332

Figure Lengend Snippet: Silencing ASNS and using an ASNS inhibitor significantly reduced RCC growth and metastasis in vitro and in vivo . The impact of ASNS overexpression and knockdown on the growth of HK2, Caki-1 and 786-O cell lines was evaluated using a colony formation assay. (B and C) Transwell migration and invasion assays were performed to assess the effects of ASNS overexpression and knockdown on the migration and invasion capabilities of HK2, Caki-1, and 786-O cell lines. (D) The influence of ASNS overexpression and knockdown on the growth and spontaneous lung metastasis of orthotopic renal tumors in mice was analyzed. (E) The effects of ASNS overexpression and knockdown on EdU, TUNEL, and Cleaved-Caspase 3 expression in mouse renal tumors were examined. (F) The impact of ASNS overexpression and knockdown on E-cadherin and N-cadherin expression in mouse lung metastases was assessed. (G) The effect of the ASNS inhibitor (Bisabosqual A) on the growth of Caki-1 and 786-O cell lines was evaluated through cell plate cloning experiments. (H and I) The influence of ASNS inhibitor on the migration and invasion of Caki-1 and 786-O cell lines was analyzed through migration and invasion assays. (J) The effect of ASNS inhibitor on the growth and spontaneous lung metastasis of mouse renal tumors was assessed. (K) The effects of ASNS inhibitor on EdU, TUNEL, and Cleaved-Caspase 3 expression in mouse renal tumors were examined. (L) The impact of ASNS inhibitor on E-cadherin and N-cadherin expression in mouse lung metastases was analyzed.

Article Snippet: Six RCC cell lines A498, OSRC2, Caki-1 ( VHL wild-type, metastatic RCC cell line), 769-P, 786-O ( VHL mutant), RCC4 ( VHL mutant) and two control cell lines HK2 (Epithelial cells of human renal cortex proximal tubules), HEK-293 (Human embryonic kidney cells) were purchased from the ATCC (American Type Culture Collection) cell bank.

Techniques: In Vitro, In Vivo, Over Expression, Knockdown, Colony Assay, Migration, TUNEL Assay, Expressing, Cloning

Journal: The Journal of Clinical Investigation

Article Title: HIF-2 α expression and metabolic signaling require ACSS2 in clear cell renal cell carcinoma

doi: 10.1172/JCI164249

Figure Lengend Snippet: ( A ) Left: Representative images of crystal violet staining of HEK293FT cells 72 hours after transfection with an empty vector control (EV), WT-ACSS2, or ΔT376K catalytically inactive mutant. Right: Bar graph showing quantification of 3 independent experiments in HEK293FT cells. Statistical significance was determined using a 1-way ANOVA and Tukey’s multiple-comparison test (* P < 0.05; ** P < 0.01). Data are represented as mean ± SD. ( B ) Western blot images showing expression of HIF-2α ( n = 3), ACSS2 ( n = 3), and actin ( n = 3) in 786-O Cas9 sgControl and sgACSS2 cells transfected with EV, WT-ACSS2, or ΔT376K. ( C ) Left: Representative images of crystal violet staining of 786-O Cas9 cells stably transduced to express sgControl or sgACSS2 72 hours after transfection with an empty vector control (EV), WT-ACSS2, or ΔT376K catalytically inactive mutant. Right: Bar graph showing quantification of 3 independent experiments in 786-O Cas9 cells. Statistical significance was determined using a 2-way ANOVA and Tukey’s multiple-comparison test (* P < 0.05; ** P < 0.01). Data are represented as mean ± SD. ( D ) Representative images from day 4 of a tumor sphere formation assay where 786-O Cas9 sgControl and sgACSS2 cells were transfected with EV, WT-ACSS2, or ΔT376K.

Article Snippet: 786-O Cas9 cells were purchased from Genecopoeia.

Techniques: Staining, Transfection, Plasmid Preparation, Control, Mutagenesis, Comparison, Western Blot, Expressing, Stable Transfection, Tube Formation Assay

Journal: Frontiers in Immunology

Article Title: Development of a polyamine gene expression score for predicting prognosis and treatment response in clear cell renal cell carcinoma

doi: 10.3389/fimmu.2022.1048204

Figure Lengend Snippet: Identification of key PMRGs in ccRCC and in vitro cell function experiments. (A) Screening of soft threshold. (B) Hierarchical clustering dendrogram of 16 modules. (C) Heatmap of correlations between modules and tumor. (D) K–M curves of SRM in protein levels. (E–F) Knockdown efficiency of SRM in 786-0 and 769-P. (G) Effects of SRM knockdown on CCK8 and clone formation ability of ccRCC cells. (H) Transwell was used to detect the effect of SRM on the migration and invasion ability of 786-0 and 769-P. PMRGs, polyamine metabolism-related genes; ccRCC, clear cell renal cell carcinoma. ***p < 0.001, ****p < 0.0001.

Article Snippet: ccRCC cells (786-0 and 769-P) were purchased from the China Centre for Type Culture Collection (CCTCC, Wuhan,China).

Techniques: In Vitro, Cell Function Assay, Knockdown, Migration

Journal: Scientific Reports

Article Title: ORM1 promotes tumor progression of kidney renal clear cell carcinoma (KIRC) through CALR-mediated apoptosis

doi: 10.1038/s41598-023-42962-w

Figure Lengend Snippet: ORM1 was essential to cell proliferation. ( a ) The expression of ORM1 protein in A498, 786-O, and Caki-2 cells was much higher than the 293 T cells. ( b ) The gray value analysis of protein in ( a ). ( c ) ORM1 was knocked down in 786-O and Caki-2 cells. ( d ) The gray value analysis of protein in ( c ). ( e ) Cell proliferation of 786-O cells was inhibited in ORM1-KD group compared to NC group. ( f ) Cell proliferation of Caki-2 cells was inhibited in ORM1-KD group compared to NC group. # p < 0.05 showed statistically difference.

Article Snippet: KIRC cell lines including 786-O, A498, and Caki-2 cells were obtained from ICell Bioscience Inc, Shanghai (Shanghai, China).

Techniques: Expressing

Journal: Scientific Reports

Article Title: ORM1 promotes tumor progression of kidney renal clear cell carcinoma (KIRC) through CALR-mediated apoptosis

doi: 10.1038/s41598-023-42962-w

Figure Lengend Snippet: ORM1 was essential to cell migration and invasion. ( a ) Cell migration and invasion was suppressed in ORM1-KD group compared to NC group in 786-O and Caki-2 cells in transwell assay with/without Matrigel. ( b ) The statistical analysis of cells in cell migration in ( a ). ( c ) The statistical analysis of cells in cell invasion in a. # p < 0.05 showed statistically difference.

Article Snippet: KIRC cell lines including 786-O, A498, and Caki-2 cells were obtained from ICell Bioscience Inc, Shanghai (Shanghai, China).

Techniques: Migration, Transwell Assay

Journal: Scientific Reports

Article Title: ORM1 promotes tumor progression of kidney renal clear cell carcinoma (KIRC) through CALR-mediated apoptosis

doi: 10.1038/s41598-023-42962-w

Figure Lengend Snippet: ORM1 enhanced the efficiency of sorafenib in KIRC. ( a ) Sorafenib inhibited cell proliferation in concentration-dependent manner. ( b ) The efficiency of sorafenib was enhanced in ORM1-KD group but relieved in ORM1-OE group in 786-O cells. ( c ) The efficiency of sorafenib was enhanced in ORM1-KD group but relieved in ORM1-OE group in Caki-2 cells. # p < 0.05 showed statistically difference.

Article Snippet: KIRC cell lines including 786-O, A498, and Caki-2 cells were obtained from ICell Bioscience Inc, Shanghai (Shanghai, China).

Techniques: Concentration Assay