Journal: Cell metabolism

Article Title: Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity

doi: 10.1016/j.cmet.2018.04.009

Figure Lengend Snippet: (A) Schematic representation of polyamine biosynthesis. Each enzyme contains a corresponding box plot with TCGA mRNA data in normal (N) and ccRCC (T) samples. Each y-axis represents the normalized RNA-seq reads of the gene; n = 69 normal kidney and 480 tumor samples. *** p< 0.001 (B) 786-O cells grown under hypoxia (0.5% O2) and supplemented with various concentrations of putrescine (Put), andHK-2 cells grown in 1 mM glucose, 1% FBS conditions, supplemented with Put. Cell growth was assessed by WST-1 assays, and error bars represent SEM from 10 replicate wells. Prostate cancer (VCaP) cells were supplemented with indicated concentrations of putrescine, and cell counts performed over the indicated times. Error bars represent SEM from 4 replicate wells. (C) Polyamine metabolite abundance in primary ccRCC combining two independent datasets, n = 158 (Hakimi et al., 2016; Li et al., 2014). Data are displayed as the fold change in metabolite abundance of each tumor, normalized to its control sample. (D) Quantification of immunohistochemistry staining of primary ccRCC tissue microarray,demonstrating reduced ODC protein in ccRCC tumors. *** p <0.001, Welch’s t-test. See image in Figure S6B. (E) Kaplan-Meier survival analysis of OAZ1 expression (from TCGA data) in ccRCC. ‘Low’ versus ‘High’ data are segregated based on the median expression of OAZ1. Mantel-Cox log-rank test was performed. Abbreviations: OAZ, ornithine decarboxylase antizyme; AZIN1, ornithine decarboxylase antizyme inhibitor; ODC1, ornithine decarboxylase1; AMD1, adenosylmethionine decarboxylase1; SRM, spermidine synthase, SMS, spermine synthase. See also Figures S6 and S7.

Article Snippet: Normal and ccRCC kidney tissue sections (from Cooperative Human Tissue Network) and tissue microarray slides (KD802, KD804, KD481, KD482, KD244, KD901, KD601, KD701 from US Biomax) were deparaffinized by baking slides at 50°C for 20 min.

Techniques: RNA Sequencing Assay, Immunohistochemistry, Staining, Microarray, Expressing

Journal: Oncology Letters

Article Title: Identification of ATP6V0A4 as a potential biomarker in renal cell carcinoma using integrated bioinformatics analysis

doi: 10.3892/ol.2023.13952

Figure Lengend Snippet: Construction of the PPI network and identification of hub genes. (A) Intersectional plot of the top 50 genes evaluated using 12 methods in the 8 datasets. (B) PPI network of the hub genes. (C-I) Receiver operating characteristic curves of the hub genes, (C) SERPINE1, (D) CXCR4, (E) VEGFA, (F) VCAN, (G) ATP6V0A4, (H) SUCNR1 and (I) CASR. AUC, area under the curve; MNC, Maximum Neighborhood Component; DMNC, Density of Maximum Neighborhood Component; MCC, Maximal Clique Centrality; EPC, Edge Percolated Component.

Article Snippet: Single spot tissue microarray (TMA) slides (30 points of ccRCC, 30 points of adjacent normal kidney tissues; cat. no. HKid-CRC060CS-01, Shanghai Outdo Biotech Company) were prepared and incubated with the ATP6V0A4 rabbit antibody (1:500; cat. no. 21570-1-AP; ProteinTech Group, Inc.) at 4°C overnight.

Techniques:

Journal: Oncology Letters

Article Title: Identification of ATP6V0A4 as a potential biomarker in renal cell carcinoma using integrated bioinformatics analysis

doi: 10.3892/ol.2023.13952

Figure Lengend Snippet: Validation of the expression of the differentially expressed hub genes using reverse transcription-quantitative PCR. (A and B) The expression of ATP6V0A4 was significantly downregulated in ccRCC tissues. (C and D) The expression of SUCNR1 was significantly downregulated in ccRCC tissues. (E and F) The expression of SERPINE1 did not differ between ccRCC tissues and paracancerous tissues. ccRCC, clear cell renal cell carcinoma; ***P<0.001 vs. normal; ns, not significant.

Article Snippet: Single spot tissue microarray (TMA) slides (30 points of ccRCC, 30 points of adjacent normal kidney tissues; cat. no. HKid-CRC060CS-01, Shanghai Outdo Biotech Company) were prepared and incubated with the ATP6V0A4 rabbit antibody (1:500; cat. no. 21570-1-AP; ProteinTech Group, Inc.) at 4°C overnight.

Techniques: Biomarker Discovery, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: Identification of ATP6V0A4 as a potential biomarker in renal cell carcinoma using integrated bioinformatics analysis

doi: 10.3892/ol.2023.13952

Figure Lengend Snippet: ATP6V0A4 is downregulated in ccRCC. (A) The expression of ATP6V0A4 in ccRCC tissues was significantly lower than that in adjacent renal tissues in eight Gene Expression Omnibus datasets (GSE76351, GES6344, GSE15641, GSE16449, GSE47032, GSE66270, GSE53000 and GSE53757). ***P<0.001 vs. normal. (B) The results of immunohistochemistry staining revealed that ATP6V0A4 protein expression was lower in ccRCC tissues than that in the adjacent renal tissues. (C) ATP6V0A4 was significantly downregulated in 769-P, ACHN and CAKI-2 cell lines compared with that in HK-2 cells at the transcription level. (D) Combined western blotting assay and densitometry indicated that ATP6V0A4 was remarkably decreased in all 5 RCC cell lines compared with that in HK-2 cells at the translational level. ccRCC, clear cell renal cell carcinoma; ***P<0.001 vs. HK-2; ns, not significant.

Article Snippet: Single spot tissue microarray (TMA) slides (30 points of ccRCC, 30 points of adjacent normal kidney tissues; cat. no. HKid-CRC060CS-01, Shanghai Outdo Biotech Company) were prepared and incubated with the ATP6V0A4 rabbit antibody (1:500; cat. no. 21570-1-AP; ProteinTech Group, Inc.) at 4°C overnight.

Techniques: Expressing, Gene Expression, Immunohistochemistry, Staining, Western Blot

Journal: Oncology Letters

Article Title: Identification of ATP6V0A4 as a potential biomarker in renal cell carcinoma using integrated bioinformatics analysis

doi: 10.3892/ol.2023.13952

Figure Lengend Snippet: Expression of ATP6V0A4 in the cancerous and paracancerous tissues.

Article Snippet: Single spot tissue microarray (TMA) slides (30 points of ccRCC, 30 points of adjacent normal kidney tissues; cat. no. HKid-CRC060CS-01, Shanghai Outdo Biotech Company) were prepared and incubated with the ATP6V0A4 rabbit antibody (1:500; cat. no. 21570-1-AP; ProteinTech Group, Inc.) at 4°C overnight.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Identification of ATP6V0A4 as a potential biomarker in renal cell carcinoma using integrated bioinformatics analysis

doi: 10.3892/ol.2023.13952

Figure Lengend Snippet: Association between ATP6V0A4 expression and clinicopathological characteristics of patients with clear cell renal cell carcinoma based on data obtained from The Cancer Genome Atlas.8800240497.

Article Snippet: Single spot tissue microarray (TMA) slides (30 points of ccRCC, 30 points of adjacent normal kidney tissues; cat. no. HKid-CRC060CS-01, Shanghai Outdo Biotech Company) were prepared and incubated with the ATP6V0A4 rabbit antibody (1:500; cat. no. 21570-1-AP; ProteinTech Group, Inc.) at 4°C overnight.

Techniques: Expressing

Journal: Oncogene

Article Title: ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma.

doi: 10.1038/s41388-022-02460-1

Figure Lengend Snippet: Fig. 5 ISCA2 inhibition induces ferroptosis. A Effect of co-treatment of ISCA2 siRNA and DMSO or liproxstatin (2.5 μM) on cell viability determined using resazurin in ACHN cells. siRNA transfection was performed for a total of 5 days with liproxstatin (or DMSO) added for the last 24 h. B, C Cell viability assays (resazurin) of 786-0 cells treated with (B) #1 or (C) #25, ± DFO (100 μM), liproxstatin (1 μM) or ZVAD-FMK (20 μM); or (H) #25 ± DFO (100 μM), NAC (1 mM), liproxstatin (1 μM) or ZVAD-FMK (20 μM). Treatments were performed for 24 h. Average IC50 values (µM) are shown in brackets. D Impact of #25 treatment (48 h) on BODIPY 581/591 fluorescence detected by flow cytometry in 786-0 cells. E Impact of 48 hours’ treatment with #25, PT2385 (PT) or 6 hours’ treatment with RSL3 on malondialdehyde (MDA), an indicator of lipid peroxidation in RCC10 cells. F Transmission electron microscopy of RCC10 cells treated with DMSO or #25 for 24 h (2 representative micrographs of each condition). DMSO-treated cells show normal mitochondria whereas #25-treated cells show damaged mitochondria including lost or irregular cristae (white arrows) and irregular matrix with voids (yellow arrows).

Article Snippet: TMAs and tissue were stained for ISCA2 (HPA030492 Atlas antibodies, Bromma, Sweden) using conditions optimized using normal kidney according to the manufacturer’s protocols using the BenchMark Ultra automated slide stainer (Ventana Medical Systems, Roche, Oro Valley, AZ).

Techniques: Inhibition, Transfection, Cytometry, Transmission Assay, Electron Microscopy

Journal: Oncogene

Article Title: ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma.

doi: 10.1038/s41388-022-02460-1

Figure Lengend Snippet: Fig. 7 ISCA2 is decreased in ccRCC and low ISCA2 is associated with poor patient prognosis. A Immunohistochemistry showing representative expression of ISCA2 in normal kidney (top) and in ccRCC (bottom). B Quantitation of ISCA2 H-Score in paired uninvolved and ccRCC cores from 19 patients. ****p < 0.0001 determined via Mann Whitney test. C, D Kaplan–Meier curves showing associations of (C) ISCA2 protein, or (D) ISCA2 transcript levels above (high) or below (low) the median with overall survival using a tumor microarray (TMA; 94 cases) or data from TCGA-KIRC (522 cases) respectively.

Article Snippet: TMAs and tissue were stained for ISCA2 (HPA030492 Atlas antibodies, Bromma, Sweden) using conditions optimized using normal kidney according to the manufacturer’s protocols using the BenchMark Ultra automated slide stainer (Ventana Medical Systems, Roche, Oro Valley, AZ).

Techniques: Immunohistochemistry, Expressing, Quantitation Assay, MANN-WHITNEY, Microarray

Journal: Oncogene

Article Title: ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma.

doi: 10.1038/s41388-022-02460-1

Figure Lengend Snippet: Fig. 8 ISCA2 inhibition inhibits xenograft growth in vivo. A, B Effect of treatment of 786-0 subcutaneous xenografts with vehicle or indicated doses of compound #25 (8 mice/group) administered orally once daily, on tumor volume (A), or mouse body weights (B). **p < 0.01; ***p < 0.001; ****p < 0.0001 from Students’ T-tests of vehicle versus 60 mg/kg treated group. p was not significant (NS) for 30 mg/kg treated group versus vehicle. C Effect of treatment of RENCA subcutaneous xenografts in syngeneic Balb/c mice with vehicle or 60 mg/kg #25 (8 mice/ group) once daily. D Western blots showing HIF-1α and GAPDH with densitometric values normalized to GAPDH below. E MDA content of RENCA tumors from mice treated with vehicle or 60 mg/kg #25. F Proposed model for effects of ISCA2 blockade. Inhibition of ISCA2 using either small molecules or siRNA results in perceived iron deprivation, which induces upregulation of IRP2, TFRC and downregulation of FTH1, that together promote iron/metals accumulation that triggers cell death via ferroptosis, and also drives the later downregulation of IRP1, IRP2 and TFRC. This perceived iron deprivation also drives a shift to the IRE-binding form of IRP1 which inhibits HIF-2α translation. ISCA2 inhibition may also cause other mitochondrial/extramitochondrial defects dependent or independent of perceived iron deprivation that inhibits the translation of HIF-1α through unknown mechanisms. Thus, ISCA2 inhibition depletes both HIF-1α and HIF-2α and promotes cell death via ferroptosis.

Article Snippet: TMAs and tissue were stained for ISCA2 (HPA030492 Atlas antibodies, Bromma, Sweden) using conditions optimized using normal kidney according to the manufacturer’s protocols using the BenchMark Ultra automated slide stainer (Ventana Medical Systems, Roche, Oro Valley, AZ).

Techniques: Inhibition, In Vivo, Western Blot, Binding Assay