Journal: Journal of Advanced Research

Article Title: Epigenetically silenced KAT2B suppresses de novo lipogenesis through destroying HDAC5/LSD1 complex assembly in renal cell carcinoma

doi: 10.1016/j.jare.2025.08.007

Figure Lengend Snippet: Therapeutic targeting of KAT2B-low RCC with a FASN inhibitor (A-B) Representative images of IHC staining of FASN in RCC cohort and statistical analysis. (C) Representative images of IHC staining for FASN and KAT2B in RCC tissues with high and low KAT2B expression. (D) Scatter plot of the relationship among KAT2B expression and FASN expression in advanced RCC tumors (n = 53). (E) The cell viability of ACHN and Caki-1 cells after treated with TVB-2640 (n = 4). (F) The cell viability of 786O and 769P cells after treated with TVB-2640 (n = 10). Proteins from three independent sites in RCC tissues were extracted to detect KAT2B expression. (H) Representative images of Caki-1 and ACHN organoids after treatment with TVB-2640 (7.5 μM). 15 organoids were randomly selected from each group for statistical analysis. (I) Representative images of Caki-1 and ACHN organoids after treatment with TVB-2640 (7.5 μM) (n = 15). (J) Representative images of two PDOs with different KAT2B expression after treatment with TVB-2640 (n = 10). (K) Representative images of PRO-1 staining of PDOs. (L-M) The cell viability of ACHN cells (L) and case 1 primary RCC cells (M) with KAT2B knockdown after treated with TVB-2640 (n = 4). (N-O) The picture (N) of xenograft using 786O cells with KAT2B knockdown after treated with TVB-2640, and tumor growth curve (n = 4). Data were analyzed by unpaired t test (B, H, I, J), one-way ANOVA (O) or two-way ANOVA (E, F, G, L, M).

Article Snippet: The HK‐2, 293 T, A549, PC9, T47D, MCF7, A498, Caki-1, OSRC-2, 786O, 769P and ACHN cell lines were obtained from the American Type Culture Collection (ATCC, USA) and were cultivated under proper conditions according to the manufacturer’s protocols.

Techniques: Immunohistochemistry, Expressing, Staining, Knockdown

Journal: Frontiers in Genetics

Article Title: Identification of IGF2BP3 and CENPA as key regulators of immunophenotypes in renal clear cell carcinoma

doi: 10.3389/fgene.2025.1749780

Figure Lengend Snippet: Construction of the risk prognostic classifier. (A) LASSO coefficient profiles of the risk genes. (B) The risk scores and heatmap of expression profiles in high-risk and low-risk groups. (C) AUC of time-dependent ROC curves of the 3-risk genes prognostic signature. (D) OS of KIRC patients in high-risk and low-risk groups. (E) Univariate and (F) multivariate independent prognostic analysis in patients with KIRC.

Article Snippet: The KIRC 769P cells (Cat# CL-0009, RRID: CVCL_1050) purchased from Procell (China) were cultured in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin, and 1% streptomycin, and cells were maintained in a 37 °C incubator with 5% CO 2 . siRNAs targeting CENPA, IGF2BP3 or a negative control (NC) were designed and synthesized by GenePharma (China).

Techniques: Expressing

Journal: Frontiers in Genetics

Article Title: Identification of IGF2BP3 and CENPA as key regulators of immunophenotypes in renal clear cell carcinoma

doi: 10.3389/fgene.2025.1749780

Figure Lengend Snippet: DEGs and Immune characteristics between the high-risk and the low-risk groups. (A) Volcano plot of the DEGs. (B) Significantly enriched KEGG pathways of the DEGs. (C) Significantly enriched Gene Ontology terms of the DEGs. (D) Immune characteristics in terms of risk core. (E) OS analysis of KIRC patients with different ImmuneScore.

Article Snippet: The KIRC 769P cells (Cat# CL-0009, RRID: CVCL_1050) purchased from Procell (China) were cultured in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin, and 1% streptomycin, and cells were maintained in a 37 °C incubator with 5% CO 2 . siRNAs targeting CENPA, IGF2BP3 or a negative control (NC) were designed and synthesized by GenePharma (China).

Techniques:

Journal: Frontiers in Genetics

Article Title: Identification of IGF2BP3 and CENPA as key regulators of immunophenotypes in renal clear cell carcinoma

doi: 10.3389/fgene.2025.1749780

Figure Lengend Snippet: Expression levels and Methylation status of the two mRNAs. (A) Expression of IGF2BP3 in KIRC based on individual cancer stages. (B) Expression of CENPA in KIRC based on individual cancer stages. (C) Expression of IGF2BP3 based on nodal metastasis status. (D) Expression of CENPA based on nodal metastasis status. (E) Methylation difference of genes in KIRC. (F) Spearman correlation between IGF2BP3 methylation and mRNA expression in KIRC.

Article Snippet: The KIRC 769P cells (Cat# CL-0009, RRID: CVCL_1050) purchased from Procell (China) were cultured in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin, and 1% streptomycin, and cells were maintained in a 37 °C incubator with 5% CO 2 . siRNAs targeting CENPA, IGF2BP3 or a negative control (NC) were designed and synthesized by GenePharma (China).

Techniques: Expressing, Methylation

Journal: Frontiers in Genetics

Article Title: Identification of IGF2BP3 and CENPA as key regulators of immunophenotypes in renal clear cell carcinoma

doi: 10.3389/fgene.2025.1749780

Figure Lengend Snippet: Inhibition of CENPA expression leads to growth inhibition and death of KIRC cells. (A) The expression of IGF2BP3 and CENPA were detected by Western blot. (B) Calcein-AM/PI assay was used to explore the effect of CENPA expression on cell viability. (C) Representative images and quantification of colony formation assay, * p < 0.05. (D) Mitochondrial membrane potential was analyzed by JC1. (E) Reactive oxygen species assay.

Article Snippet: The KIRC 769P cells (Cat# CL-0009, RRID: CVCL_1050) purchased from Procell (China) were cultured in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin, and 1% streptomycin, and cells were maintained in a 37 °C incubator with 5% CO 2 . siRNAs targeting CENPA, IGF2BP3 or a negative control (NC) were designed and synthesized by GenePharma (China).

Techniques: Inhibition, Expressing, Western Blot, Colony Assay, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Mechanism of NUMBL enhancing axitinib resistance in clear cell renal cell carcinoma by UCHL1-mediated deubiquitination of MMP9

doi: 10.1016/j.jbc.2025.110873

Figure Lengend Snippet: NUMBL promotes migration, invasion, and proliferation of ccRCC cells. A , the expression level of NUMBL in all RCCs in the CCLE database. B and C , detecting the differential expression of NUMBL between ccRCC cells and renal tubular epithelial cells (HK2) using WB. D and E , knockdown of NUMBL in OSRC2 and 769P. F , overexpression of NUMBL in ACHN. G – I , the migration abilities of OSRC2, 769P, and ACHN were evaluated using a wound healing assay following NUMBL expression interference. J – L , the migration and invasion abilities of OSRC2, 769P, and ACHN cells were assessed using Transwell assays with interferencing NUMBL expression. M – O , proliferation was assessed using colony formation assays. Data were shown as mean ± SD of at least three independent experiments for every cell line. Statistical differences were determined using Tukey's post hoc one-way ANOVA test. NS, nonstatistically significant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. ccRCC, clear cell renal cell carcinoma; CCLE, Cancer Cell Line Encyclopedia; NUMBL, NUMB-like endocytic adaptor protein; WB, Western blot.

Article Snippet: The human renal cancer cell lines 769P, OSRC2, and ACHN (obtained from the American Type Culture Collection) were cultured using standard methods.

Techniques: Migration, Expressing, Quantitative Proteomics, Knockdown, Over Expression, Wound Healing Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Mechanism of NUMBL enhancing axitinib resistance in clear cell renal cell carcinoma by UCHL1-mediated deubiquitination of MMP9

doi: 10.1016/j.jbc.2025.110873

Figure Lengend Snippet: NUMBL promotes VM in ccRCC cells. A , VM (PAS+/CD31-) exists in ccRCC tissues. B , tube formation assay detects VM in OSRC2, 769P, and ACHN. C – H , based on , D – F , the VM capacity of ccRCC cells was assessed using tube formation assays. I – K , based on , D – F , the drug sensitivity of ccRCC cells to axitinib was evaluated using CCK-8 assays. CCK-8, Cell Counting Kit-8; ccRCC, clear cell renal cell carcinoma; NUMBL, NUMB-like endocytic adaptor protein; PAS, Periodic Acid-Schiff; VM, vasculogenic mimicry.

Article Snippet: The human renal cancer cell lines 769P, OSRC2, and ACHN (obtained from the American Type Culture Collection) were cultured using standard methods.

Techniques: Tube Formation Assay, CCK-8 Assay, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: Mechanism of NUMBL enhancing axitinib resistance in clear cell renal cell carcinoma by UCHL1-mediated deubiquitination of MMP9

doi: 10.1016/j.jbc.2025.110873

Figure Lengend Snippet: Upregulated NUMBL enhances the mRNA stability of UCHL1. A , transcriptome sequencing following NUMBL knockdown in 769P cells. B , proteome sequencing after NUMBL knockdown in 769P cells. C and D , validate the mRNA changes of UCHL1 after interfering with NUMBL expression in 769P and ACHN cells. E – F , confirm via WB that UCHL1 protein levels change with NUMBL expression after NUMBL interference in 769P and ACHN cells. G , the correlation between NUMBL and UCHL1 in the TCGA database. H , the correlation between NUMBL and UCHL1 in the CPTAC database. I , based on the treatment in D , after treating 769P cells with stable NUMBL knockdown with actinomycin D , UCHL1 mRNA levels were measured at specific time points. Data were shown as mean ± SD of at least three independent experiments for every cell line. Statistical differences were determined using Tukey's post hoc one-way ANOVA test. NS, nonstatistically significant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. CPTAC, Clinical Proteomic Tumor Analysis Consortium; NUMBL, NUMB-like endocytic adaptor protein; TCGA, The Cancer Genome Atlas; UCHL1, ubiquitin C-terminal hydrolase L1; WB, Western blot.

Article Snippet: The human renal cancer cell lines 769P, OSRC2, and ACHN (obtained from the American Type Culture Collection) were cultured using standard methods.

Techniques: Sequencing, Knockdown, Expressing, Ubiquitin Proteomics, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Mechanism of NUMBL enhancing axitinib resistance in clear cell renal cell carcinoma by UCHL1-mediated deubiquitination of MMP9

doi: 10.1016/j.jbc.2025.110873

Figure Lengend Snippet: UCHL1 stabilizes the expression of MMP9 protein. A , heatmap of the correlation between NUMBL and the VM gene set. B , identify the intersection between the ECM gene set and the VM gene set. C – F , correlation analysis of the four genes in the intersection with UCHL1 in the CPTAC database. G – H , knocking down NUMBL in 769P cells results in decreased expression of MMP9. I – J , treating 769P cells with 20 μM of MG-132 for 12 h results in increased expression of MMP9. K , the co-IP experiment detects an interaction between MMP9 and UCHL1. L , MMP9 undergoes ubiquitination modification. M and P , WB is used to detect the efficiency of knocking down UCHL1. N , knocking down UCHL1 in 769P cells results in increased ubiquitination levels of MMP9. O , immunofluorescence staining reveals colocalization of UCHL1 and MMP9 in the cytoplasm of 769P cells. Data were shown as mean ± SD of at least three independent experiments for every cell line. Statistical differences were determined using Tukey's post hoc one-way ANOVA test. NS, nonstatistically significant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. co-IP, coimmunoprecipitation; CPTAC, Clinical Proteomic Tumor Analysis Consortium; ECM, extracellular matrix; MMP9, matrix metalloproteinase 9; UCHL1, ubiquitin C-terminal hydrolase L1; VM, vasculogenic mimicry; WB, Western blot.

Article Snippet: The human renal cancer cell lines 769P, OSRC2, and ACHN (obtained from the American Type Culture Collection) were cultured using standard methods.

Techniques: Expressing, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Modification, Immunofluorescence, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Mechanism of NUMBL enhancing axitinib resistance in clear cell renal cell carcinoma by UCHL1-mediated deubiquitination of MMP9

doi: 10.1016/j.jbc.2025.110873

Figure Lengend Snippet: The NUMBL–UCHL1–MMP9 pathway is involved in the development of ccRCC in vivo . A , photographs of tumors at the final time point (15 days after subcutaneous transplantation of 769P cells). B and C , tumor volumes and weights were measured following resection in each group. D and E , immunohistochemical staining was used to detect MMP9 and Ki-67 expression in tumor tissues. F , immunofluorescence staining is used to examine the expression of Ki-67 in tumor tissues. The scale bar represents 100 μm. Original magnification, 200×. Data were shown as mean ± SD of at least three independent experiments for every cell line. Statistical differences were determined using Tukey's post hoc one-way ANOVA test. NS, nonstatistically significant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. ccRCC, clear cell renal cell carcinoma; MMP9, matrix metalloproteinase 9; NUMBL, NUMB-like endocytic adaptor protein; UCHL1, ubiquitin C-terminal hydrolase L1.

Article Snippet: The human renal cancer cell lines 769P, OSRC2, and ACHN (obtained from the American Type Culture Collection) were cultured using standard methods.

Techniques: In Vivo, Transplantation Assay, Immunohistochemical staining, Staining, Expressing, Immunofluorescence, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Mechanism of NUMBL enhancing axitinib resistance in clear cell renal cell carcinoma by UCHL1-mediated deubiquitination of MMP9

doi: 10.1016/j.jbc.2025.110873

Figure Lengend Snippet: Inhibiting the expression of NUMBL enhances the sensitivity of 769P R to axitinib. A , using the CCK-8 assay to verify the standardization of drug-resistant strain construction (DRI = 9.75). B and C , WB analysis was conducted to detect changes in NUMBL expression levels between 769P R and 769P. D and E , WB analysis was performed to detect the knockdown of NUMBL in 769P R cells. F and G , proliferation was assessed using colony formation assays. H , tube formation assay was conducted to detect the tubulogenesis ability of 769P R cells after NUMBL knockdown. I – L , based on D , a wound healing assay was performed to detect the migration ability of 769P R cells after NUMBL knockdown, whereas a Transwell assay was conducted to assess both migration and invasion capabilities. M , a CCK-8 assay was conducted to detect the sensitivity of 769P R cells to axitinib after NUMBL knockdown. Data were shown as mean ± SD of at least three independent experiments for every cell line. Statistical differences were determined using Tukey's post hoc one-way ANOVA test. NS, nonstatistically significant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. CCK-8, Cell Counting Kit-8; DRI, drug resistance index; NUMBL, NUMB-like endocytic adaptor protein; WB, Western blot.

Article Snippet: The human renal cancer cell lines 769P, OSRC2, and ACHN (obtained from the American Type Culture Collection) were cultured using standard methods.

Techniques: Expressing, CCK-8 Assay, Knockdown, Tube Formation Assay, Wound Healing Assay, Migration, Transwell Assay, Cell Counting, Western Blot