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

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: Experimental & Molecular Medicine

Article Title: Gut microbiota modulation of epigenetic target EHMT2: Lacticaseibacillus rhamnosus Fb7-311 regulated renal cell carcinoma apoptosis and metastasis

doi: 10.1038/s12276-026-01659-6

Figure Lengend Snippet: a EHMT2 expression in normal and RCC samples derived from TCGA database. P values were calculated using Student’s t -test (** P < 0.01). b Kaplan‒Meier plot showing that the overall survival rates of patients with low EHMT2 expression were substantially higher than those of patients with high EHMT2 expression in RCC tissues. P values were calculated using Student’s t -test (*** P < 0.001). c Immunohistochemical staining for EHMT2. Kidney cancer tissues were purchased from TissueArray ( https://www.tissuearray.com ). Scale bar, 200 μm. d DAVID-based GO analysis of the RNA-seq results from the siEHMT2 (#1) and siCont groups, which included 1207 DEGs. e , f Cell growth assay after transfection with siEHMT2 and siCont for 48 h. e A498 and Caki-1 cells were fixed with 100% methanol and stained with the CV solution. Scale bar, 500 μm. f CCK-8 solution was added to the culture medium and the cells were incubated for 5 min at 37 °C. Cell growth was measured using a microplate reader (450 nm). The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001). g Western blot analysis of cells transfected with siEHMT2 transfection using anti-EHMT2, anti-PARP and anti-ACTB antibodies. ACTB was used as the internal control in A498 and Caki-1 cells. h FACS analysis of Annexin V staining was performed after the cells were transfected with siEHMT2 or siCont. The lower right and upper right quadrants indicate early apoptotic cells and late apoptotic cells, respectively (top). Quantification of apoptosis: the data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001) (bottom). i FACS analysis using the Muse Caspase-3/7 working solution was performed after the cells were transfected with siEHMT2 or siCont. The upper right image shows the proportions of apoptotic and dead cells (top). Quantification of caspase-3/7 activity: the data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001) (bottom).

Article Snippet: The human RCC cell lines A498 and Caki-1 were purchased from the Korean Cell Line Bank and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO 2 at 37 °C.

Techniques: Expressing, Derivative Assay, Immunohistochemical staining, Staining, RNA Sequencing, Growth Assay, Transfection, CCK-8 Assay, Incubation, Western Blot, Control, Activity Assay

Journal: Experimental & Molecular Medicine

Article Title: Gut microbiota modulation of epigenetic target EHMT2: Lacticaseibacillus rhamnosus Fb7-311 regulated renal cell carcinoma apoptosis and metastasis

doi: 10.1038/s12276-026-01659-6

Figure Lengend Snippet: a Migration and invasion assays were performed using the A498 and Caki-1 cell lines after EHMT2 knockdown. Cell migration and invasion assays were performed after 24 h (A498) and 48 h (Caki-1). The migrating/invading cells were stained with CV. Scale bar, 500 μm (left). Quantification of migrating/invading cells: the data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001) (right). b Migration assay of A498 and Caki-1 cells after treatment with TGF-β. The cell migration assay was performed after 24 h (A498) and 48 h (Caki-1). The migrating cells were stained with CV. Scale bar, 500 μm (left). Quantification of migrating cells: the data are presented as the means ± SDs of three independent experiments. P values were calculated using Student’s t -tests (** P < 0.01, *** P < 0.001) (right). c RT‒qPCR analysis of E-cadherin and N-cadherin expression in cells transfected with siEHMT2. The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001). d Migration and invasion assays were performed using the A498 and Caki-1 cell lines after treatment with TGF-β and EHMT2 knockdown. Cell migration and invasion assays were performed after 24 h (A498) and 48 h (Caki-1). The migrating/invading cells were stained with CV. Scale bar, 500 μm (left). Quantification of migrating/invading cells: the data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t- tests (** P < 0.01, *** P < 0.001) (right). e RT‒qPCR analysis of E-cadherin and N-cadherin expression in cells transfected with siEHMT2. The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -tests (** P < 0.01, *** P < 0.001).

Article Snippet: The human RCC cell lines A498 and Caki-1 were purchased from the Korean Cell Line Bank and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO 2 at 37 °C.

Techniques: Migration, Knockdown, Staining, Cell Migration Assay, Expressing, Transfection

Journal: Experimental & Molecular Medicine

Article Title: Gut microbiota modulation of epigenetic target EHMT2: Lacticaseibacillus rhamnosus Fb7-311 regulated renal cell carcinoma apoptosis and metastasis

doi: 10.1038/s12276-026-01659-6

Figure Lengend Snippet: a , b Cell growth assay after treatment with BIX for 48 h: A498 and Caki-1 cells were fixed with 100% methanol and stained with a CV solution, scale bar, 500 μm ( a ); CCK-8 solution was added to the culture medium and the cells were incubated for 5 min at 37 °C. Cell growth was measured using a microplate reader (450 nm) ( b ). The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001). c Western blot analysis of cells treated with BIX using anti-EHMT2, anti-PARP and anti-ACTB antibodies. ACTB was used as the internal control in A498 and Caki-1 cells. d FACS analysis of Annexin V staining was performed after BIX treatment. The lower right and upper right quadrants indicate early apoptotic cells and late apoptotic cells, respectively (left). Quantification of apoptosis: the data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001) (right). e FACS analysis using the Muse Caspase-3/7 working solution was performed after BIX treatment. The upper right image shows the proportions of apoptotic and dead cells (left). Quantification of caspase-3/7 activity: the data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001) (right). f Migration and invasion assays were performed in A498 and Caki-1 cells after BIX treatment. Cell migration and invasion assays were performed after 24 h (A498) and 48 h (Caki-1). The migrating/invading cells were stained with CV. Scale bar, 500 μm (left). Quantification of migrating/invading cells: the data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -tests (** P < 0.01, *** P < 0.001) (right). g Migration and invasion assays were performed after the A498 and Caki-1 cell lines were treated with TGF-β and BIX. Cell migration and invasion assays were performed after 24 h (A498) and 48 h (Caki-1). The migrating/invading cells were stained with CV. Scale bar, 500 μm (left). Quantification of migrating/invading cells. The data are presented as the means ± s.d. of three independent experiments: P values were calculated using Student’s t -tests (* P < 0.05, *** P < 0.001) (right). h RT‒qPCR analysis of E-cadherin and N-cadherin expression in cells after BIX treatment. The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -tests (* P < 0.05, *** P < 0.001).

Article Snippet: The human RCC cell lines A498 and Caki-1 were purchased from the Korean Cell Line Bank and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO 2 at 37 °C.

Techniques: Growth Assay, Staining, CCK-8 Assay, Incubation, Western Blot, Control, Activity Assay, Migration, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Gut microbiota modulation of epigenetic target EHMT2: Lacticaseibacillus rhamnosus Fb7-311 regulated renal cell carcinoma apoptosis and metastasis

doi: 10.1038/s12276-026-01659-6

Figure Lengend Snippet: a A heat map of RNA-seq data from siEHMT2- and siCont-transfected cells. b RNA-seq results for DDIT3 expression after EHMT2 knockdown. c RT‒qPCR analysis of DDIT3 expression in cells transfected with siEHMT2. The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -tests (** P < 0.01, *** P < 0.001). d Correlation analysis of the expression of the EHMT2 and DDIT3 genes derived from TCGA portal and using analysis of variance (ANOVA). e Immunohistochemical staining for EHMT2 and DDIT3. Kidney cancer tissues were purchased from TissueArray ( https://www.tissuearray.com ). Scale bar, 200 μm. f Immunocytochemical staining for DDIT3. A498 and Caki-1 cells transfected with siEHMT2 and siCont were fixed with 100% methanol and stained with an anti-DDIT3 antibody (Alexa Fluor 488, green) and DAPI (blue). Scale bar, 150 μm. g Graphical abstract of the ChIP primer design for the DDIT3 promoter region. h The ChIP assay was performed with an anti-H3K9me2 antibody. The result is shown as relative enrichment compared to the control in A498 and Caki-1 cells after siEHMT2 transfection. The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -tests (* P < 0.05, ** P < 0.01).

Article Snippet: The human RCC cell lines A498 and Caki-1 were purchased from the Korean Cell Line Bank and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO 2 at 37 °C.

Techniques: RNA Sequencing, Transfection, Expressing, Knockdown, Derivative Assay, Immunohistochemical staining, Staining, Control

Journal: Experimental & Molecular Medicine

Article Title: Gut microbiota modulation of epigenetic target EHMT2: Lacticaseibacillus rhamnosus Fb7-311 regulated renal cell carcinoma apoptosis and metastasis

doi: 10.1038/s12276-026-01659-6

Figure Lengend Snippet: a Immunocytochemical staining for DDIT3. A498 and Caki-1 cells were treated with BIX, fixed with 100% methanol and stained with an anti-DDIT3 antibody (Alexa Fluor 488, green) and DAPI (blue). Scale bar, 300 μm. b The ChIP assay was performed with an anti-H3K9me2 antibody. The results are shown as relative enrichment compared to the control in A498 and Caki-1 cells after BIX treatment. The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001). c Cell growth assay after cotransfection with siDDIT3 and siEHMT2 for 48 h. A498 and Caki-1 cells were fixed with 100% methanol and stained with a CV solution. Scale bar, 500 μm (top). CCK-8 solution was added to the culture medium and the cells were incubated for 5 min at 37 °C. Cell growth was measured using a microplate reader (450 nm). The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001) (bottom). d FACS analysis of Annexin V staining was performed after cells were cotransfected with siDDIT3 and siEHMT2. Quantification of apoptosis: the data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001). e FACS analysis using the Muse Caspase-3/7 working solution was performed after cells were cotransfected with siDDIT3 and siEHMT2. Quantification of caspase-3/7 activity: the data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001). f Western blot analysis of cells cotransfected with siDDIT3 and siEHMT2 using anti-EHMT2, anti-PARP and anti-ACTB antibodies. ACTB was used as the internal control in A498 and Caki-1 cells.

Article Snippet: The human RCC cell lines A498 and Caki-1 were purchased from the Korean Cell Line Bank and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO 2 at 37 °C.

Techniques: Staining, Control, Growth Assay, Cotransfection, CCK-8 Assay, Incubation, Activity Assay, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: Gut microbiota modulation of epigenetic target EHMT2: Lacticaseibacillus rhamnosus Fb7-311 regulated renal cell carcinoma apoptosis and metastasis

doi: 10.1038/s12276-026-01659-6

Figure Lengend Snippet: a Cell growth assay after treatment with Fb7-311 for 24 h. A498 and Caki-1 cells were fixed with 100% methanol and stained with a CV solution. Scale bar, 500 μm (top). CCK-8 solution was added to the culture medium and the cells were incubated for 5 min at 37 °C. Cell growth was measured using a microplate reader (450 nm). The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001) (bottom). b FACS analysis of Annexin V staining was performed after cells were treated with Fb7-311. The lower right and upper right quadrants indicate early apoptotic cells and late apoptotic cells, respectively (top). Quantification of apoptosis: the data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001) (bottom). c FACS analysis using the Muse Caspase-3/7 working solution was performed after cells were treated with Fb7-311. The upper right image shows the proportions of apoptotic and dead cells (top). Quantification of caspase-3/7 activity: the data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -tests (* P < 0.05, *** P < 0.001) (bottom). d RT‒qPCR analysis of EHMT2 and DDIT3 expression after cells were treated with Fb7-311. The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001). e Western blot analysis of cells treated with Fb7-311 using anti-EHMT2, anti-PARP and anti-ACTB antibodies. ACTB was used as the internal control in A498 and Caki-1 cells. f Immunocytochemical staining for EHMT2 and DDIT3. A498 and Caki-1 cells were treated with Fb7-311 fixed with 100% methanol and stained with an anti-DDIT3 antibody (Alexa Fluor 488, green) and DAPI (blue). Scale bar, 150 μm. g The ChIP assay was performed with an anti-H3K9me2 antibody on the DDIT3 promoter region. The result is shown as relative enrichment compared to the control in A498 and Caki-1 cells after Fb7-311 treatment. The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (** P < 0.01). h Cell growth assay after treatment with indole-3-carbinol for 72 h. A498 cells were fixed with 100% methanol and stained with a CV solution. Scale bar, 500 μm (left). CCK-8 solution was added to the culture medium and the cells were incubated for 5 min at 37 °C. Cell growth was measured using a microplate reader (450 nm). The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001) (right). i RT‒qPCR analysis of EHMT2 and DDIT3 expression after cells were treated with indole-3-carbinol. The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (** P < 0.01).

Article Snippet: The human RCC cell lines A498 and Caki-1 were purchased from the Korean Cell Line Bank and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO 2 at 37 °C.

Techniques: Growth Assay, Staining, CCK-8 Assay, Incubation, Activity Assay, Expressing, Western Blot, Control

Journal: Experimental & Molecular Medicine

Article Title: Gut microbiota modulation of epigenetic target EHMT2: Lacticaseibacillus rhamnosus Fb7-311 regulated renal cell carcinoma apoptosis and metastasis

doi: 10.1038/s12276-026-01659-6

Figure Lengend Snippet: a The 3D spheroid formation assay. The cells transfected with siEHMT2 and siCont were loaded onto ULA plates and incubated for 48 h. The cells were photographed under a microscope each day. Scale bar, 500 μm. b Western blot analysis of cells after EHMT2 knockdown using anti-PARP and anti-ACTB antibodies. ACTB was used as the internal control in A498 cell. c RT‒qPCR analysis of EHMT2 and DDIT3 expression after EHMT2 knockdown. The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001). d The 3D spheroid formation assay. Cells cotransfected with siEHMT2 and siDDIT3 were loaded onto ULA plates and incubated for 48 h. The cells were photographed under a microscope each day. Scale bar, 500 μm. e Western blot analysis of cells cotransfected with siEHMT2 and siDDIT3 using anti-PARP and anti-ACTB antibodies. ACTB was used as the internal control in A498 cell. f RT‒qPCR analysis of EHMT2 and DDIT3 expression in cells cotransfected with siEHMT2 and siDDIT3. The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -tests (** P < 0.01, *** P < 0.001). g The 3D spheroid formation assay. After BIX was added to ULA plates, the cells were incubated for 48 h. The cells were then photographed under a microscope each day. Scale bar, 500 μm. h Western blot analysis of cells treated with BIX using anti-PARP and anti-ACTB antibodies. ACTB was used as the internal control in A498 cell. i RT‒qPCR analysis of DDIT3 expression in cells treated with BIX. The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -test (*** P < 0.001). j The 3D spheroid formation assay. The cells were loaded onto ULA plates after treatment with Fb7-311 and incubated for 48 h. The cells were photographed under a microscope each day. Scale bar, 500 μm. k Western blot analysis of Fb7-311-treated cells using anti-PARP and anti-ACTB antibodies. ACTB was used as the internal control in A498 cell. l RT‒qPCR analysis of EHMT2 and DDIT3 expression in Fb7-311-treated cells. The data are presented as the means ± s.d. of three independent experiments. P values were calculated using Student’s t -tests (** P < 0.01, *** P < 0.001). m , n BIX treatment suppressed the growth of xenograft tumors in nude mice. Both the control and BIX were intraperitoneally injected three times a week after A498 cell implantation: tumor volumes ( P values were calculated using two-way ANOVA (** P < 0.01)) (m) and macroscopic image of tumors on day 24 ( n ). o Representative H&E-stained mouse tumor sections. Scale bars, 200 μm. p Immunohistochemical staining for DDIT3 in mouse tumor sections. Scale bar, 200 μm.

Article Snippet: The human RCC cell lines A498 and Caki-1 were purchased from the Korean Cell Line Bank and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO 2 at 37 °C.

Techniques: Tube Formation Assay, Transfection, Incubation, Microscopy, Western Blot, Knockdown, Control, Expressing, Injection, Staining, Immunohistochemical staining

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