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