hk2 cells  (TargetMol)

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA NKILA regulates the JAK2/STAT3 pathway to exacerbate TGF-β1-mediated renal fibrosis

doi: 10.3892/mmr.2026.13839

Figure Lengend Snippet: Heat map analysis of the top 20 cis-regulated lncRNAs in the lncRNA high-throughput sequencing and the binding and site results of the NKILA promoter region predicted by JASPAR database. (A) Heatmap demonstrating the top 20 cis-regulated lncRNAs in the control group compared with the normal group. ‘Normal’ indicates blank groups and ‘control’ indicates TGF-β1 (10 ng/ml) induced HK-2 cells. Y-axis shows cis-regulated lncRNA corresponding names. Color changes represent the expression level of the control group changes after normalization treatment compared with the blank group. The change from blue to red after normalization ranges from 0–2. (B) STAT3 binding site information predicted by the JASPAR database for the promoter region from 2000-99 bp upstream of NKILA. lncRNA, long non-coding RNA.

Article Snippet: HK-2 cells (cat. no. CL-0109) were cultured in RPMI 1640 medium (cat. no. L220KJ; Shanghai Basal Media Technologies Co., Ltd), supplemented with 10% FBS (cat. no. FBSSR-01021-5, Suzhou Cyagen Biosciences Inc.) at 37°C in a humified 5% CO 2 atmosphere in a CO 2 incubator.

Techniques: Next-Generation Sequencing, Binding Assay, Control, Expressing

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA NKILA regulates the JAK2/STAT3 pathway to exacerbate TGF-β1-mediated renal fibrosis

doi: 10.3892/mmr.2026.13839

Figure Lengend Snippet: lncRNA NKILA overexpression lentivirus and negative control lentivirus transfection experiments. (A) Relative expression level of NKILA after transfection with Lv-NKILA detected by RT-qPCR (n=3). (B) Relative expression levels of NKILA after transfection with Lv-NKILA-shRNA and Lv-NKILA-shRNA (n=3) detected by RT-qPCR. LVCON313 indicates transfection of the knockdown negative control virus LVCON313. LVCON335 indicates transfection of the overexpressed negative control virus LVCON335. Lv-NKILA (76304) indicates transfection of overexpressing Lv-NKILA (76304) in normally cultured HK-2 cells (MOI=5). Lv-NKILA-shRNA (108126) indicates transfection of the knockdown virus Lv-NKILA-shRNA (108126) in normally cultured HK-2 cells (MOI=5). Lv-NKILA-shRNA (108127) indicates transfection of the knockdown virus Lv-NKILA-shRNA (108127) in normally cultured HK-2 cells (MOI=5). ## P<0.01 compared with the normal group. RT-qPCR, reverse transcription quantitative PCR; MOI, multiplicity of infection; shRNA, short hairpin; Lv, lentivirus.

Article Snippet: HK-2 cells (cat. no. CL-0109) were cultured in RPMI 1640 medium (cat. no. L220KJ; Shanghai Basal Media Technologies Co., Ltd), supplemented with 10% FBS (cat. no. FBSSR-01021-5, Suzhou Cyagen Biosciences Inc.) at 37°C in a humified 5% CO 2 atmosphere in a CO 2 incubator.

Techniques: Over Expression, Negative Control, Transfection, Expressing, Quantitative RT-PCR, shRNA, Knockdown, Virus, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Infection

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA NKILA regulates the JAK2/STAT3 pathway to exacerbate TGF-β1-mediated renal fibrosis

doi: 10.3892/mmr.2026.13839

Figure Lengend Snippet: Partial EMT phenotype proteins in HK-2 cells transfected with lncRNA NKILA overexpression virus (A) Representative western blotting images and (B) quantification of EMT phenotypic indicators. (C) Statistical analysis results of E-cad fluorescence intensity (n=3). (D) RT-qPCR statistical results of EMT phenotype indicators (n=3). ‘Normal’ indicates after starvation treatment, HK-2 cells were replaced with fresh complete medium and continued to be cultured for 24 h. Control, following starvation treatment, 10 ng/ml TGF-β1 cytokine diluent was added to induce HK-2 cells to construct the renal interstitial fibrosis model for 24 h. ‘OE-NKILA’ indicates after starvation, overexpressed Lv-NKILA (76304) was used for transfection and cells were collected after 24 h of lentivirus transfection. Compared with OE-NC ## P<0.01. E-cad; epithelial-cadherin; RT-qPCR, reverse transcription quantitative PCR; EMT, epithelial-mesenchymal transition; NC, negative control; Lv, lentivirus; OE, overexpression; FN, fibronectin; Col1, collagen I; α-SMA, α-smooth muscle actin; Vim; vimentin; ns, not significant.

Article Snippet: HK-2 cells (cat. no. CL-0109) were cultured in RPMI 1640 medium (cat. no. L220KJ; Shanghai Basal Media Technologies Co., Ltd), supplemented with 10% FBS (cat. no. FBSSR-01021-5, Suzhou Cyagen Biosciences Inc.) at 37°C in a humified 5% CO 2 atmosphere in a CO 2 incubator.

Techniques: Transfection, Over Expression, Virus, Western Blot, Fluorescence, Quantitative RT-PCR, Cell Culture, Control, Construct, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA NKILA regulates the JAK2/STAT3 pathway to exacerbate TGF-β1-mediated renal fibrosis

doi: 10.3892/mmr.2026.13839

Figure Lengend Snippet: JKA2/STAT3 pathway protein detection in HK-2 cells transfected with LncRNA NKILA overexpression virus (A) Representative western blotting bands and (B) semi-quantification of results of protein expression of phosphorylated JAK2 and STAT3 (n=3). (C) RT-qPCR statistical results (n=3). ## P<0.01 and # P<0.05 compared with the normal group. ns, not significant; p-, phosphorylated; OE, overexpression; NC, negative control; JAK, Janus kinase; RT-qPCR, reverse transcription quantitative PCR.

Article Snippet: HK-2 cells (cat. no. CL-0109) were cultured in RPMI 1640 medium (cat. no. L220KJ; Shanghai Basal Media Technologies Co., Ltd), supplemented with 10% FBS (cat. no. FBSSR-01021-5, Suzhou Cyagen Biosciences Inc.) at 37°C in a humified 5% CO 2 atmosphere in a CO 2 incubator.

Techniques: Transfection, Over Expression, Virus, Western Blot, Expressing, Quantitative RT-PCR, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA NKILA regulates the JAK2/STAT3 pathway to exacerbate TGF-β1-mediated renal fibrosis

doi: 10.3892/mmr.2026.13839

Figure Lengend Snippet: Partial EMT phenotype proteins in HK-2 cells transfected with lncRNA NKILA knockdown lentivirus (A) Representative western blotting bands and (B) semi-quantification results of EMT phenotypic indicators. (C) E-cad fluorescence intensity and (D) EMT phenotypic index RT-qPCR (n=3). ‘Normal’ indicates starvation treatment. Compared with normal group ## P<0.01. Compared with control + KD-NC group, **P<0.01 and *P<0.05. EMT, epithelial-mesenchymal transition; E-cad, epithelial-cadherin; RT-qPCR, reverse transcription quantitative PCR; KD, knockdown; Lv, lentivirus; shRNA, short hairpin RNA; NC, negative control; FN, fibronectin; Col1, collagen I; α-SMA, α-smooth muscle actin; Vim; vimentin.

Article Snippet: HK-2 cells (cat. no. CL-0109) were cultured in RPMI 1640 medium (cat. no. L220KJ; Shanghai Basal Media Technologies Co., Ltd), supplemented with 10% FBS (cat. no. FBSSR-01021-5, Suzhou Cyagen Biosciences Inc.) at 37°C in a humified 5% CO 2 atmosphere in a CO 2 incubator.

Techniques: Transfection, Knockdown, Western Blot, Fluorescence, Quantitative RT-PCR, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, shRNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA NKILA regulates the JAK2/STAT3 pathway to exacerbate TGF-β1-mediated renal fibrosis

doi: 10.3892/mmr.2026.13839

Figure Lengend Snippet: JAK2/STAT3 signaling pathway-associated proteins in HK-2 cells transfected with LncRNA NKILA knockdown lentivirus (A) Representative western blotting bands and (B) semi-quantification of JAK2/STAT3 pathway protein expression (n=3). (C) Statistical analysis results of reverse transcription-quantitative PCR for channel indicators. ## P<0.01 compared with the normal group (n=3), **P<0.01 and *P<0.05 compared with the control + KD-NC group. JAK, Janus kinase; KD, knockdown; NC, negative control; p-, phosphorylated.

Article Snippet: HK-2 cells (cat. no. CL-0109) were cultured in RPMI 1640 medium (cat. no. L220KJ; Shanghai Basal Media Technologies Co., Ltd), supplemented with 10% FBS (cat. no. FBSSR-01021-5, Suzhou Cyagen Biosciences Inc.) at 37°C in a humified 5% CO 2 atmosphere in a CO 2 incubator.

Techniques: Transfection, Knockdown, Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Negative Control

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA NKILA regulates the JAK2/STAT3 pathway to exacerbate TGF-β1-mediated renal fibrosis

doi: 10.3892/mmr.2026.13839

Figure Lengend Snippet: JAK2/STAT3 pathway protein detection in the recovery experiment of HK-2 cells treated with AG490. (A) Representative western blotting bands and (B) semi-quantification of JAK2/STAT3 pathway protein expression (n=3). (C) Reverse transcription-quantitative PCR for channel indicators (n=3). ## P<0.01 compared with the normal group, **P<0.01 and *P<0.05 compared with control + DMSO group and △△ P<0.01 compared with OE-NKILA group. OE, overexpression; p-, phosphorylated; JAK, Janus kinase; ns, not significant.

Article Snippet: HK-2 cells (cat. no. CL-0109) were cultured in RPMI 1640 medium (cat. no. L220KJ; Shanghai Basal Media Technologies Co., Ltd), supplemented with 10% FBS (cat. no. FBSSR-01021-5, Suzhou Cyagen Biosciences Inc.) at 37°C in a humified 5% CO 2 atmosphere in a CO 2 incubator.

Techniques: Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Over Expression

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA NKILA regulates the JAK2/STAT3 pathway to exacerbate TGF-β1-mediated renal fibrosis

doi: 10.3892/mmr.2026.13839

Figure Lengend Snippet: Detection of EMT-related phenotypic proteins in the AG490 intervention HK-2 cell recovery experiment (A) Representative western blotting bands and (B) semi-quantification of EMT phenotypic protein changes (n=3). (C) Statistical analysis results of E-cad fluorescence intensity (n=3) and (D) EMT phenotypic index through reverse transcription quantitative PCR (n=3). ‘Normal’ indicates starvation treatment, HK-2 cells were replaced with fresh complete medium and continued to be cultured for 24 h. ## P<0.01 compared with the normal group, ** P<0.01 and *P<0.05 compared with the control + DMSO group and △△ P<0.01 and △ P<0.05 compared with the OE-NKILA group. EMT, epithelial-mesenchymal transition; E-cad, epithelial cadherin; OE, overexpression; Lv, lentivirus; FN, fibronectin; Col1, collagen I; α-SMA, α-smooth muscle actin; Vim; vimentin.

Article Snippet: HK-2 cells (cat. no. CL-0109) were cultured in RPMI 1640 medium (cat. no. L220KJ; Shanghai Basal Media Technologies Co., Ltd), supplemented with 10% FBS (cat. no. FBSSR-01021-5, Suzhou Cyagen Biosciences Inc.) at 37°C in a humified 5% CO 2 atmosphere in a CO 2 incubator.

Techniques: Cell Recovery, Western Blot, Fluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Culture, Control, Over Expression

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA NKILA regulates the JAK2/STAT3 pathway to exacerbate TGF-β1-mediated renal fibrosis

doi: 10.3892/mmr.2026.13839

Figure Lengend Snippet: Immunofluorescence detection of E-CAD protein in the recovery experiment of HK-2 cells treated with AG490. E-cad immunofluorescence staining (magnification, ×400; scale bar, 20 µm; n=3) compared with the normal group. E-cad, epithelial cadherin; OE, overexpression.

Article Snippet: HK-2 cells (cat. no. CL-0109) were cultured in RPMI 1640 medium (cat. no. L220KJ; Shanghai Basal Media Technologies Co., Ltd), supplemented with 10% FBS (cat. no. FBSSR-01021-5, Suzhou Cyagen Biosciences Inc.) at 37°C in a humified 5% CO 2 atmosphere in a CO 2 incubator.

Techniques: Immunofluorescence, Staining, Over Expression

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification of NLRC5 promotes renal fibrosis in chronic kidney disease through Keap1/Nrf2/ARE signaling pathway

doi: 10.3389/fimmu.2026.1739011

Figure Lengend Snippet: Analysis of m 6 A RNA methylation and NLRC5 expression in the TGF-β1-induced cell fibrosis model. (A) Western blot showing dose-dependent changes in fibrosis markers after 48 h treatment with increasing TGF-β1 concentrations (0, 1, 5, 10 ng/mL). (B) Time-course Western blot analysis of HK-2 cells exposed to 10 ng/mL TGF-β1 for 12, 24, and 48 (h) * p < 0.05, ** p < 0.01, *** p < 0.001, vs. 0 ng/ml or 0 h; (C) Quantification of global m 6 A RNA methylation in HK-2 cells treated with 10 ng/mL TGF-β1 for 48 h, demonstrating a significant increase versus control (** p < 0.01, vs. control). (D) Quantitative reverse transcription PCR quantitative reverse transcription PCR analysis of METTL3 and NLRC5 mRNA levels after 48 h TGF-β1 treatment, indicating significant upregulation (*** p < 0.001, vs. control). (E) Representative Western blots depicting METTL3 and NLRC5 protein expression in control and TGF-β1-treated cells (10 ng/mL, 48 h). Data are presented as mean ± SD from three independent experiments.

Article Snippet: Pro-inflammatory cytokines IL-1β and TNF-α were measured in the supernatant of cultured HK-2 cells and mouse serum using species-specific ELISA kits (IL-1β: #CSB-E08053h for human, #CSB-E08054m for mouse; TNF-α: #CSB-E04740h for human, #CSB-E04741m for mouse; Cusabio Biotech, Wuhan, China), in accordance with the manufacturer’s protocols.

Techniques: Methylation, Expressing, Western Blot, Control, Reverse Transcription

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification of NLRC5 promotes renal fibrosis in chronic kidney disease through Keap1/Nrf2/ARE signaling pathway

doi: 10.3389/fimmu.2026.1739011

Figure Lengend Snippet: METTL3 regulated m 6 A modification and expression of NLRC5 in TGF-β1-induced cell fibrosis model. (A) Quantitative reverse transcription PCR analysis of METTL3 mRNA levels in HK-2 cells transfected with OE-METTL3 or si-METTL3, followed by TGF-β1 (10 ng/mL) treatment for 48 h (*** p < 0.001, vs. Vector; ### p < 0.001, vs. si-NC). (B) Western blot confirming METTL3 protein expression in the same conditions as (A, C) Quantitative reverse transcription PCR showing NLRC5 mRNA levels after METTL3 overexpression or knockdown (** p < 0.01, *** p < 0.001, vs. vector or si-NC). (D) Western blot analysis of NLRC5 protein expression under the indicated conditions (*** p < 0.001, vs. vector; ### p < 0.001, vs. si-NC). (E) NLRC5 mRNA stability assessed by actinomycin D chase assay in METTL3-modulated cells, measured by quantitative reverse transcription PCR at 0, 4, and 8 h. (F) Predicted m6A methylation sites in the NLRC5 transcript analyzed by SRAMP. The graph illustrates the distribution of m6A sites across the transcript, with confidence levels indicated by different color thresholds. (G) MeRIP-qPCR showing reduced m 6 A enrichment on NLRC5 mRNA upon METTL3 knockdown. (H) RIP-qPCR analysis indicating direct interaction between METTL3 and NLRC5 mRNA, with IgG as control (** p < 0.01, *** p < 0.001, vs. indicated controls). (I) Western blot of fibrosis markers (α-SMA, Collagen I, and E-cadherin) following METTL3 overexpression or knockdown, with or without STM2457 treatment (*** p < 0.001, vs. vector; ### p < 0.001, vs. OE-METTL3; && p < 0.01, &&& p < 0.001, vs. si-NC). Data are presented as mean ± SD from three independent experiments.

Article Snippet: Pro-inflammatory cytokines IL-1β and TNF-α were measured in the supernatant of cultured HK-2 cells and mouse serum using species-specific ELISA kits (IL-1β: #CSB-E08053h for human, #CSB-E08054m for mouse; TNF-α: #CSB-E04740h for human, #CSB-E04741m for mouse; Cusabio Biotech, Wuhan, China), in accordance with the manufacturer’s protocols.

Techniques: Modification, Expressing, Reverse Transcription, Transfection, Plasmid Preparation, Western Blot, Over Expression, Knockdown, Methylation, Control

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification of NLRC5 promotes renal fibrosis in chronic kidney disease through Keap1/Nrf2/ARE signaling pathway

doi: 10.3389/fimmu.2026.1739011

Figure Lengend Snippet: Effects of NLRC5 knockdown on inflammation, oxidative stress, and fibrosis in TGF-β1–treated HK-2 cells. (A, B) Quantitative reverse transcription PCR (A) and Western blot analysis (B) confirming the efficient knockdown of NLRC5 expression in TGF-β1–stimulated HK-2 cells transfected with si-NLRC5 (*** p < 0.001, vs. si-NC). (C) ELISA detection of IL-1β and TNF-α levels in cell culture supernatants following TGF-β1 treatment and/or NLRC5 silencing. (D) Measurement of oxidative stress indicators, including MDA content and SOD activity, under different experimental conditions. (E) Representative fluorescence images and quantitative analysis of intracellular ROS levels detected using the DCFDA probe in the indicated groups. (F) Western blot analysis of fibrosis-related proteins, including α-SMA, Collagen I, and E-cadherin, in TGF-β1–treated HK-2 cells with or without NLRC5 knockdown (*** p < 0.001, vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001, vs. TGF-β1 + si-NC). Data are presented as mean ± SD from three independent experiments.

Article Snippet: Pro-inflammatory cytokines IL-1β and TNF-α were measured in the supernatant of cultured HK-2 cells and mouse serum using species-specific ELISA kits (IL-1β: #CSB-E08053h for human, #CSB-E08054m for mouse; TNF-α: #CSB-E04740h for human, #CSB-E04741m for mouse; Cusabio Biotech, Wuhan, China), in accordance with the manufacturer’s protocols.

Techniques: Knockdown, Reverse Transcription, Western Blot, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Activity Assay, Fluorescence, Control

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification of NLRC5 promotes renal fibrosis in chronic kidney disease through Keap1/Nrf2/ARE signaling pathway

doi: 10.3389/fimmu.2026.1739011

Figure Lengend Snippet: Involvement of the Keap1/Nrf2/ARE signaling pathway in the regulatory effects of NLRC5 knockdown in TGF-β1–induced HK-2 cells. (A) Western blot analysis of Keap1, nuclear Nrf2, and the Nrf2 downstream targets HO-1 and NQO-1 in HK-2 cells under the indicated conditions (Control, TGF-β1, TGF-β1 + si-NC, TGF-β1 + si-NLRC5, TGF-β1 + si-NLRC5 + OE-Keap1, and TGF-β1 + si-NLRC5 + ML385). Statistical significance is indicated as follows: *** p < 0.001, vs. control; # p < 0.05, ### p < 0.001, vs . TGF-β1 + si-NC; & p < 0.05, &&& p < 0.001, vs . TGF-β1 + si-NLRC5. (B) ELISA determination of IL-1β and TNF-α levels in cell culture supernatants following the indicated treatments (*** p < 0.001, vs. si-NC; # p < 0.05, ## p < 0.01, vs. si-NLRC5). (C) Quantitative analysis of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in different groups (*** p < 0.001, vs. si-NC; # p < 0.05, ## p < 0.01, vs. si-NLRC5). (D) Representative fluorescence images and quantitative measurement of intracellular reactive oxygen species (ROS) using the DCFDA probe (*** p < 0.001, vs. si-NC; ### p < 0.001, vs. si-NLRC5). (E) Western blot analysis of fibrosis-associated proteins, including α-SMA, Collagen I, and E-cadherin, in HK-2 cells under the indicated conditions (*** p < 0.001, vs. TGF-β1 + si-NC, ## p < 0.01, ### p < 0.001, vs. TGF-β1 + si-NLRC5). Data are presented as mean ± SD from three independent experiments.

Article Snippet: Pro-inflammatory cytokines IL-1β and TNF-α were measured in the supernatant of cultured HK-2 cells and mouse serum using species-specific ELISA kits (IL-1β: #CSB-E08053h for human, #CSB-E08054m for mouse; TNF-α: #CSB-E04740h for human, #CSB-E04741m for mouse; Cusabio Biotech, Wuhan, China), in accordance with the manufacturer’s protocols.

Techniques: Knockdown, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Activity Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: METTL3-driven m 6 A modification of NLRC5 promotes renal fibrosis in chronic kidney disease through Keap1/Nrf2/ARE signaling pathway

doi: 10.3389/fimmu.2026.1739011

Figure Lengend Snippet: Effects of METTL3 knockdown and NLRC5 modulation on Keap1/Nrf2/ARE signaling and fibrotic responses in TGF-β1–induced HK-2 cells. (A) Western blot analysis of Keap1, nuclear Nrf2, and Nrf2 downstream targets HO-1 and NQO-1 in TGF-β1–stimulated HK-2 cells transfected with si-NC, si-METTL3, si-METTL3 + ML385, or si-METTL3 + OE-NLRC5. (B) ELISA analysis of IL-1β and TNF-α levels in the culture supernatants of the indicated groups. (C) Quantitative assessment of oxidative stress markers, including MDA content and SOD activity, under the indicated treatments. (D) Representative fluorescence images and quantitative analysis of intracellular ROS levels detected by the DCFDA probe. (E) Western blot analysis of fibrosis-related proteins, including α-SMA, Collagen I, and E-cadherin, in TGF-β1–treated HK-2 cells transfected as indicated. Data are presented as mean ± SD from three independent experiments. *** p < 0.001, versus si-NC; # p < 0.05, ## p < 0.01, ### p < 0.001, versus si-METTL3.

Article Snippet: Pro-inflammatory cytokines IL-1β and TNF-α were measured in the supernatant of cultured HK-2 cells and mouse serum using species-specific ELISA kits (IL-1β: #CSB-E08053h for human, #CSB-E08054m for mouse; TNF-α: #CSB-E04740h for human, #CSB-E04741m for mouse; Cusabio Biotech, Wuhan, China), in accordance with the manufacturer’s protocols.

Techniques: Knockdown, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Activity Assay, Fluorescence