Journal: Human Mutation

Article Title: Cross‐Cohort Transcriptomic Integration Identifies IFIT2 as a Translational Diagnostic Biomarker and Functional Driver of Inflammation‐Linked Tubular Injury in Chronic Kidney Disease

doi: 10.1155/humu/8282277

Figure Lengend Snippet: Induction and knockdown of IFIT2 in renal tubular epithelial cells. (A–B) IFN‐ γ –induced IFIT2 expression in HK‐2 and RPTEC cells. (C–D) TGF‐ β 1–induced IFIT2 expression in HK‐2 and RPTEC cells. (E–F) Validation of IFIT2 knockdown efficiency by qPCR. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Human renal proximal tubular epithelial cells, including the HK‐2 cell line (ATCC, RRID: CVCL_0302) and primary RPTEC cells (ATCC, RRID: CVCL_K278), were used in this study.

Techniques: Knockdown, Expressing, Biomarker Discovery

Journal: Human Mutation

Article Title: Cross‐Cohort Transcriptomic Integration Identifies IFIT2 as a Translational Diagnostic Biomarker and Functional Driver of Inflammation‐Linked Tubular Injury in Chronic Kidney Disease

doi: 10.1155/humu/8282277

Figure Lengend Snippet: IFIT2 knockdown attenuates IFN‐ γ –induced injury and apoptosis in renal tubular epithelial cells. (A–B) CCK‐8 assay showing that IFIT2 knockdown alleviates IFN‐ γ –induced reduction of cell viability in HK‐2 and RPTEC cells. (C–F) Annexin V/PI flow cytometry analysis showing that IFIT2 knockdown reduces IFN‐ γ –induced apoptosis in (C, E) HK‐2 and (D, F) RPTEC cells. Data are presented as mean ± SD from three independent experiments. ∗∗∗ p < 0.001.

Article Snippet: Human renal proximal tubular epithelial cells, including the HK‐2 cell line (ATCC, RRID: CVCL_0302) and primary RPTEC cells (ATCC, RRID: CVCL_K278), were used in this study.

Techniques: Knockdown, CCK-8 Assay, Flow Cytometry

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Negative regulatory responses to metabolically triggered inflammation impair renal epithelial immunity in diabetes mellitus

doi: 10.1007/s00109-012-0969-x

Figure Lengend Snippet: Renal tubular epithelial cells, but not mesangial cells, modulate overt inflammation via negative feedback mechanisms that impair immunity. a Human renal primary tubular epithelial cells ( RPTEC ) and mesangial cells ( NHMC ; Lonza) were cultured with diabetes-relevant concentrations of cytokines (TNF-α, 50 pg/ml; IL-6, 20 pg/ml; and IL-1β, 20 pg/ml), high glucose ( HG ; 20 mM) or methylglyoxal ( MG ; 10 μM) for 3 h or 5 days (120 h). Protein lysates from each condition ( n = 4) were prepared for western blot analysis using the indicated antibodies ( n ≥ 3 for each antibody). b RPTEC were first exposed to a combination of cytokines, high glucose, and methylglyoxal at concentrations stated in ( a ) for 5 days. The cells were further cultured in fresh medium in the presence of either diphenyleneiodonium chloride ( DPI ; 10 μM) or combined with Gö6983 (10 nM) for 1 day before challenge with lipopolysaccharide ( LPS ; 1 μg/ml) for 2 h. Protein lysates ( n = 4 for each condition) were analyzed by western blotting (≥3 immunoblots for each antibody). c RPTEC cultured in the combined presence of cytokines, HG and MG at the aforementioned concentrations for 5 days were challenged with a higher dose of LPS (10 μg/ml) for the indicated time points ( n = 4 for each time point). Minus 1 h denotes cells that were not exposed to LPS but were lysed for protein isolation one hour before similarly cultured cells were challenged with LPS. Protein lysates from each condition were prepared for western blotting as described (Figs. and legends). d RPTEC and NHMC were treated as follows before harvesting for protein and total RNA isolations ( n = 4 for each condition). I Untreated control cells; II incubated with cytokines (TNF-α, 50 pg/ml; IL-6, 20 pg/ml; and IL-1β, 20 pg/ml), HG (20 mM), and MG (10 μM) for 5 days; III incubated with MG (10 μM) only for 5 days; IV challenged with LPS (10 μg/ml), lipoteichoic acid ( LTA ; 10 μg/ml), and poly(I:C) (10 μg/ml) for 2 h; V treated as in ( II ) before returning to normal culture medium for 1 day and subsequently challenged with microbial stimulants as in ( IV ); VI cultured as in ( II ) before treatment with DPI (10 μM) and Gö6983 (10 nM) for 1 day in fresh culture medium; VII cultured as in ( VI ) before challenge as in ( IV ). Protein lysates were assayed using quantitative protein arrays. e Results of qPCR array plotted as fold change (RPTEC of each condition cf. untreated RPTEC). Genes with similar functions or involvement in the same biological process were grouped. A Diabetes related, B inflammatory pathways, C antioxidant system, D negative immune regulators, E interleukin and cytokine receptors, F TLR signaling, and G antimicrobial molecules and adaptive immunity. Roman numerals ( I–V ) indicate the type of treatment. I RPTEC incubated with cytokines (TNF-α, 50 pg/ml; IL-6, 20 pg/ml; and IL-1β, 20 pg/ml), HG (20 mM), and MG (10 μM) for 5 days; II RPTEC challenged with LPS (10 μg/ml), LTA (10 μg/ml) and poly I/C (10 μg/ml) for 2 h; III RPTEC treated as in ( I ), returned to normal culture medium for 1 day and then challenged with microbial analogs as in ( II ); IV RPTEC cultured as in ( I ) before combined treatment with DPI (10 μM) and Gö6983 (10 nM) for 1 day in fresh culture medium; V RPTEC cultured as in ( IV ) before being challenged as in ( II ). The qPCR array experiment for each of the conditions was performed twice

Article Snippet: Clonetics® human primary renal tubular epithelial cells (RPTEC) and mesangial cells (NHMC), purchased from Lonza BioSciences, were cultured in REGMTM Renal Epithelial Cell Growth Medium and NHMC in MsGMTM Mesangial Cell Growth Medium, respectively.

Techniques: Cell Culture, Western Blot, Isolation, Control, Incubation