Journal: Open Medicine

Article Title: NTN1 regulates autophagy through the MAP1B/DAPK1 axis to ameliorate acute kidney injury in vitro

doi: 10.1515/med-2025-1374

Figure Lengend Snippet: The effects of NTN1 on the apoptosis, inflammatory levels and ROS generation in LPS-treated HK-2 cells (A–B) after the transfection of NTN1 overexpression plasmid or si-NTN1, the expression of NTN1 in the blank, OE-NC, OE-NTN1, si-NC, and si-NTN1 groups was determined by qRT-PCR. GAPDH served as the internal control. (C–D) the apoptosis rate of HK-2 cells or RPTEC/TERT1 in the OE-NC, OE-NTN1, si-NC, and si-NTN1 groups was assessed by flow cytometry. (E–G) the levels of TNF-α, IL-6 and IL-1β in the OE-NC, OE-NTN1, si-NC, and si-NTN1 groups were determined by ELISA. (H) The level of ROS generation in each group was detected by DCFH-DA reagent. The data are presented as the mean±standard deviation of three independent experiments; *** p<0.001 vs. OE-NC; ### p<0.001 vs. si-NC. Abbreviation: NTN1, netrin 1; ROS, reactive oxygen species; LPS, lipopolysaccharides; OE-NTN1, NTN1 overexpression; si-NTN1, silenced NTN1; NC, negative control; qRT-PCR, quantitative real-time PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; ELISA, enzyme-linked immunosorbent assay; DCFH-DA, 2,7-Dichlorodi-hydrofluorescein diacetate.

Article Snippet: Renal tubular epithelial cell line HK-2 (AW-CELLS-H0142, Anweisci, China), and RPTEC/TERT1 cells (CRL-4031, ATCC, Virginia, MA, USA) were placed in Minimum Essential Medium (MEM, BC-M-019, Sbjbio, China) enriched with 10 % fetal bovine serum (FBS, FCS500, ExCell, China) at 37 °C with 5 % CO 2 .

Techniques: Transfection, Over Expression, Plasmid Preparation, Expressing, Quantitative RT-PCR, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation, Negative Control, Real-time Polymerase Chain Reaction

Journal: Open Medicine

Article Title: NTN1 regulates autophagy through the MAP1B/DAPK1 axis to ameliorate acute kidney injury in vitro

doi: 10.1515/med-2025-1374

Figure Lengend Snippet: The effects of MAP1B phosphorylation inhibitor on the apoptosis and autophagy in LPS-treated HK-2 cells (A) after the treatment of MAP1B phosphorylation inhibitor, the apoptosis of HK-2 cells in the OE-NC, OE-NTN1 and OE-NTN1+inhibitor groups was evaluated by flow cytometry. (B) The protein expression of Atg5 and the ratio of LC3II/LC3I were determined by western blot. GAPDH served as the internal control. The data are presented as the mean ± standard deviation of three independent experiments; *** p<0.001 vs. OE-NC; &&& p<0.001 vs. OE-NTN1. Abbreviation: MAP1B, microtubule associated protein 1B; LPS, lipopolysaccharides; NTN1, netrin 1; OE-NTN1, NTN1 overexpression; NC, negative control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Renal tubular epithelial cell line HK-2 (AW-CELLS-H0142, Anweisci, China), and RPTEC/TERT1 cells (CRL-4031, ATCC, Virginia, MA, USA) were placed in Minimum Essential Medium (MEM, BC-M-019, Sbjbio, China) enriched with 10 % fetal bovine serum (FBS, FCS500, ExCell, China) at 37 °C with 5 % CO 2 .

Techniques: Phospho-proteomics, Flow Cytometry, Expressing, Western Blot, Control, Standard Deviation, Over Expression, Negative Control

Journal: Open Medicine

Article Title: NTN1 regulates autophagy through the MAP1B/DAPK1 axis to ameliorate acute kidney injury in vitro

doi: 10.1515/med-2025-1374

Figure Lengend Snippet: The effects of MAP1B and DAPK1 on the apoptosis and ROS generation of LPS-treated HK-2 cells (A) Co-IP assay was performed to detect the relationship between MAP1B and DAPK1. (B–C) after the transfection of si-DAPK1 (B) or OE-MAP1B (C), the mRNA expression of DAPK1 or MAP1B in HK-2 cells was tested by qRT-PCR. GAPDH served as the internal control. (D) flow cytometry was used to determine the apoptosis rate of LPS-treated HK-2 cells in the si-NC+OE-NC, si-DAPK1+OE-NC, si-NC+OE-MAP1B and si-DAPK1+OE-MAP1B groups. (E) the ROS generation in each group was detected by DCFH-DA reagent. The data are presented as the mean±standard deviation of three independent experiments; ### p<0.001 vs. si-NC; +++ p<0.001 vs. OE-NC; *** p<0.001 vs. si-NC+OE-NC; ˆˆˆ p<0.001 vs. si-DAPK1+OE-NC; &&& p<0.001 vs. si-NC+OE-MAP1B. Abbreviation: MAP1B, microtubule associated protein 1B; DAPK1, death associated protein kinase 1; ROS, reactive oxygen species; LPS, lipopolysaccharides; si-DAPK1, silenced DAPK1; NC, negative control; qRT-PCR, quantitative real-time PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DCFH-DA, 2,7-Dichlorodi-hydrofluorescein diacetate.

Article Snippet: Renal tubular epithelial cell line HK-2 (AW-CELLS-H0142, Anweisci, China), and RPTEC/TERT1 cells (CRL-4031, ATCC, Virginia, MA, USA) were placed in Minimum Essential Medium (MEM, BC-M-019, Sbjbio, China) enriched with 10 % fetal bovine serum (FBS, FCS500, ExCell, China) at 37 °C with 5 % CO 2 .

Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Quantitative RT-PCR, Control, Flow Cytometry, Standard Deviation, Negative Control, Real-time Polymerase Chain Reaction

Journal: Open Medicine

Article Title: NTN1 regulates autophagy through the MAP1B/DAPK1 axis to ameliorate acute kidney injury in vitro

doi: 10.1515/med-2025-1374

Figure Lengend Snippet: The effects of MAP1B and DAPK1 on the membrane blebbing and autophagy of LPS-treated HK-2 cells (A) the membrane blebbing of LPS-treated HK-2 cells in the si-NC+OE-NC, si-DAPK1+OE-NC, si-NC+OE-MAP1B and si-DAPK1+OE-MAP1B groups was observed under the microscope (magnification × 400, scale bar=100 μm). (B) The protein expression of Atg5 and the ratio of LC3II/LC3I in each group were determined by western blot. GAPDH served as the internal control. The data are presented as the mean±standard deviation of three independent experiments; ** p<0.01, *** p<0.001 vs. si-NC+OE-NC; ˆˆ p<0.01, ˆˆˆ p<0.001 vs. si-DAPK1+OE-NC; &&& p<0.001 vs. si-NC+OE-MAP1B; ++ p<0.01, +++ p<0.001 vs si-NC+OE-NC+BafA1; ΔΔ p <0.01, ΔΔΔ p <0.001 vs. si-DAPK1+OE-NC+BafA1; ### p<0.001 vs. si-NC+OE-MAP1B+BafA1; $$$ p<0.001vs. si-DAPK1+OE-MAP1B. Abbreviation: MAP1B, microtubule associated protein 1B; DAPK1, death associated protein kinase 1; LPS, lipopolysaccharides; si-DAPK1, silenced DAPK1; NC, negative control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Renal tubular epithelial cell line HK-2 (AW-CELLS-H0142, Anweisci, China), and RPTEC/TERT1 cells (CRL-4031, ATCC, Virginia, MA, USA) were placed in Minimum Essential Medium (MEM, BC-M-019, Sbjbio, China) enriched with 10 % fetal bovine serum (FBS, FCS500, ExCell, China) at 37 °C with 5 % CO 2 .

Techniques: Membrane, Microscopy, Expressing, Western Blot, Control, Standard Deviation, Negative Control

Journal: Frontiers in Pharmacology

Article Title: Formononetin alleviated cisplatin-induced acute kidney injury by orchestrating renal tubular cell ferroptosis via PI3K/AKT/NRF2 pathway

doi: 10.3389/fphar.2026.1777367

Figure Lengend Snippet: FN Alleviated Cisplatin-Induced Ferroptosis in HK-2 Cells (A) Cell viability was assessed by CCK-8 assay after treatment with increasing concentrations of RSL3 for 24 h; (B) Cell viability was measured after pretreatment with increasing concentrations of Fer-1 for 24 h followed by 20 μmol/L cisplatin for an additional 24 h; (C) Cell viability was measured after pretreatment with increasing concentrations of FN for 24 h followed by 20 μmol/L cisplatin for an additional 24 h; (D) Cell viability was assessed after pretreatment with 40 μmol/L FN for 24 h followed by 20 μmol/L cisplatin or 0.5 μmol/L RSL3 for an additional 24 h; (E) Intracellular ROS levels were measured using the DCFH-DA fluorescent probe after FN pretreatment followed by cisplatin exposure; (F) Cellular MDA levels were measured after FN pretreatment followed by cisplatin exposure; (G) Intracellular Fe 2+ levels in HK-2 cells were assessed using the FerroOrange fluorescent probe. Left, representative fluorescence images (scale bar, 100 μm); right, quantitative analysis of intracellular Fe 2+ levels; (H) Lipid peroxidation in HK-2 cells was evaluated using the BODIPY 581/591 C11 fluorescent probe. Left, representative fluorescence images (scale bar, 100 μm). Middle, quantitative analysis of red-labeled non-oxidized lipids. Right, quantitative analysis of green-labeled oxidized lipids; (I) Western blot analysis of FTL and FTH protein expression after pretreatment with 40 μmol/L FN or 10 μmol/L DFO for 24 h followed by cisplatin for an additional 24 h. Top, representative immunoblots; bottom, densitometric quantification of FTL and FTH protein levels; (J) Western blot analysis of SLC7A11 and GPX4 protein expression after pretreatment with 40 μmol/L FN or 10 μmol/L Fer-1 for 24 h followed by cisplatin for an additional 24 h. Top, representative immunoblots; bottom, densitometric quantification of SLC7A11 and GPX4 protein levels; (K) Western blot analysis of SLC7A11 and GPX4 protein expression after pretreatment with 40 μmol/L FN for 24 h followed by 20 μmol/L cisplatin or 0.5 μmol/L RSL3 for an additional 24 h. Top, representative immunoblots; bottom, densitometric quantification of SLC7A11 and GPX4 protein levels. *** P < 0.001; # P < 0.05, ## P < 0.01, ### P < 0.001; & P < 0.05 vs. indicated group. CTL, control; CIS, HK-2 cells treated with 20 μmol/L cisplatin; CIS + FN, HK-2 cells treated with cisplatin plus 40 μmol/L FN; RSL3, HK-2 cells treated with 0.5 μmol/L RSL3; RSL3+FN, HK-2 cells treated with RSL3 plus 40 μmol/L FN; CIS + FN + RSL3, HK-2 cells treated with cisplatin, FN, and RSL3; Fer-1, ferrostatin-1; ROS, reactive oxygen species; MDA, malondialdehyde.

Article Snippet: The human proximal tubular cell line (HK-2) was obtained from the American Type Culture Collection and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) (BasalMedia, L310KJ) supplemented with 10% fetal bovine serum (FBS, Excell, FSP500) at 37 °C in a 5% CO 2 humidified incubator.

Techniques: CCK-8 Assay, Fluorescence, Labeling, Western Blot, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Formononetin alleviated cisplatin-induced acute kidney injury by orchestrating renal tubular cell ferroptosis via PI3K/AKT/NRF2 pathway

doi: 10.3389/fphar.2026.1777367

Figure Lengend Snippet: FN Inhibited Ferroptosis by Regulating the PI3K/AKT/NRF2 Pathway (A) Representative immunoblots of NRF2 and HO-1 protein expression in HK-2 cells pretreated with 40 μmol/L FN for 24 h followed by 20 μmol/L cisplatin for an additional 24 h; (B) Densitometric quantification of NRF2 and HO-1 protein levels shown in (A) ; (C) Evaluation of NRF2 knockdown efficiency by Western blot after transient transfection with si NRF2 ; (D) Densitometric quantification of NRF2 protein expression in NC and NRF2 -knockdown HK-2 cells shown in (C) ; (E) Western blot analysis of NRF2, SLC7A11, and GPX4 protein expression in NC and NRF2 -knockdown HK-2 cells treated with 40 μmol/L formononetin for 24 h followed by 20 μmol/L cisplatin for an additional 24 h; (F) Densitometric quantification of NRF2, SLC7A11, and GPX4 protein levels shown in (E) ; (G) Western blot analysis of FTL and FTH protein expression in NC and NRF2-knockdown HK-2 cells under the same treatment conditions as in (E) ; (H) Densitometric quantification of FTL and FTH protein levels shown in (G) ; (I) Western blot analysis of p-PI3K/PI3K, p-AKT/AKT, NRF2, and GPX4 protein expression in HK-2 cells treated with 20 μmol/L LY294002 and/or 40 μmol/L formononetin for 24 h followed by 20 μmol/L cisplatin for an additional 24 h; (J) Quantitative analysis of the p-PI3K/PI3K and p-AKT/AKT ratios shown in (I) ; (K) Quantitative analysis of NRF2 and GPX4 protein levels shown in (I) . ** P < 0.01, *** P < 0.001; # P < 0.05, ## P < 0.01, ### P < 0.001; & P < 0.05 vs. indicated group. CTL, control; CIS, HK-2 cells treated with 20 μmol/L cisplatin; FN, HK-2 cells treated with 40 μmol/L FN alone; CIS + FN, HK-2 cells treated with cisplatin plus 40 μmol/L FN; CIS + FN + LY294002, HK-2 cells treated with cisplatin, 40 μmol/L FN, and 20 μmol/L LY294002; NC, Negative control; NRF2 -KD, NRF2 -knockdown.

Article Snippet: The human proximal tubular cell line (HK-2) was obtained from the American Type Culture Collection and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) (BasalMedia, L310KJ) supplemented with 10% fetal bovine serum (FBS, Excell, FSP500) at 37 °C in a 5% CO 2 humidified incubator.

Techniques: Western Blot, Expressing, Knockdown, Transfection, Control, Negative Control

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