transfection wild type human renal tubular epithelial cell line hk 2  (ATCC)

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