Journal: Biomedicines

Article Title: Omega-3 Fatty Acids Attenuate Renal Fibrosis via AMPK-Mediated Autophagy Flux Activation

doi: 10.3390/biomedicines11092553

Figure Lengend Snippet: DHA/EPA treatment inhibited the expression of EMT in TGF-β1-treated (10 ng/mL) HK2 cells in a dose-dependent manner. ( A , B ) Representative Western blot images: DHA/EPA inhibited the expression of α-SMA in a dose-dependent manner in TGF-β1-treated HK2 cells. ( C , D ) Representative Western blot images: DHA/EPA increased E-cadherin levels in a dose-dependent manner in TGF-β1-treated HK2 cells. * p < 0.05 vs. control HK2 cells, # p < 0.05 vs. TGF-β1-treated HK2 cells in the absence of DHA/EPA. All experiments were performed in triplicate. All values are expressed as the mean ± SD. HK2: human kidney 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; EMT: epithelial–mesenchymal transition; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; TGF: transforming growth factor; SMA: α-smooth muscle actin.

Article Snippet: Human kidney-2 (HK-2) cells, an immortalized human PTC line, purchased from American Type Culture Collection (Mannasas, VA, USA), were grown in Dulbecco’s Modified Eagle Medium (DMEM)/F12 (Gibco, New York, NY, USA).

Techniques: Expressing, Western Blot

Journal: Biomedicines

Article Title: Omega-3 Fatty Acids Attenuate Renal Fibrosis via AMPK-Mediated Autophagy Flux Activation

doi: 10.3390/biomedicines11092553

Figure Lengend Snippet: DHA/EPA enhanced autophagic flux via AMPK phosphorylation ( A , B ). Representative Western blot images: the p-AMPKα/AMPK ratio was decreased in TGF-β-treated (10 ng/mL) HK2 cells compared to control HK2 cells. DHA/EPA increased the p-AMPKα/AMPKα ratio in TGF-beta-treated HK2 cells, in a dose-dependent manner. ( C , D ) Representative Western blot images: DHA/EPA increased the expression of LC3-II in TGF-beta-treated HK2 cells. in dose-dependent manner. ( E , F ) Representative Western blot images: DHA/EPA decreased p62 expression in TGF-β1-treated HK2 cells. All experiments were performed in triplicate. All values are expressed as the mean ± SD. * p < 0.05 vs. control HK2 cells; # p < 0.05 vs. TGF-β1-treated HK2 cells in the absence of DHA/EPA. HK2: human kidney 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; EMT: epithelial–mesenchymal transition; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; TGF: transforming growth factor; SMA: α-smooth muscle actin.

Article Snippet: Human kidney-2 (HK-2) cells, an immortalized human PTC line, purchased from American Type Culture Collection (Mannasas, VA, USA), were grown in Dulbecco’s Modified Eagle Medium (DMEM)/F12 (Gibco, New York, NY, USA).

Techniques: Western Blot, Expressing

Journal: International Journal of Medical Sciences

Article Title: TREM-1 promoted apoptosis and inhibited autophagy in LPS-treated HK-2 cells through the NF-κB pathway

doi: 10.7150/ijms.50893

Figure Lengend Snippet: TREM-1 expression in HK-2 cells treated with a TREM-1 vector or shRNA with or without LPS. HK-2 cells were transfected with a control vector, plasmid for TREM-1 expression, control siRNA or TREM-1-specific siRNA as described in the Materials and Methods section. Panel A: Representative PT-PCR results indicating suppression of TREM-1 by siRNA (TREM-1 siRNA) or overexpression of TREM-1 (TREM-1 DNA). Panels B & C: Representative immunoblotting results indicating suppression of TREM-1 by siRNA (TREM-1 siRNA) or overexpression of TREM-1 (TREM-1 DNA) from qualitative or quantitative analysis, respectively. * P < 0.05 compared with control cells without LPS; # P < 0.05 compared with control cells with LPS. The data presented are one representative assessment of triplicate assays with similar results.

Article Snippet: For TREM-1 depletion studies, TREM-1-specific siRNA (Santa Cruz, Cat# sc-42999), control (CON) shRNA plasmid (Santa Cruz, Cat# sc-108060), TREM-1 cDNA ORF clone (Sino Bio, Cat# HG10511-UT), or negative control vector (Sino Bio, Cat# CV011) was transfected into HK-2 cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Expressing, Plasmid Preparation, shRNA, Transfection, Over Expression, Western Blot

Journal: International Journal of Medical Sciences

Article Title: TREM-1 promoted apoptosis and inhibited autophagy in LPS-treated HK-2 cells through the NF-κB pathway

doi: 10.7150/ijms.50893

Figure Lengend Snippet: The roles of TREM-1 in modulating HK-2 cell proliferation and apoptosis in response to LPS. HK-2 cells were transfected with a control vector, TREM-1 vector, control shRNA, or TREM-1-specific shRNA as described in the Materials and Methods section. The cells were then cultured for 72 h with or without LPS (10 µg/mL). Panel A: Quantitative comparison of cell proliferation in cells with or without LPS exposure. Panel B: Quantitative comparison of cell apoptosis in cells with or without LPS exposure. Panel C: Flow cytometry of cell apoptosis in cells with or without LPS exposure. * P < 0.05 compared with control cells without LPS; # P < 0.05 compared with control cells with LPS. The data presented are a representative assessment of triplicate assays with similar results.

Article Snippet: For TREM-1 depletion studies, TREM-1-specific siRNA (Santa Cruz, Cat# sc-42999), control (CON) shRNA plasmid (Santa Cruz, Cat# sc-108060), TREM-1 cDNA ORF clone (Sino Bio, Cat# HG10511-UT), or negative control vector (Sino Bio, Cat# CV011) was transfected into HK-2 cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Transfection, Plasmid Preparation, shRNA, Cell Culture, Flow Cytometry

Journal: International Journal of Medical Sciences

Article Title: TREM-1 promoted apoptosis and inhibited autophagy in LPS-treated HK-2 cells through the NF-κB pathway

doi: 10.7150/ijms.50893

Figure Lengend Snippet: Inflammation and cytokine levels in supernatants of HK-2 cells treated with a TREM-1 vector or shRNA with or without LPS. The concentrations of sTREM-1 (panel A), IL-1β (panel B), TNFα (panel C), and IL-6 (panel D) were measured. HK-2 cells were transfected with a control vector, plasmid for TREM-1 expression, control siRNA or TREM-1-specific siRNA as described in the Materials and Methods section. * P < 0.05 compared with control cells without LPS; # P < 0.05 compared with control cells with LPS. The data presented are a representative assessment of triplicate assays with similar results.

Article Snippet: For TREM-1 depletion studies, TREM-1-specific siRNA (Santa Cruz, Cat# sc-42999), control (CON) shRNA plasmid (Santa Cruz, Cat# sc-108060), TREM-1 cDNA ORF clone (Sino Bio, Cat# HG10511-UT), or negative control vector (Sino Bio, Cat# CV011) was transfected into HK-2 cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Plasmid Preparation, shRNA, Transfection, Expressing

Journal: International Journal of Medical Sciences

Article Title: TREM-1 promoted apoptosis and inhibited autophagy in LPS-treated HK-2 cells through the NF-κB pathway

doi: 10.7150/ijms.50893

Figure Lengend Snippet: LPS-induced HK-2 cell apoptosis was mediated by TREM-1. HK-2 cells were transfected with a plasmid containing a negative control vector/TREM-1 gene or negative control shRNA/TREM-1-specific shRNA, followed by exposure to LPS (10 µg/mL). Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-9 were assessed by immunoblotting as described in the Materials and Methods section. Panel A: Representative images showing the results of immunoblotting. Panel B: Average of three separate immunoblots for Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-9. Vertical axis: ratio to control; horizontal axis: cells transfected with plasmids containing negative control vector/TREM-1 gene or negative control shRNA/TREM-1 shRNA. * P< 0.05 .

Article Snippet: For TREM-1 depletion studies, TREM-1-specific siRNA (Santa Cruz, Cat# sc-42999), control (CON) shRNA plasmid (Santa Cruz, Cat# sc-108060), TREM-1 cDNA ORF clone (Sino Bio, Cat# HG10511-UT), or negative control vector (Sino Bio, Cat# CV011) was transfected into HK-2 cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Transfection, Plasmid Preparation, Negative Control, shRNA, Western Blot

Journal: International Journal of Medical Sciences

Article Title: TREM-1 promoted apoptosis and inhibited autophagy in LPS-treated HK-2 cells through the NF-κB pathway

doi: 10.7150/ijms.50893

Figure Lengend Snippet: LPS-induced HK-2 cell autophagy was mediated by TREM-1. HK-2 cells were transfected with a plasmid containing a negative control vector/TREM-1 gene or negative control shRNA/TREM-1-specific shRNA, followed by exposure to LPS (10 µg/mL). Beclin-1, Atg-5, LC-3b, and p62 were assessed by immunoblotting as described in the Materials and Methods section. Panel A: Representative images of immunoblots. Panel B: Averages of three separate immunoblots for Beclin-1, Atg-5, LC-3b, and p62. Vertical axis: ratio to control; horizontal axis: cells transfected with plasmids containing a negative control vector/TREM-1 gene or negative control shRNA/TREM-1-specific shRNA. * P< 0.05 .

Article Snippet: For TREM-1 depletion studies, TREM-1-specific siRNA (Santa Cruz, Cat# sc-42999), control (CON) shRNA plasmid (Santa Cruz, Cat# sc-108060), TREM-1 cDNA ORF clone (Sino Bio, Cat# HG10511-UT), or negative control vector (Sino Bio, Cat# CV011) was transfected into HK-2 cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Transfection, Plasmid Preparation, Negative Control, shRNA, Western Blot

Journal: International Journal of Medical Sciences

Article Title: TREM-1 promoted apoptosis and inhibited autophagy in LPS-treated HK-2 cells through the NF-κB pathway

doi: 10.7150/ijms.50893

Figure Lengend Snippet: The NF-κB signaling pathway in LPS-treated HK-2 cells was regulated by TREM-1. HK-2 cells were transfected with plasmid containing a negative control vector/TREM-1 gene or negative control shRNA/TREM-1-specific shRNA, followed by exposure to LPS (10 µg/mL). P-p65, p65, P-IκBα, and IκBα were assessed by immunoblotting as described in the Materials and Methods section. Panel A: Representative images of immunoblots. Panel B: Average of three separate immunoblots for P-p65/p65 and P-IκBα/IκBα. Vertical axis: ratio to control; horizontal axis: cells transfected with plasmids containing negative control vector/TREM-1 gene or negative control shRNA/TREM-1 shRNA. * P< 0.05 .

Article Snippet: For TREM-1 depletion studies, TREM-1-specific siRNA (Santa Cruz, Cat# sc-42999), control (CON) shRNA plasmid (Santa Cruz, Cat# sc-108060), TREM-1 cDNA ORF clone (Sino Bio, Cat# HG10511-UT), or negative control vector (Sino Bio, Cat# CV011) was transfected into HK-2 cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Transfection, Plasmid Preparation, Negative Control, shRNA, Western Blot

Journal: Advanced Science

Article Title: Deletion of Pyruvate Carboxylase in Tubular Epithelial Cell Promotes Renal Fibrosis by Regulating SQOR/cGAS/STING‐Mediated Glycolysis

doi: 10.1002/advs.202408753

Figure Lengend Snippet: Pyruvate carboxylase (PC) knockout increased glycolysis levels. A,B) Targeted metabolomics showed the metabolites of glycolysis in HK‐2 cells of the WT+TGF‐β1 and PCKO+TGF‐β1 groups and mouse kidney of Pcx flox/flox +UUO and Pcx cKO +UUO groups. The color in the heatmaps from blue to red shows the progression from low expression to high expression, respectively. C) Mitochondrial extracellular acidification rate (ECAR) in HK‐2 cells of different groups ( n = 6/ea.). D) Basal glycolysis, maximal glycolysis, and glycolytic reserve in HK‐2 cells of different groups ( n = 6/ea.). E) Mitochondrial oxygen consumption rate (OCR) in HK‐2 cells of different groups ( n = 6/ea.). F) Basal respiration, maximal respiration, and spare respiratory capacity in HK‐2 cells of different groups ( n = 6/ea.). G,H) Western blot analysis and densitometric quantification of HK2, PKM2, ATP5A, UQCRC2, MTCO1, SDHB, and NUDFB8 kidney tissues from control and Pcx cKO mice subjected to sham operation or 14 days after the UUO operation ( n = 3/ea.). I–L) Enzymatic activity assay results of hexokinase (HK), pyruvate kinase (PK), succinate dehydrogenase (SDH), and mitochondrial complex III in kidney tissues from control and Pcx cKO mice subjected to sham operation or 14 days after the UUO operation ( n = 6/ea.). Results are expressed as mean ± SEM. * P < 0.05; ** P < 0.01; and *** P < 0.001; ns, non‐significant. WT, wildtype.

Article Snippet: Control or PC knockdown HK‐2 cells were treated with cycloheximide (catalog S7418, Selleck, USA) or DMSO for the indicated durations (0, 1, 3, and 6 h).

Techniques: Knock-Out, Expressing, Western Blot, Control, Enzyme Activity Assay