Journal: Cell Death & Disease

Article Title: LincRNA-Gm4419 knockdown ameliorates NF- κ B/NLRP3 inflammasome-mediated inflammation in diabetic nephropathy

doi: 10.1038/cddis.2016.451

Figure Lengend Snippet: Gm4419 regulates inflammation, fibrosis and proliferation of MCs under high-glucose condition. ( a ) The mRNA levels of pro-inflammatory cytokines (mcp-1, TNF- α and IL-1 β ) and fibrosis biomarkers (Fn and Col.IV) in MCs that were stimulated by high or low glucose by qRT-PCR analysis. ( b ) The mRNA levels of pro-inflammatory cytokines and fibrosis biomarkers by qRT-PCR analysis in cells overexpressed or downexpressed Gm4419. ( c ) The protein levels of pro-inflammatory cytokines and fibrosis biomarkers in untransfected or transfected MCs by western blot and quantitative analysis. ( d ) The expressions of pro-inflammatory cytokines and fibrosis biomarkers in untransfected or transfected MCs by immunofluorescent (× 400). ( e ) Proliferative capability of untransfecting or transfecting MCs was analyzed by EdU (5-ethynyl-2′-deoxyuridine) incorporation assay, and the EdU incorporation rate was shown as the ratio of EdU-positive cells to total Hoechst33342-positive cells. ( f ) Untransfected or transfected MCs were analyzed by flow cytometry and quantitative analysis. The percentage of cells in the G0/G1, S and G2/M phases of the cell cycle were calculated. The data are representative of the results of three independent experiments, and the data were presented as means±S.E.M. (* P <0.05, ** P <0.01, NS, no significant)

Article Snippet: Both non-transfected and transfected MCs were fixed with 4% paraformaldehyde for 30 min at room temperature and then permeabilized in PBS containing 0.1% Triton X-100 on ice for 10 min. And then samples were confined by 3% goat serum (Beyotime, Nantong, China) for 1 h at room temperature and incubated by overnight at 4 °C using anti-p50 antibody (Abcam, 1:100), anti-p65 antibody (Abcam, 1:100), anti-NLRP3 inflammasome antibody (Sangon Bio Tech, 1:50), anti-mcp-1 antibody (Sangon Bio Tech, 1:50), anti-TNF- α antibody (Bioworld Tech, Minnesote, CA, USA, 1:50), anti-IL-1 β antibody (Bioworld Tech, 1:50), anti-Fn antibody (Sangon Bio Tech, 1:50) or anti-Col4 antibody (Proteintech, Wuhan, China, 1:50).

Techniques: Quantitative RT-PCR, Transfection, Western Blot, Flow Cytometry

Journal: iScience

Article Title: Tmem30a protects against podocyte injury through suppression of pyroptosis

doi: 10.1016/j.isci.2024.109976

Figure Lengend Snippet: Tmem30a expression in patients with FSGS and controls (A) Overview of the bioinformatics analysis flow and collected renal tissues from clinical patients. (B and C) Heatmap of DEGs between the FSGS and Ctrl groups in the GSE121233 and GSE129973 datasets. (D and E) Tmem30a expression levels in FSGS and Ctrl groups in the GSE121233 and GSE129973 datasets. n = 5/group. Data are represented as mean ± SEM. (F) PAS and Masson's trichrome staining of kidney tissue in FSGS group, IgAN group, and Ctrl group (scale bar: 20 μm). (G) Transmission electron microscopy images of kidney tissues in patients with FSGS and IgAN. Black arrowheads: foot process; red arrowheads: membrane disruption. (H) Immunofluorescence images of Tmem30a expression in glomerular of FSGS group, IgAN group, and Ctrl group (scale bar: 20 μm). DEG, differentially expressed gene; Ctrl, control; FSGS, focal segmental glomerulosclerosis; IgAN, IgA nephropathy; PAS, periodic acid-Schiff; Tmem30a, transmembrane protein 30a. ∗∗∗ p < 0.001.

Article Snippet: The membranes were blocked with 5% nonfat milk or 5% bovine serum albumin in Tris buffered-saline with Tween 20 for 2 h and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-Tmem30a (Cat#: bs-16576R-1, Bioss, 1:1000), rabbit anti-Nephrin (Cat#: NBP1-77303, Novusbio, 1:2000), rabbit anti-WT1 (Cat#: 12609-1-AP, Proteintech, 1:500), rabbit anti-NLRP3 (Cat#: ab214185, Abcam, 1:500), rabbit anti caspase-1 (Cat#: ab179515, Abcam, 1:1000), rabbit anti-GSDMD (Cat#: ab209845, Abcam, 1:1000), and rabbit anti-IL-1β (Cat#: 516288, ZEN BIO, 1:1000).

Techniques: Expressing, Staining, Transmission Assay, Electron Microscopy, Membrane, Disruption, Immunofluorescence

Journal: iScience

Article Title: Tmem30a protects against podocyte injury through suppression of pyroptosis

doi: 10.1016/j.isci.2024.109976

Figure Lengend Snippet: Tmem30a expression levels in mice and cell models of ADR-induced podocyte injury (A) Construction of the ADR-induced mice and cell models. (B) PAS staining of kidney tissue in ADR-induced mice and WT mice (scale bar: 20 μm). (C) UACR levels in ADR-induced mice and WT mice. n = 5/group. Data are represented as mean ± SEM. (D) Transmission electron microscopy images of glomeruli in ADR-induced mice and WT mice. (E) Representative images of immunohistochemical staining for Tmem30a in ADR-induced mice and WT mice (scale bar: 20 μm). (F) ADR was used at different concentrations and time points to observe mouse podocyte viability. n = 3/group. Data are represented as mean ± SEM. (G) The expression of Tmem30a, nephrin, and WT1 in ADR-induced mouse podocytes and normal mouse podocytes using western blotting. (H–J) Immunoblot quantification of nephrin, WT1, and Tmem30a in (G). n = 3/group. Data are represented as mean ± SEM. ADR, adriamycin; WT, wild type; Ctrl, control; PAS, periodic acid-Schiff; Tmem30a, transmembrane protein 30a; UACR, urinary albumin/creatinine ratio; WT1, Wilms tumor 1. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The membranes were blocked with 5% nonfat milk or 5% bovine serum albumin in Tris buffered-saline with Tween 20 for 2 h and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-Tmem30a (Cat#: bs-16576R-1, Bioss, 1:1000), rabbit anti-Nephrin (Cat#: NBP1-77303, Novusbio, 1:2000), rabbit anti-WT1 (Cat#: 12609-1-AP, Proteintech, 1:500), rabbit anti-NLRP3 (Cat#: ab214185, Abcam, 1:500), rabbit anti caspase-1 (Cat#: ab179515, Abcam, 1:1000), rabbit anti-GSDMD (Cat#: ab209845, Abcam, 1:1000), and rabbit anti-IL-1β (Cat#: 516288, ZEN BIO, 1:1000).

Techniques: Expressing, Staining, Transmission Assay, Electron Microscopy, Immunohistochemical staining, Western Blot, Wilms Tumor Assay

Journal: iScience

Article Title: Tmem30a protects against podocyte injury through suppression of pyroptosis

doi: 10.1016/j.isci.2024.109976

Figure Lengend Snippet: Construction of podocyte-specific Tmem30a lox P /loxP ; NPHS2-Cre mice and Tmem30a KD mouse podocytes (A) Flowchart of the construction of podocyte-specific Tmem30a lox P /loxP ; NPHS2-Cre mice and Tmem30a KD mouse podocytes. (B) PAS staining of kidney sections in Tmem30a lox P /loxP ; NPHS2-Cre mice and WT mice (scale bar: 20 μm). (C) Representative immunofluorescence staining images of Tmem30a in kidney samples from Tmem30a lox P /loxP ; NPHS2-Cre mice and WT mice (scale bar: 20 μm). (D) Real-time PCR showing the relative mRNA expression of Tmem30a in the Ctrl group, NC group and Tmem30a KD group in vitro . n = 3/group. Data are represented as mean ± SEM. (E) The expression of nephrin, WT1, and Tmem30a in the Ctrl group, NC group and Tmem30a KD group using western blotting. (F–H) Immunoblot quantification of nephrin, WT1, and Tmem30a in (E). n = 3/group. Data are represented as mean ± SEM. Ctrl, control; NC, negative control; KD, knockdown; Real-time PCR, reverse-transcription polymerase chain reaction; Tmem30a, transmembrane protein 30a; WT, wild type; WT1, Wilms tumor 1. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: The membranes were blocked with 5% nonfat milk or 5% bovine serum albumin in Tris buffered-saline with Tween 20 for 2 h and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-Tmem30a (Cat#: bs-16576R-1, Bioss, 1:1000), rabbit anti-Nephrin (Cat#: NBP1-77303, Novusbio, 1:2000), rabbit anti-WT1 (Cat#: 12609-1-AP, Proteintech, 1:500), rabbit anti-NLRP3 (Cat#: ab214185, Abcam, 1:500), rabbit anti caspase-1 (Cat#: ab179515, Abcam, 1:1000), rabbit anti-GSDMD (Cat#: ab209845, Abcam, 1:1000), and rabbit anti-IL-1β (Cat#: 516288, ZEN BIO, 1:1000).

Techniques: Staining, Immunofluorescence, Real-time Polymerase Chain Reaction, Expressing, In Vitro, Western Blot, Negative Control, Reverse Transcription, Polymerase Chain Reaction, Wilms Tumor Assay

Journal: iScience

Article Title: Tmem30a protects against podocyte injury through suppression of pyroptosis

doi: 10.1016/j.isci.2024.109976

Figure Lengend Snippet: MCC950 alleviates podocyte injury by inhibiting ADR-induced pyroptosis (A) Bioinformatic analysis flowchart between normal human podocytes and ADR-induced human podocytes, and construct ADR-induced mouse podocytes. (B) Heatmap of DEGs in ADR-induced human podocytes and normal human podocytes in the GSE124622 dataset. (C) Cell viability of mouse podocytes after ADR treatment for 24 h and then MCC950 treatment for 48 h. n = 3/group. Data are represented as mean ± SEM. (D) The expression levels of nephrin, WT1, Tmem30a, NLRP3, pro-caspase-1, cleaved caspase-1, GSDMD, N-GSDMD, pro-IL-1β, and IL-1β in the Ctrl group, ADR group, MCC950 group, and ADR+MCC950 group using western blotting. (E–K) Immunoblot quantification of nephrin, WT1, Tmem30a, NLRP3, cleaved caspase-1/pro-caspase-1, N-GSDMD/GSDMD, and IL-1β/pro-IL-1β in (D). n = 3/group. Data are represented as mean ± SEM. ADR, adriamycin; Ctrl, control; DEG, differentially expressed gene; GSDMD, gasdermin D; NLRP3, NLR family pyrin domain containing 3; Tmem30a, transmembrane protein 30a; WT1, Wilms tumor 1. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: The membranes were blocked with 5% nonfat milk or 5% bovine serum albumin in Tris buffered-saline with Tween 20 for 2 h and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-Tmem30a (Cat#: bs-16576R-1, Bioss, 1:1000), rabbit anti-Nephrin (Cat#: NBP1-77303, Novusbio, 1:2000), rabbit anti-WT1 (Cat#: 12609-1-AP, Proteintech, 1:500), rabbit anti-NLRP3 (Cat#: ab214185, Abcam, 1:500), rabbit anti caspase-1 (Cat#: ab179515, Abcam, 1:1000), rabbit anti-GSDMD (Cat#: ab209845, Abcam, 1:1000), and rabbit anti-IL-1β (Cat#: 516288, ZEN BIO, 1:1000).

Techniques: Construct, Expressing, Western Blot, Wilms Tumor Assay

Journal: iScience

Article Title: Tmem30a protects against podocyte injury through suppression of pyroptosis

doi: 10.1016/j.isci.2024.109976

Figure Lengend Snippet: DSF alleviates podocyte injury by inhibiting ADR-induced pyroptosis (A) Constructed ADR-induced mouse podocytes and treated with DSF. (B) Cell viability of mouse podocytes after ADR treatment for 24 h and DSF treatment for 1 h. n = 3/group. Data are represented as mean ± SEM. (C) The expression levels of nephrin, WT1, Tmem30a, NLRP3, pro-caspase-1, cleaved caspase-1, GSDMD, N-GSDMD, pro-IL-1β, and IL-1β in the Ctrl group, ADR group, DSF group, and ADR+DSF group using western blotting. (D–J) Immunoblot quantification of nephrin, WT1, Tmem30a, NLRP3, cleaved caspase-1/pro-caspase-1, N-GSDMD/GSDMD, and IL-1β/pro-IL-1β in (C). n = 3/group. Data are represented as mean ± SEM. ADR, adriamycin; Ctrl, control; GSDMD, gasdermin D; NLRP3, NLR family pyrin domain containing 3; Tmem30a, transmembrane protein 30a; WT1, Wilms tumor 1. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: The membranes were blocked with 5% nonfat milk or 5% bovine serum albumin in Tris buffered-saline with Tween 20 for 2 h and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-Tmem30a (Cat#: bs-16576R-1, Bioss, 1:1000), rabbit anti-Nephrin (Cat#: NBP1-77303, Novusbio, 1:2000), rabbit anti-WT1 (Cat#: 12609-1-AP, Proteintech, 1:500), rabbit anti-NLRP3 (Cat#: ab214185, Abcam, 1:500), rabbit anti caspase-1 (Cat#: ab179515, Abcam, 1:1000), rabbit anti-GSDMD (Cat#: ab209845, Abcam, 1:1000), and rabbit anti-IL-1β (Cat#: 516288, ZEN BIO, 1:1000).

Techniques: Construct, Expressing, Western Blot, Wilms Tumor Assay

Journal: iScience

Article Title: Tmem30a protects against podocyte injury through suppression of pyroptosis

doi: 10.1016/j.isci.2024.109976

Figure Lengend Snippet: ADR aggravates podocyte injury and pyroptosis in Tmem30a KD mouse podocytes (A) Experimental design for ADR intervention in Tmem30a KD mouse podocytes. (B) Expression levels of nephrin, WT1, Tmem30a, NLRP3, pro-caspase-1, cleaved caspase-1, GSDMD, N-GSDMD, pro-IL-1β, and IL-1β in the Ctrl group, NC group, Tmem30a KD group, Ctrl + ADR group, NC + ADR group, and Tmem30a KD + ADR group using western blotting. (C–I) Immunoblot quantification of nephrin, WT1, Tmem30a, NLRP3, cleaved caspase-1/pro-caspase-1, N-GSDMD/GSDMD, and IL-1β/pro-IL-1β in (B). n = 3/group. Data are represented as mean ± SEM. ADR, adriamycin; Ctrl, control; KD, knockdown; GSDMD, gasdermin D; NC, negative control; NLRP3, NLR family pyrin domain containing 3; Tmem30a, transmembrane protein 30a; WT1, Wilms tumor 1. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: The membranes were blocked with 5% nonfat milk or 5% bovine serum albumin in Tris buffered-saline with Tween 20 for 2 h and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-Tmem30a (Cat#: bs-16576R-1, Bioss, 1:1000), rabbit anti-Nephrin (Cat#: NBP1-77303, Novusbio, 1:2000), rabbit anti-WT1 (Cat#: 12609-1-AP, Proteintech, 1:500), rabbit anti-NLRP3 (Cat#: ab214185, Abcam, 1:500), rabbit anti caspase-1 (Cat#: ab179515, Abcam, 1:1000), rabbit anti-GSDMD (Cat#: ab209845, Abcam, 1:1000), and rabbit anti-IL-1β (Cat#: 516288, ZEN BIO, 1:1000).

Techniques: Expressing, Western Blot, Negative Control, Wilms Tumor Assay

Journal: iScience

Article Title: Tmem30a protects against podocyte injury through suppression of pyroptosis

doi: 10.1016/j.isci.2024.109976

Figure Lengend Snippet: MCC950 alleviates podocyte injury by inhibiting Tmem30a KD induced pyroptosis (A) Experimental design for MCC950 intervention in Tmem30a KD mouse podocytes. (B) Expression levels of nephrin, WT1, Tmem30a, NLRP3, pro-caspase-1, cleaved caspase-1, GSDMD, N-GSDMD, pro-IL-1β, and IL-1β in the Ctrl group, NC group, Tmem30a KD group, Ctrl + MCC950 group, NC + MCC950 group, and Tmem30a KD + MCC950 group using western blotting. (C–I) Immunoblot quantification of nephrin, WT1, Tmem30a, NLRP3, cleaved caspase-1/pro-caspase-1, N-GSDMD/GSDMD, and IL-1β/pro-IL-1β in (B). n = 3/group. Data are represented as mean ± SEM. Ctrl, control; NC, negative control; KD, knockdown; GSDMD, gasdermin D; NLRP3, NLR family pyrin domain containing 3; Tmem30a, transmembrane protein 30a; WT1, Wilms tumor 1. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: The membranes were blocked with 5% nonfat milk or 5% bovine serum albumin in Tris buffered-saline with Tween 20 for 2 h and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-Tmem30a (Cat#: bs-16576R-1, Bioss, 1:1000), rabbit anti-Nephrin (Cat#: NBP1-77303, Novusbio, 1:2000), rabbit anti-WT1 (Cat#: 12609-1-AP, Proteintech, 1:500), rabbit anti-NLRP3 (Cat#: ab214185, Abcam, 1:500), rabbit anti caspase-1 (Cat#: ab179515, Abcam, 1:1000), rabbit anti-GSDMD (Cat#: ab209845, Abcam, 1:1000), and rabbit anti-IL-1β (Cat#: 516288, ZEN BIO, 1:1000).

Techniques: Expressing, Western Blot, Negative Control, Wilms Tumor Assay

Journal: iScience

Article Title: Tmem30a protects against podocyte injury through suppression of pyroptosis

doi: 10.1016/j.isci.2024.109976

Figure Lengend Snippet: DSF alleviates podocyte injury by inhibiting Tmem30a KD induced pyroptosis (A) Experimental design for DSF intervention in Tmem30a KD mouse podocytes. (B) Expression levels of nephrin, WT1, Tmem30a, NLRP3, pro-caspase-1, cleaved caspase-1, GSDMD, N-GSDMD, pro-IL-1β, and IL-1β in the Ctrl group, NC group, Tmem30a KD group, Ctrl + DSF group, NC + DSF group, and Tmem30a KD + DSF group using western blotting. (C–I) Immunoblot quantification of nephrin, WT1, Tmem30a, NLRP3, cleaved caspase-1/pro-caspase-1, N-GSDMD/GSDMD, and IL-1β/pro-IL-1β in (B). n = 3/group. Data are represented as mean ± SEM. Ctrl, control; NC, negative control; KD, knockdown; GSDMD, gasdermin D; NLRP3, NLR family pyrin domain containing 3; Tmem30a, transmembrane protein 30a; WT1, Wilms tumor 1. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: The membranes were blocked with 5% nonfat milk or 5% bovine serum albumin in Tris buffered-saline with Tween 20 for 2 h and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-Tmem30a (Cat#: bs-16576R-1, Bioss, 1:1000), rabbit anti-Nephrin (Cat#: NBP1-77303, Novusbio, 1:2000), rabbit anti-WT1 (Cat#: 12609-1-AP, Proteintech, 1:500), rabbit anti-NLRP3 (Cat#: ab214185, Abcam, 1:500), rabbit anti caspase-1 (Cat#: ab179515, Abcam, 1:1000), rabbit anti-GSDMD (Cat#: ab209845, Abcam, 1:1000), and rabbit anti-IL-1β (Cat#: 516288, ZEN BIO, 1:1000).

Techniques: Expressing, Western Blot, Negative Control, Wilms Tumor Assay

Journal: iScience

Article Title: Tmem30a protects against podocyte injury through suppression of pyroptosis

doi: 10.1016/j.isci.2024.109976

Figure Lengend Snippet:

Article Snippet: The membranes were blocked with 5% nonfat milk or 5% bovine serum albumin in Tris buffered-saline with Tween 20 for 2 h and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-Tmem30a (Cat#: bs-16576R-1, Bioss, 1:1000), rabbit anti-Nephrin (Cat#: NBP1-77303, Novusbio, 1:2000), rabbit anti-WT1 (Cat#: 12609-1-AP, Proteintech, 1:500), rabbit anti-NLRP3 (Cat#: ab214185, Abcam, 1:500), rabbit anti caspase-1 (Cat#: ab179515, Abcam, 1:1000), rabbit anti-GSDMD (Cat#: ab209845, Abcam, 1:1000), and rabbit anti-IL-1β (Cat#: 516288, ZEN BIO, 1:1000).

Techniques: Recombinant, Saline, Lysis, Western Blot, Marker, Membrane, Immunohistochemistry, Protein Concentration, Staining, Modification, Microarray, Software

Journal: Foods

Article Title: Soluble Dietary Fiber of Hawthorn Relieves Constipation Induced by Loperamide Hydrochloride by Improving Intestinal Flora and Inflammation, Thereby Regulating the Aquaporin Ion Pathway in Mice

doi: 10.3390/foods13142220

Figure Lengend Snippet: The effect of HSDF on the expression of inflammatory factor proteins in various groups of mice. ( A ) WB results of the expressions of inflammatory factors IL-6, IL-1β, and TNF-α. ( B ) Relative expression of TNF-α in each group. ( C ) Relative expression of IL-1β in each group. ( D ) Relative expression of IL-6 in each group. C: blank control group; M: loperamide hydrochloride model group; HS: maren soft capsule (0.4 g/kg bw); LHDF: low-dose HSDF (1.0 g/kg bw); HHDF: high-dose HSDF (2.0 g/kg bw). Results are expressed as mean ± SD ( n = 3) **** p < 0.0001, ** p < 0.01, * p < 0.05.

Article Snippet: Male Kunming (KM) 3-week-old mice were obtained from Beijing Vital River laboratory animal technology Co., Ltd., Beijing, China; loperamide hydrochloride from Xian Janssen Pharmaceutical Ltd., Xi’an, China; maren soft capsules from the Central Pharmaceutical Co., Tianjin, China; ELISA kits NO(AKNM005M), MDA(AKFA013M), MTL(EKMO25001), GAS(EKMO25002), VIP(EKMO1033), SP(EKMO29001), and SS(EKMO30011) from Boxbio, Beijing, China; BCA protein concentration determination kit (P0010) from Beyotime Biotechnology, China, Shanghai, China; mouse McAb β-Action (66009-1 Ig) from Proteintech Group, Inc., Wuhan, China; rabbit polyclonal antibody AQP3 (BS3671), rabbit polyclonal antibody AQP8 (BS71,279), rabbit polyclonal antibody AQP9 (BS71,280), rabbit polyclonal antibody IL-1β (A00101-1), rabbit polyclonal antibody IL-6 (BA4339-2), and rabbit polyclonal antibody TNF-α (A00002-2) from NovaTeinbio, Inc., Boston, MA, USA; HRP-labeled sheep anti-mouse secondary antibody (BA1051) and HRP-labeled sheep anti-rabbit secondary antibody (BA1054) from Wuhan Boster Biological Technology, Ltd, Wuhan, China; acetic, propionic, butyric, isobutyric, valeric, isovaleric, caproic, and isohexanoic acids (standard) from sigma, Shanghai, China; and α-amylase (100,000 U/g), glucoamylase (100,000 U/g), neutral protease (100,000 U/g), glucosidase (200 U/g), and cellulase (10,000 U/g) from Shanghai yuanye Bio Technology Co., Ltd., Shanghai, China.

Techniques: Expressing, Control