Journal: Frontiers in Immunology

Article Title: TLR3 and GLUL orchestrate inflammatory and homeostatic imbalance in osteoarthritis

doi: 10.3389/fimmu.2025.1650375

Figure Lengend Snippet: Expression profiling and correlation analysis of NAFRGs in osteoarthritis. (A) Heatmap showing differentially expressed genes between OA and healthy control samples in the test datasets ( GSE51588 and GSE117999 ). (B) Correlation matrix of differentially expressed NAFRGs, indicating potential gene–gene interactions relevant to OA pathogenesis.

Article Snippet: Primary antibodies used included: β-actin (1:10,000, Proteintech), IL-1β (1:2,000, Affinity), TNF-α (1:2,000, Affinity), IL-10 (1:2,000, Affinity).

Techniques: Expressing, Control

Journal: Frontiers in Immunology

Article Title: TLR3 and GLUL orchestrate inflammatory and homeostatic imbalance in osteoarthritis

doi: 10.3389/fimmu.2025.1650375

Figure Lengend Snippet: Functional enrichment analysis of differentially expressed genes in OA and NAFRGs. (A) Gene Ontology (GO) analysis of OA-related differentially expressed genes. In biological processes (BP), genes were enriched in leukocyte migration, humoral immune response, and neutrophil migration. In cellular components (CC), enrichment was observed in cytoplasmic vesicle lumen, azurophil granule lumen, and related structures. In molecular functions (MF), genes were associated with receptor–ligand activity and cytokine receptor binding. (B) KEGG pathway analysis of OA-related genes showing enrichment in cytokine–cytokine receptor interaction, chemokine signaling pathway, neutrophil extracellular trap formation, viral protein interactions with cytokines and cytokine receptors, and nitrogen metabolism. (C) GO analysis of differentially expressed NAFRGs. BP terms included cellular response to external stimulus and vascular transport; CC terms included basal plasma membrane and plasma membrane raft; MF terms included iron ion binding and NADP binding. (D) KEGG pathway analysis of NAFRGs showing enrichment in the HIF-1 signaling pathway, central carbon metabolism in cancer, glutathione metabolism, and ferroptosis.

Article Snippet: Primary antibodies used included: β-actin (1:10,000, Proteintech), IL-1β (1:2,000, Affinity), TNF-α (1:2,000, Affinity), IL-10 (1:2,000, Affinity).

Techniques: Functional Assay, Migration, Activity Assay, Binding Assay, Clinical Proteomics, Membrane

Journal: Frontiers in Immunology

Article Title: TLR3 and GLUL orchestrate inflammatory and homeostatic imbalance in osteoarthritis

doi: 10.3389/fimmu.2025.1650375

Figure Lengend Snippet: Validation and functional analysis of TLR3 in osteoarthritis. (A) Immunohistochemical staining of TLR3 expression in cartilage and synovial tissues from OA patients and healthy controls. (B) Quantitative expression of TLR3 in cartilage tissues in Figure 6A . (C) Quantitative expression of TLR3 in synovial tissues in Figure 6A . (D) Fluorescence microscopy images showing transduction efficiency of three TLR3-targeting shRNAs in HC-a cells; shRNA-2 exhibited the strongest knockdown effect. (E) Western blot analysis confirming reduced TLR3 expression following shRNA-2 transduction in HC-a cells. (F) Quantitative expression of intensity ration of TLR3/β-actin. (G–I) ELISA results showing the levels of IL-1β, TNF-α, and IL-10 in the supernatant of four experimental groups. (J) Western blot analysis of inflammatory cytokine protein levels (IL-1β, TNF-α, IL-10) in the same groups. (K, L) Colony formation assays comparing proliferative capacity among control, OA model, and TLR3 knockdown groups. (M) TUNEL staining to assess apoptosis in chondrocytes across experimental groups. (N) Alcian Blue staining to evaluate acidic mucopolysaccharide content and cartilage matrix integrity. (O) Alizarin Red staining to assess calcium deposition and mineralized nodule formation in chondrocytes. Quantitative data are presented as mean ± standard deviation. Statistical comparisons were made using one-way ANOVA followed by post hoc tests. P < 0.05 was considered statistically significant.

Article Snippet: Primary antibodies used included: β-actin (1:10,000, Proteintech), IL-1β (1:2,000, Affinity), TNF-α (1:2,000, Affinity), IL-10 (1:2,000, Affinity).

Techniques: Biomarker Discovery, Functional Assay, Immunohistochemical staining, Staining, Expressing, Fluorescence, Microscopy, Transduction, shRNA, Knockdown, Western Blot, Enzyme-linked Immunosorbent Assay, Control, TUNEL Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: TLR3 and GLUL orchestrate inflammatory and homeostatic imbalance in osteoarthritis

doi: 10.3389/fimmu.2025.1650375

Figure Lengend Snippet: Validation and functional analysis of GLUL in osteoarthritis in patient samples and three cell lines. (A) Immunohistochemical staining of GLUL expression in cartilage and synovial tissues from OA patients and healthy controls. (B) Quantitative expression of GLUL in cartilage tissues in Figure 7A . (C) Quantitative expression of GLUL in synovial tissues in Figure 7A . (D) Fluorescence microscopy images showing transduction efficiency of GLUL-overexpression in HC-a cells. (E) Western blot analysis shows the overexpression of GLUL in HC-a cells. (F) Quantitative expression of intensity ration of GLUL/β-actin. (G–I) ELISA results showing the levels of IL-1β, TNF-α, and IL-10 in the supernatant of four experimental groups. (J) Western blot analysis of inflammatory cytokine protein levels (IL-1β, TNF-α, IL-10) in the same groups. (K, L) Quantitative analysis of J. (M–O) Colony formation assays comparing proliferative capacity among control, OA model, and GLUL overexpression groups. (P) TUNEL staining to assess apoptosis in chondrocytes across experimental groups. (Q) Alcian Blue staining to evaluate acidic mucopolysaccharide content and cartilage matrix integrity by using HC-a cell line. (R) Alizarin Red staining to assess calcium deposition and mineralized nodule formation in chondrocytes by using HC-a cell line. (S, T) RT-qPCR analysis of TLR3 and GLUL mRNA in OA-like chondrocytes after TLR3 knockdown (TLR3i) or PRP treatment. (S) TLR3 expression is significantly reduced in TLR3i and PRP groups compared to OA controls. (T) GLUL expression is significantly increased following TLR3 knockdown and also elevated by PRP treatment, indicating TLR3 negatively regulates GLUL in chondrocytes. Data are mean ± SD; *p < 0.05, p < 0.01 vs OA group.

Article Snippet: Primary antibodies used included: β-actin (1:10,000, Proteintech), IL-1β (1:2,000, Affinity), TNF-α (1:2,000, Affinity), IL-10 (1:2,000, Affinity).

Techniques: Biomarker Discovery, Functional Assay, Immunohistochemical staining, Staining, Expressing, Fluorescence, Microscopy, Transduction, Over Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Control, TUNEL Assay, Quantitative RT-PCR, Knockdown

Journal: Frontiers in Immunology

Article Title: TLR3 and GLUL orchestrate inflammatory and homeostatic imbalance in osteoarthritis

doi: 10.3389/fimmu.2025.1650375

Figure Lengend Snippet: In vivo validation of the TLR3/GLUL axis and therapeutic intervention in an OA rat model. (A) Schematic illustration of osteoarthritis (OA) induction in Sprague-Dawley rats (~200 g) via anterior cruciate ligament transection (ACLT). Rats were anesthetized using sodium pentobarbital (40–60 mg/kg, i.p.), and a longitudinal incision was made to expose the knee joint. The anterior cruciate ligament was transected, followed by wound closure and postoperative care. OA was established over a 6–8 weeks period. (B) Assessment of knee joint range of motion from week 0 to week 8 in sham, model, TLR3 inhibitor, and PRP groups. (C) Measurement of knee joint circumference across the 8-week period. (D) Quantification of hind limb stride length as a measure of joint mobility. (E) Step counts recorded during a 20-second locomotor activity test. (F) OARSI scores of knee joints from sham, OA model, TLR3 inhibitor, and PRP-treated groups. OA model group showed significantly higher scores compared with sham controls, indicating cartilage degeneration. Treatment with TLR3 inhibitor or PRP reduced OARSI scores, demonstrating protective effects on cartilage integrity. (G–I) Serum cytokine levels assessed by ELISA, including IL-1β (G) , TNF-α (H) , and IL-10 (I) . (J) Western blot analysis of inflammatory and anti-inflammatory protein expression in joint tissues. (K–M) Protein expression levels of COX2 (K) , TNF-α (M) , and IL-1β (M) in joint tissues. (O, P) IL-10 protein expression in joint tissues assessed by Western blot (O) and corresponding quantification (P) . (P–R) Expression of MMP-13 (Q) and MMP-3 (R) in joint tissues as detected by Western blot. (S) Protein expression of TLR3 and GLUL in joint tissues analyzed by Western blot. (T, U) Quantitative analysis of TLR3 (T) and GLUL (U) expression levels. (V) Representative immunohistochemistry images of TLR3 expression in knee joint tissues from sham, OA model, TLR3 knockdown, and PRP-treated groups. The images show localization and relative intensity of TLR3 expression in cartilage and synovium. Scale bars: 100 μm. Data are presented as mean ± SD; Statistical significance: P < 0.05, P < 0.01, P < 0.001; comparisons were made among sham, OA model, TLR3 inhibitor, and PRP-treated groups using one-way ANOVA followed by post hoc testing.

Article Snippet: Primary antibodies used included: β-actin (1:10,000, Proteintech), IL-1β (1:2,000, Affinity), TNF-α (1:2,000, Affinity), IL-10 (1:2,000, Affinity).

Techniques: In Vivo, Biomarker Discovery, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunohistochemistry, Knockdown

Journal: Frontiers in Immunology

Article Title: TLR3 and GLUL orchestrate inflammatory and homeostatic imbalance in osteoarthritis

doi: 10.3389/fimmu.2025.1650375

Figure Lengend Snippet: Graphical summary of the TLR3–GLUL axis in osteoarthritis. OA joints exhibit an inflammatory microenvironment characterized by elevated IL-1β, TNF-α, and ECM degradation. TLR3 activation triggers NF-κB signaling, leading to increased inflammatory cytokines, apoptosis, ECM breakdown, mineralization, and chondrocyte damage, accompanied by reduced GLUL. In vivo inhibition of TLR3 (CU-CPT4a) combined with PRP injection restores GLUL, suppresses IL-1β/TNF-α and MMP3/MMP13, elevates IL-10, and improves cartilage integrity, reflected by reduced OARSI scores.

Article Snippet: Primary antibodies used included: β-actin (1:10,000, Proteintech), IL-1β (1:2,000, Affinity), TNF-α (1:2,000, Affinity), IL-10 (1:2,000, Affinity).

Techniques: Activation Assay, In Vivo, Inhibition, Injection

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: Journal of Cellular and Molecular Medicine

Article Title: Phloretin attenuates hyperuricemia‐induced endothelial dysfunction through co‐inhibiting inflammation and GLUT 9‐mediated uric acid uptake

doi: 10.1111/jcmm.13176

Figure Lengend Snippet: Effects of UA on cell viability and inflammation in HUVEC s. ( A ) Cell viability of HUVEC s assayed by CCK 8. ( B‐C ) Real‐time PCR analysis of MCP ‐1 , VCAM ‐1 , ICAM ‐1 and IL ‐1 β mRNA . ( D ) Western blot of ICAM ‐1, IL ‐1β and MCP ‐1 protein, cell treated with UA for 24h. ( E ) Quantification analysis of protein level related to control (* P < 0.05, UA group versus control). HUVECs: human umbilical vein endothelial cells, NFκB/ERK: nuclear factor‐kappa B/extracellular regulated protein kinases.

Article Snippet: The membranes were blocked with 5% non‐fat milk and incubated with primary antibodies against p‐NFκB (1:1000, Abcam, MA, USA) and NFκB (1:500, Santa Cruz Biotechnology, CA, USA), GLUT9 (1:1000, Abcam), IL‐1β (1:500, ABclonal Biotechnology), OAT1 (1:1000, ABclonal ), URAT1 (1:1000,ABclonal) ICAM‐1 (1:1000, Cell Signaling Technology, MA, USA), MCP‐1 (1:1000, Abcam), VCAM‐1 (1:500, Proteintech, USA) overnight at 4°C.

Techniques: CCK-8 Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Phloretin attenuates hyperuricemia‐induced endothelial dysfunction through co‐inhibiting inflammation and GLUT 9‐mediated uric acid uptake

doi: 10.1111/jcmm.13176

Figure Lengend Snippet: Effect of phloretin on UA ‐induced inflammation in HUVEC s. ( A ) Cell viability of HUVEC s assayed by CCK 8, cell treated with phloretin for 24h. ( B‐C ) Real‐time PCR analysis of MCP ‐1 , VCAM ‐1 , ICAM ‐1 and IL ‐1 β mRNA , and cell treated with UA with or without phloretin for 24h. ( D ) Western blot and ( E ) quantification analysis of ICAM ‐1, IL ‐1β and MCP ‐1 protein (* P < 0.05, UA group versus control, # P < 0.05, phloretin group versus UA group). HUVECs: human umbilical vein endothelial cells, NFκB/ERK: nuclear factor‐kappa B/extracellular regulated protein kinases.

Article Snippet: The membranes were blocked with 5% non‐fat milk and incubated with primary antibodies against p‐NFκB (1:1000, Abcam, MA, USA) and NFκB (1:500, Santa Cruz Biotechnology, CA, USA), GLUT9 (1:1000, Abcam), IL‐1β (1:500, ABclonal Biotechnology), OAT1 (1:1000, ABclonal ), URAT1 (1:1000,ABclonal) ICAM‐1 (1:1000, Cell Signaling Technology, MA, USA), MCP‐1 (1:1000, Abcam), VCAM‐1 (1:500, Proteintech, USA) overnight at 4°C.

Techniques: CCK-8 Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Phloretin attenuates hyperuricemia‐induced endothelial dysfunction through co‐inhibiting inflammation and GLUT 9‐mediated uric acid uptake

doi: 10.1111/jcmm.13176

Figure Lengend Snippet: Effect of phloretin on UA / TNF ‐α‐induced inflammation and NF κB/ ERK activation. ( A‐B ) Western blot and quantification analysis of ICAM ‐1, IL ‐1β and MCP ‐1 protein, cell treated with TNF ‐α and phloretin. ( C‐D ) Western blot and quantification analysis of p‐ ERK / ERK and p‐ NF κB p65/ NF κB p65 levels. ( E ) Immunofluorescent staining and ( F ) quantification results of NF κB p65 nuclear translocation in HUVEC s (scale bar = 20 μm) (* P < 0.05, UA or TNF group versus control, # P < 0.05, phloretin group versus UA or TNF group). HUVECs: human umbilical vein endothelial cells, NFκB/ERK: nuclear factor‐kappa B/extracellular regulated protein kinases.

Article Snippet: The membranes were blocked with 5% non‐fat milk and incubated with primary antibodies against p‐NFκB (1:1000, Abcam, MA, USA) and NFκB (1:500, Santa Cruz Biotechnology, CA, USA), GLUT9 (1:1000, Abcam), IL‐1β (1:500, ABclonal Biotechnology), OAT1 (1:1000, ABclonal ), URAT1 (1:1000,ABclonal) ICAM‐1 (1:1000, Cell Signaling Technology, MA, USA), MCP‐1 (1:1000, Abcam), VCAM‐1 (1:500, Proteintech, USA) overnight at 4°C.

Techniques: Activation Assay, Western Blot, Staining, Translocation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Phloretin attenuates hyperuricemia‐induced endothelial dysfunction through co‐inhibiting inflammation and GLUT 9‐mediated uric acid uptake

doi: 10.1111/jcmm.13176

Figure Lengend Snippet: Effect of GLUT 9 inhibition on UA uptake and inflammatory response in HUVEC s. ( A ) Western blot analysis of GLUT 9 expression in si RNA treated HUVEC s (24 hrs). ( B‐C ) Western blot analysis of SLC 2A9 si RNA on ICAM ‐1, IL ‐1β and NF κB/ ERK activation in UA ‐treated HUVEC s (* P < 0.05, UA group versus control, # P < 0.05, si RNA group versus UA group). HUVECs: human umbilical vein endothelial cells, NFκB/ERK: nuclear factor‐kappa B/extracellular regulated protein kinases.

Article Snippet: The membranes were blocked with 5% non‐fat milk and incubated with primary antibodies against p‐NFκB (1:1000, Abcam, MA, USA) and NFκB (1:500, Santa Cruz Biotechnology, CA, USA), GLUT9 (1:1000, Abcam), IL‐1β (1:500, ABclonal Biotechnology), OAT1 (1:1000, ABclonal ), URAT1 (1:1000,ABclonal) ICAM‐1 (1:1000, Cell Signaling Technology, MA, USA), MCP‐1 (1:1000, Abcam), VCAM‐1 (1:500, Proteintech, USA) overnight at 4°C.

Techniques: Inhibition, Western Blot, Expressing, Activation Assay

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