Journal: Journal of Advanced Research

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation

doi: 10.1016/j.jare.2025.07.013

Figure Lengend Snippet: TLR4/MyD88/PKCδ/SHP-1 signaling cascade induced podocyte damage through regulating ER stress in DKD and podocyte damage. (A) The protein levels of ER stress markers BIP, sXBP-1, ATF4 and ATF6 were determined in renal tissues from mice at 12 and 16 weeks by western blot. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; # P < 0.05; ## P < 0.01; ### P < 0.01 ( n = 8). (B) Representative double IF staining of the expression ATF4 and podocyte marker synaptopodin in mice at 16 weeks. Scale bar, 20 μm ( n = 8). (C) Representative western blot to assess the levels of ER stress markers BIP, sXBP-1, ATF4 and ATF6 in cultured mouse podocytes from treatments of LG or HG for 48 h with or without transfection of TLR4, MyD88 or scrambled shRNAs. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; ### P < 0.01; ### P < 0.01 ( n = 3 independent experiments). (D) ER morphological alterations and ER stress illustrated by ER-tracker staining in podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, or MCP-1 shRNAs, or pretreatment of 4-PBA. Scale bar, 10 μm. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; ## P < 0.01; # P < 0.05 ( n = 3 independent experiments). (E) Representative western blot to detect the expression of ER stress proteins BIP, sXBP-1, ATF4 and ATF6 in cultured podocytes after HG stimulation for 48 h with or without transfection of PKCδ, SHP-1 or scrambled shRNAs, or pretreatment of 4-PBA. P values were determined by one-way ANOVA and data are presented as mean ± SD. ** P < 0.01; *** P < 0.01; **** P < 0.01; ## P < 0.01; ### P < 0.01; #### P < 0.01 ( n = 3 independent experiments).

Article Snippet: The following primary antibodies were used: f4/80 (29414–1-AP), desmin (16520–1-AP), MCP-1 (26161–1-AP), podocin (20384–1-AP), synaptopodin (21064–1-AP), TLR4 (66350–1-Ig), β-actin (66009–1-Ig), PKCδ(14188–1-AP), ATF6 (24169–1-AP), p-cadherin (13773–1-AP) were from Proteintech (IL, USA); phospho-PKCδ (Y311) (ab76181), PKCdelta (ab182126), SHP-1 (ab227503), HA (ab9110) were from Abcam (MA, USA); BIP (M010300), MyD88 (P012343) were from Epizyme, Shanghai, China); and ATF4 (11815), sXBP-1 (40435) were from Cell Signaling Technology (MA, USA).

Techniques: Western Blot, Staining, Expressing, Marker, Cell Culture, Transfection

Journal: Journal of Advanced Research

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation

doi: 10.1016/j.jare.2025.07.013

Figure Lengend Snippet: Further validation of PKCδ activation being indispensable for ER stress and MCP-1 production through SHP-1 in diabetic podocyte injury. (A) Schematic representation of PKCδ plasmids to express PKCδ proteins in DN (tyrosine mutation at amino acid 311), CA or WT form. (B) Representative western blot to validate the expression of DN-PKCδ, CA-PKCδ and WT-PKCδ plasmids in HEK-293 T cells by using antibodies specific for HA-tag, with GAPDH detection as a loading control. ( n = 3 independent experiments). (CDE) Determination of protein levels of SHP-1 (C), ER stress proteins BIP, sXBP-1, ATF4 and ATF6 (D), and MCP-1 (E) in cultured mouse podocytes transfected with DN-, CA- and WT-PKCδ plasmids, or control plasmids under LG or HG conditions. P values were determined by one-way ANOVA and data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.01; **** P < 0.01; ## P < 0.01; # P < 0.05; ## P < 0.01; ### P < 0.01; #### P < 0.01 ( n = 3 independent experiments).

Article Snippet: The following primary antibodies were used: f4/80 (29414–1-AP), desmin (16520–1-AP), MCP-1 (26161–1-AP), podocin (20384–1-AP), synaptopodin (21064–1-AP), TLR4 (66350–1-Ig), β-actin (66009–1-Ig), PKCδ(14188–1-AP), ATF6 (24169–1-AP), p-cadherin (13773–1-AP) were from Proteintech (IL, USA); phospho-PKCδ (Y311) (ab76181), PKCdelta (ab182126), SHP-1 (ab227503), HA (ab9110) were from Abcam (MA, USA); BIP (M010300), MyD88 (P012343) were from Epizyme, Shanghai, China); and ATF4 (11815), sXBP-1 (40435) were from Cell Signaling Technology (MA, USA).

Techniques: Biomarker Discovery, Activation Assay, Mutagenesis, Western Blot, Expressing, Control, Cell Culture, Transfection

Journal: Journal of Advanced Research

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation

doi: 10.1016/j.jare.2025.07.013

Figure Lengend Snippet: TLR4/MyD88/PKCδ/SHP-1 signaling-dependent ER stress stimulated MCP-1 production via enhanced transcription activity by ATF4 binding to its promoter in hyperglycemic podocytes. (A) The altered expression of MCP-1 in renal tissue from mice at 12 and 16 weeks by western blot. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; # P < 0.05; ## P < 0.01 ( n = 8 samples). (B) Representative western blot to assess the levels of MCP-1 in cultured mouse podocytes from treatments of LG or HG for 48 h with or without transfection of TLR4, MyD88 or scrambled shRNAs. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; ### P < 0.01 ( n = 3 independent experiments). (C) Representative western blot to show the expression of MCP-1 in podocytes after HG stimulation for 48 h with or without transfection of PKCδ, SHP-1 or scrambled shRNAs, or pretreatment of 4-PBA. P values were determined by one-way ANOVA and data are presented as mean ± SD. ** P < 0.01; *** P < 0.01; ### P < 0.01 ( n = 3 independent experiments). (DE) The expression of MCP-1 by western blot (D), and ER stress markers BIP, sXBP-1, ATF4 and ATF6 (E) after HG stimulation for 48 h with or without MCP-1 or scrambled shRNA transfection. P values were determined by one-way ANOVA and data are presented as mean ± SD. ## P < 0.01; #### P < 0.01 ( n = 3 independent experiments). (F) Schematic description of primers spanning the distal, medial, and proximal regions of the MCP-1 promoter, encompassing predicted binding sites in the present study. (GH) ChIP analysis using anti-ATF4 or anti-IgG antibodies were performed either in cultured mouse podocytes after LG or HG treatment for 48 h (G), or HG stimulation with transfection of TLR4, PKCδ, MCP-1 or scrambled shRNA, or pretreatment of 4-PBA (H). Real-time PCR was used for the detection of the ChIP signals. P values were determined by Student’s t -test, or by one-way ANOVA, and data are presented as mean ± SD. * P < 0.05; ** P < 0.01; **** P < 0.01; #### P < 0.01 ( n = 3 independent experiments). (I) Determination of ATF4 nuclear accumulation in podocytes under LG or HG conditions by western blot. P values were determined by Student’s t -test and data are presented as mean ± SD. ** P < 0.01; ## P < 0.01 ( n = 3 independent experiments). (J) Assessment of nuclear ATF4 protein levels in cultured podocytes transfected with TLR4, PKCδ, MCP-1 or scrambled shRNAs, or pretreated with 4-PBA under HG conditions for 48 h. P values were determined by one-way ANOVA and data are presented as mean ± SD. * P < 0.05; * P < 0.01 ( n = 3 independent experiments).

Article Snippet: The following primary antibodies were used: f4/80 (29414–1-AP), desmin (16520–1-AP), MCP-1 (26161–1-AP), podocin (20384–1-AP), synaptopodin (21064–1-AP), TLR4 (66350–1-Ig), β-actin (66009–1-Ig), PKCδ(14188–1-AP), ATF6 (24169–1-AP), p-cadherin (13773–1-AP) were from Proteintech (IL, USA); phospho-PKCδ (Y311) (ab76181), PKCdelta (ab182126), SHP-1 (ab227503), HA (ab9110) were from Abcam (MA, USA); BIP (M010300), MyD88 (P012343) were from Epizyme, Shanghai, China); and ATF4 (11815), sXBP-1 (40435) were from Cell Signaling Technology (MA, USA).

Techniques: Activity Assay, Binding Assay, Expressing, Western Blot, Cell Culture, Transfection, shRNA, Real-time Polymerase Chain Reaction

Journal: Journal of Advanced Research

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation

doi: 10.1016/j.jare.2025.07.013

Figure Lengend Snippet: Schematic diaphragm of TLR4/MyD88/PKCδ/SHP-1 signaling in podocytes in exposure of hyperglycemia. Upon the stimuli of hyperglycemia, TLR4/MyD88 signaling was activated in glomerular podocyte; and PKCδ was then phosphorylated and bound to SHP-1 to provoke downstream ER stress, leading to nuclear translocation of ATF4 and enhanced transcriptional activity of MCP-1, which exacerbated damage to SD proteins and disruption of the cytoskeletal structure, increased cell motility, promoted inflammation in podocytes, and finally induced local macrophage migration and infiltration.

Article Snippet: The following primary antibodies were used: f4/80 (29414–1-AP), desmin (16520–1-AP), MCP-1 (26161–1-AP), podocin (20384–1-AP), synaptopodin (21064–1-AP), TLR4 (66350–1-Ig), β-actin (66009–1-Ig), PKCδ(14188–1-AP), ATF6 (24169–1-AP), p-cadherin (13773–1-AP) were from Proteintech (IL, USA); phospho-PKCδ (Y311) (ab76181), PKCdelta (ab182126), SHP-1 (ab227503), HA (ab9110) were from Abcam (MA, USA); BIP (M010300), MyD88 (P012343) were from Epizyme, Shanghai, China); and ATF4 (11815), sXBP-1 (40435) were from Cell Signaling Technology (MA, USA).

Techniques: Translocation Assay, Activity Assay, Disruption, Migration

Journal: ASN NEURO

Article Title: Exposure to Frontline Antiretroviral Dolutegravir Disrupts Oligodendrocyte Development Across Differentiation Stages

doi: 10.1080/17590914.2026.2647877

Figure Lengend Snippet: Multistage 8 µM DTG significantly alters gene expression across a variety of cellular mechanisms in differentiating OLs. (a) Principle components (PC) plot of variance between DMSO-treated and 8 µM DTG-treated differentiating OL samples from bulk RNA sequencing, showing clustering of groups, n = 4/group. (b) Volcano plot of gene expression in 8 µM DTG-treated OLs vs. DMSO-treated OLs, where red indicates significantly upregulated genes and blue indicates significantly downregulated genes. Particular genes of interest are labeled. (c) Significantly altered gene ontology (GO) categories in 8 µM DTG-treated OLs vs. DMSO-treated OLs. Red arrows highlight GO categories directly related to ISR (ER stress, oxidative stress, amino acid starvation). (d) List of significantly altered genes mapping to ISR-related GO categories with their log2FoldChange in DMSO OLs vs. 8 µM DTG OLs and log2FoldChange in DMSO OPCs vs. DMSO OLs for comparison, n = 4/group. Pink = upregulated, blue = downregulated in 8 µM DTG OLs compared to DMSO OLs. qRT-PCR quantification shows significantly decreased expression of Mbp (e) and increased Trib3 (f), Atf4 (g), and Cebpb (h) in 8 µM DTG-treated vs. DMSO-treated OLs, n = 3-5/group. Unpaired t -test to compare 8 µM DTG to DMSO. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Coverslips were incubated at 4 °C overnight with rabbit anti-ATF4 antibody (1:100 in 1x PBS with 5% normal goat serum, Proteintech, 10835-1-AP, RRID: AB_2058600) overnight at 4 °C.

Techniques: Gene Expression, RNA Sequencing, Labeling, Comparison, Quantitative RT-PCR, Expressing

Journal: ASN NEURO

Article Title: Exposure to Frontline Antiretroviral Dolutegravir Disrupts Oligodendrocyte Development Across Differentiation Stages

doi: 10.1080/17590914.2026.2647877

Figure Lengend Snippet: Multistage exposure to DTG activates the ISR in differentiating OLs. (a) Representative immunoblots of phospho-eIF2α and total-eIF2α after multistage exposure to DTG. Densitometry analyses show significantly increased activated eIF2α, (b) after multistage exposure to either 8 µM or 24 µM DTG, n = 3/group. (c) Representative images of differentiated OLs after multistage treatment with DTG (800 nM, 8 µM, or 24 µM) and immunostained for ATF4 (white) and DAPI (blue). Scale bar = 50 µm. Quantification of the integrated density of nuclear ATF4, (d) shows significantly increased nuclear ATF4 in differentiated OLs after multistage exposure to all doses of DTG, n = 3/group. One-way ANOVA with Dunnett post-hoc test to compare all treatment groups to DMSO. ** p < 0.01, **** p < 0.0001.

Article Snippet: Coverslips were incubated at 4 °C overnight with rabbit anti-ATF4 antibody (1:100 in 1x PBS with 5% normal goat serum, Proteintech, 10835-1-AP, RRID: AB_2058600) overnight at 4 °C.

Techniques: Western Blot