Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: SARS-CoV-2-infected cardiomyocytes exhibit upregulated necroptosis, but no evidence of mitochondrial permeability transition

doi: 10.1016/j.jmccpl.2025.100833

Figure Lengend Snippet: Infection of iPSC-CMs by SARS-CoV-2 in vitro results in increased cellular cytotoxicity. (A) Immunofluorescence staining shows infection of iPSC-derived cardiomyocytes. Nuclei (blue), α-Actinin (grey) and Nucleocapsid (red). Scale bar: 50 μm.(B) Quantification of infected cells at 24 h post-infection. (C) Infection of iPSC-derived cardiomyocytes is confirmed by expression of SARS-CoV-2 Spike RNA. (D) Western blot analysis confirms Spike Protein expression, quantified in (E). Full western blots in A. (F) Cytotoxicity upon SARS-CoV-2 infection determined by activity of LDH released into the medium. Protein expression is normalized to β-actin, gene expression is normalized to GAPDH. n = 3 iPSC-CM differentiation rounds for all but (B), where n = 1 iPSC-CM differentiation round, statistical significance determined by Wilcoxon test (B), Kruskal-Wallis test (C, D), students t -test (F). p < 0.0001 = ****. Data are displayed as mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Infection of iPSC-CMs by SARS-CoV-2 in vitro results in increased cellular cytotoxicity. (A) Immunofluorescence staining shows infection of iPSC-derived cardiomyocytes. Nuclei (blue), α-Actinin (grey) and Nucleocapsid (red). Scale bar: 50 μm.(B) Quantification of infected cells at 24 h post-infection. (C) Infection of iPSC-derived cardiomyocytes is confirmed by expression of SARS-CoV-2 Spike RNA. (D) Western blot analysis confirms Spike Protein expression, quantified in (E). Full western blots in Suppl. Fig. 3A. (F) Cytotoxicity upon SARS-CoV-2 infection determined by activity of LDH released into the medium. Protein expression is normalized to β-actin, gene expression is normalized to GAPDH. n = 3 iPSC-CM differentiation rounds for all but (B), where n = 1 iPSC-CM differentiation round, statistical significance determined by Wilcoxon test (B), Kruskal-Wallis test (C, D), students t -test (F). p < 0.0001 = ****. Data are displayed as mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following antibodies were used in a 1:500 dilution: α-Actinin (Sigma, #A7811) and SARS-CoV-2 Nucleocapsid (SinoBiological, #40143-T62).

Techniques: Infection, In Vitro, Immunofluorescence, Staining, Derivative Assay, Expressing, Western Blot, Activity Assay, Gene Expression

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: SARS-CoV-2-infected cardiomyocytes exhibit upregulated necroptosis, but no evidence of mitochondrial permeability transition

doi: 10.1016/j.jmccpl.2025.100833

Figure Lengend Snippet: PDH phosphorylation is unaffected following SARS-CoV2 infection. (A) Cytotoxicity after Cyclosporine A (CsA) treatment [0.5 μM, 2 h] as determined by activity of LDH released into the medium. (B) Quantification of pyruvate dehydrogenase A1 (PDHA1) protein expression and its phosphorylation on serine 293. Values normalized to α-tubulin. (C) Representative Western Blot. (D) Quantification of CI = Complex I, CII = Complex II, CIII = Complex III, CIV = Complex IV and CV = Complex V normalized to α-tubulin. Full western blots in B. (E) Protein expression of the respiratory chain following SARS-CoV-2 infection. Full western blots in B. (F) Apoptosis rate determined by TUNEL staining 24 h post-infection with an MOI of 0.1. (G) Quantification of procaspase 3 and cleaved caspase 3 protein expression. Values normalized to α-tubulin. Corresponding western blots in C. n = 3 iPSC-CM differentiation rounds for all but (A, B), where n = 1 iPSC-CM differentiation round. Statistical significance determined by two-way ANOVA (A, B), Mann-Whitney test (F) or t-test (D, G). p ≤ 0.05 = *, p < 0.0001 = ****. Data are displayed as mean ± SD. PDH phosphorylation is unaffected following SARS-CoV2 infection. (A) Cytotoxicity after Cyclosporine A (CsA) treatment [0.5 μM, 2 h] as determined by activity of LDH released into the medium. (B) Quantification of pyruvate dehydrogenase A1 (PDHA1) protein expression and its phosphorylation on serine 293. Values normalized to α-tubulin. (C) Representative Western Blot. (D) Quantification of CI = Complex I, CII = Complex II, CIII = Complex III, CIV = Complex IV and CV = Complex V normalized to α-tubulin. Full western blots in Suppl. Fig. 3B. (E) Protein expression of the respiratory chain following SARS-CoV-2 infection. Full western blots in Suppl. Fig. 3B. (F) Apoptosis rate determined by TUNEL staining 24 h post-infection with an MOI of 0.1. (G) Quantification of procaspase 3 and cleaved caspase 3 protein expression. Values normalized to α-tubulin. Corresponding western blots in Suppl. Fig. 3C. n = 3 iPSC-CM differentiation rounds for all but (A, B), where n = 1 iPSC-CM differentiation round. Statistical significance determined by two-way ANOVA (A, B), Mann-Whitney test (F) or t-test (D, G). p ≤ 0.05 = *, p < 0.0001 = ****. Data are displayed as mean ± SD.

Article Snippet: The following antibodies were used in a 1:500 dilution: α-Actinin (Sigma, #A7811) and SARS-CoV-2 Nucleocapsid (SinoBiological, #40143-T62).

Techniques: Phospho-proteomics, Infection, Activity Assay, Expressing, Western Blot, TUNEL Assay, Staining, MANN-WHITNEY

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: SARS-CoV-2-infected cardiomyocytes exhibit upregulated necroptosis, but no evidence of mitochondrial permeability transition

doi: 10.1016/j.jmccpl.2025.100833

Figure Lengend Snippet: Increased necroptosis following SARS-CoV-2 infection. (A) Cytotoxicity after Necrostatin-1 (Nec-1) [2.5 μM] or MCC950 [0.05 μM] treatment as determined by activity of LDH released into the medium. (B) Spike- (C) RIP3 and (D) NLR family pyrin domain containing 3 (NLRP3) inflammasome gene expression following SARS-CoV-2 infection. Gene expression normalized to GAPDH. (E) Quantification of RIP3 and RIP3-S227 protein expression after Nec-1 treatment. (F) Representative WB for RIP3 and RIP3-S227 following SARS-CoV-2 infection. Full western blots in D. (G) Quantification of MLKL and MLKL-S358 protein expression with a representative WB (H). Full western blots in E. n = 3 iPSC-CM differentiation rounds, statistical significance determined by two-way ANOVA (A) or Kruskal-Wallis test (B–E) or Mann-Whitney (G). p < 0.01 = **, p < 0.0001 = ****. Data are displayed as mean ± SD. Increased necroptosis following SARS-CoV-2 infection. (A) Cytotoxicity after Necrostatin-1 (Nec-1) [2.5 μM] or MCC950 [0.05 μM] treatment as determined by activity of LDH released into the medium. (B) Spike- (C) RIP3 and (D) NLR family pyrin domain containing 3 (NLRP3) inflammasome gene expression following SARS-CoV-2 infection. Gene expression normalized to GAPDH. (E) Quantification of RIP3 and RIP3-S227 protein expression after Nec-1 treatment. (F) Representative WB for RIP3 and RIP3-S227 following SARS-CoV-2 infection. Full western blots in Suppl. Fig. 3D. (G) Quantification of MLKL and MLKL-S358 protein expression with a representative WB (H). Full western blots in Suppl. Fig. 3E. n = 3 iPSC-CM differentiation rounds, statistical significance determined by two-way ANOVA (A) or Kruskal-Wallis test (B–E) or Mann-Whitney (G). p < 0.01 = **, p < 0.0001 = ****. Data are displayed as mean ± SD.

Article Snippet: The following antibodies were used in a 1:500 dilution: α-Actinin (Sigma, #A7811) and SARS-CoV-2 Nucleocapsid (SinoBiological, #40143-T62).

Techniques: Infection, Activity Assay, Gene Expression, Expressing, Western Blot, MANN-WHITNEY

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: hfNCSC-sEVs are taken up by PCs in vitro and enhance their proliferation and migration. (A) Primary cultures of hfNCSCs were established from male Sprague–Dawley rats. (B) Immunofluorescence staining of the neural crest cell marker p75 (red) and the stem cell marker nestin (green) in hfNCSCs, with 4′,6-diamidino-2-phenylindole (DAPI) staining indicating the nuclei. (C) Western blot analysis demonstrated the presence of surface markers (cluster of differentiation [CD]9, CD81, and tumor susceptibility gene 101 protein [TSG101]) and the absence of an endoplasmic reticulum marker (calnexin) in hfNCSC-sEVs. (D) Nanoparticle tracking analysis was used to quantify the concentration and size distribution of hfNCSC-sEVs. (E) Transmission electron microscopy was used to visualize the characteristic morphology of hfNCSC-sEVs. (F) Immunofluorescence staining indicated that the third-generation PCs cultured in vitro were positive for claudin-1, zonula occludens 1 (ZO1), and glucose transporter 1 (GLUT1) but negative for S100, with DAPI staining marking the nuclei. (G) The internalization of PKH26-labeled hfNCSC-sEVs (red) by ZO1-positive PCs (green) was visualized using immunofluorescence staining, with DAPI staining to mark the nuclei. (H) The Cell Counting Kit-8 assay was used to evaluate the cell viability of PCs across concentrations of 0, 2 × 10 8 , 5 × 10 8 , and 10 × 10 8 particles/mL hfNCSC-sEVs at 3, 5, and 7 days of in vitro culture ( n = 5 per group). (I) The Transwell assay was used to quantify the number of migrating PCs at 6, 12, and 18 hours post-treatment with the aforementioned concentrations of hfNCSC-sEVs, in in vitro culture ( n = 6 per group). (J) Western blot and (K) statistical analyses revealed the relative protein expression levels of proliferating cell nuclear antigen (PCNA) and vimentin in PCs from the phosphate-buffered saline (PBS) and hfNCSC-sEVs groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for H and I; Student’s t -test for K). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; GLUT1: glucose transporter 1; hfNCSCs: hair follicle neural crest stem cells; ns: not significant; PCNA: proliferating cell nuclear antigen; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (1:2000, Cat# 60097-1-Ig, Proteintech, Wuhan, China), rabbit monoclonal anti-vimentin antibody (1:1000, Cat# 5741, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-claudin-1 antibody (1:1000, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-zonula occludens 1 (ZO1) antibody (1:10 000, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-mothers against decapentaplegic homolog (SMAD)7 antibody (1:500, Cat# WL02975, Wanleibio, Shenyang, China), rabbit polyclonal anti-SMAD2/3 antibody (1:1000, Cat# WL01520, Wanleibio), rabbit polyclonal anti-p-SMAD2/3 antibody (1:500, Cat# WL02305, Wanleibio), rabbit recombinant anti-hyaluronan synthase 2 (HAS2) antibody (1:500, Cat# DF13702, Affinity, Cincinnati, OH, USA), rabbit monoclonal anti-β-actin antibody (1:1000, Cat# 4970, Cell Signaling Technology), and mouse monoclonal anti-β-tubulin antibody (1:5000, Cat# M20005 , Abmart, Shanghai, China).

Techniques: In Vitro, Migration, Immunofluorescence, Staining, Marker, Western Blot, Concentration Assay, Transmission Assay, Electron Microscopy, Cell Culture, Labeling, Cell Counting, Transwell Assay, Expressing, Saline, Comparison, CCK-8 Assay

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: miR-21-5p in hfNCSC-sEVs augments cell proliferation and migration by enhancing HAS2 expression in PCs. (A, B) Western blot (A) and statistical analyses (B) revealed the relative protein expression levels of HAS2, proliferating cell nuclear antigen (PCNA), and vimentin in PCs across the –/–, –/si- Has2 , hfNCSC-sEVs/–, and hfNCSC-sEVs/si- Has2 groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (C, D) The wound healing assay (C) and statistical analysis (D) demonstrated the migration rates of PCs in the aforementioned groups ( n = 3 per group). (E) The Cell Counting Kit-8 assay was used to assess cell viability in PCs across the same groups on day 5 of in vitro culture ( n = 5 per group). (F, G) Western blot (F) and statistical analyses (G) indicated the relative protein expression levels of HAS2, PCNA, and vimentin in PCs treated with phosphate-buffered saline (PBS), hfNCSC-sEVs, or hfNCSC-sEVs + miR-21-5p inhibitor on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (H–J) Immunofluorescence staining visualized the expression of HAS2 (red) and 5-ethynyl-2′-deoxyuridine (EdU; green) in PCs (H), and statistical analysis revealed the integrated optical density (IOD) of zonula occludens 1 (ZO1; I) and the cell proliferation rates (J) in the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 of in vitro culture ( n = 3 per group). (K, L) Western blot (K) and statistical analyses (L) showed the relative protein expression levels of HAS2, PCNA, and vimentin in regenerated tissue from the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 post-operation (normalized to β-tubulin, n = 3 per group). Data are expressed as the mean ± SEM. ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for B, D, E, G, I, J, and L). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; EdU: 5-ethynyl-2′-deoxyuridine; HAS2: hyaluronan synthase 2; hfNCSCs: hair follicle neural crest stem cells; IOD: integrated optical density; PCNA: proliferating cell nuclear antigen; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (1:2000, Cat# 60097-1-Ig, Proteintech, Wuhan, China), rabbit monoclonal anti-vimentin antibody (1:1000, Cat# 5741, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-claudin-1 antibody (1:1000, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-zonula occludens 1 (ZO1) antibody (1:10 000, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-mothers against decapentaplegic homolog (SMAD)7 antibody (1:500, Cat# WL02975, Wanleibio, Shenyang, China), rabbit polyclonal anti-SMAD2/3 antibody (1:1000, Cat# WL01520, Wanleibio), rabbit polyclonal anti-p-SMAD2/3 antibody (1:500, Cat# WL02305, Wanleibio), rabbit recombinant anti-hyaluronan synthase 2 (HAS2) antibody (1:500, Cat# DF13702, Affinity, Cincinnati, OH, USA), rabbit monoclonal anti-β-actin antibody (1:1000, Cat# 4970, Cell Signaling Technology), and mouse monoclonal anti-β-tubulin antibody (1:5000, Cat# M20005 , Abmart, Shanghai, China).

Techniques: Migration, Expressing, Western Blot, In Vitro, Wound Healing Assay, Cell Counting, Saline, Immunofluorescence, Staining, Comparison, CCK-8 Assay

Journal: Alzheimer's & Dementia

Article Title: Female‐biased astrocytic priming shapes early locus coeruleus vulnerability in an Aβ oligomer milieu

doi: 10.1002/alz.71168

Figure Lengend Snippet: Astrocyte‐linked C3 elevation and lactate–MCT2–OXPHOS axis engagement in the pons of female APP/PS1 mice. Astrocyte‐enriched pons fraction shows increased C3 expression in 2‐ to 3‐month‐old female APP/PS1 mice. A, Schematic illustrating MACS‐based isolation of pontine astrocytes. Schematic created using Biorender. B, Assessment of C3 expression by qPCR followed by 1% agarose gel electrophoresis–gel image showing C3 bands at 172 bp in astrocyte‐enriched fractions (lane 1–4) and astrocyte‐depleted fractions (lane 5–8). C, D, Bar graphs demonstrating elevated C3 levels in the astrocyte‐enriched fraction of female APP/PS1 mice compared to other groups and to their corresponding negative fraction. Each bar represents pooled pons samples from 8 mice per group. Lactate–MCT2 axis and OXPHOS pathway engagement in female APP/PS1 mice. Female APP/PS1 mice showed higher (E) lactate levels in the pons measured by colorimetric assay and increased (F) MCT2 expression assessed by qPCR ( n = 6 per group; two‐way ANOVA with Tukey post hoc test; data shown as mean ± SEM; * p < 0.05, ** p < 0.01). G, H, mRNA expression profiling of mitochondrial markers by qPCR in the pons. Female APP/PS1 mice showed significantly elevated expression of the mitochondrial complex I subunit Ndufs8 and complex V subunit Atp5a1 , indicating adaptive mitochondrial activation in the pons ( n = 5‐6; two‐way ANOVA with Tukey post hoc test; data shown as mean ± SEM; * p < 0.05, ** p < 0.01). I, Graphical summary. Elevated Aβ oligomers in female APP/PS1 mice shift astrocytes from a resting to an active GFAP +ve state, with associated C3 and NF‐κB2 upregulation and engagement of lactate–OXPHOS axis, supporting increased energetic demands. Graphical summary created using Biorender. Aβ, amyloid beta; ACSA‐1, astrocyte cell surface antigen 1; ANOVA, analysis of variance; Atp5a1, ATP synthase F1 subunit α; C3, complement component 3; GLUT1, glucose transporter 1; MACS, magnetic‐activated cell sorting; MCT2, monocarboxylate transporter 2; Ndufs8, NADH dehydrogenase iron‐sulfur protein 8; NF‐κB2, nuclear factor‐kappa‐B p100/p52 subunit; OXPHOS, oxidative phosphorylation; qPCR, quantitative polymerase chain reaction; SEM, standard error of mean; WT, wild type.

Article Snippet: Cells were incubated with FcR blocking reagent (Miltenyi Biotec, 130‐092‐575) at a 1:9 dilution for 10 minutes to prevent non‐specific antibody binding, followed by incubation with Anti‐GLAST or Astrocyte Cell Surface Antigen‐1 (ACSA‐1)‐Biotin (Miltenyi Biotec, 130‐095‐826) for 10 minutes.

Techniques: Expressing, Isolation, Agarose Gel Electrophoresis, Colorimetric Assay, Activation Assay, FACS, Phospho-proteomics, Real-time Polymerase Chain Reaction