Journal: bioRxiv

Article Title: FGFR1-phosphate sensing and crystal-induced gasdermin D signaling in neutrophils drive vascular calcification in CKD

doi: 10.64898/2026.05.23.725305

Figure Lengend Snippet: (A) Neutrophils derived from HC were incubated for 3 hours in DMEM supplemented with various concentrations of phosphate buffer and calcium buffer without FBS. (B) The representative phase-contrast and immunofluorescence images of neutrophils stained by SYTOX green. (C) The quantification of SYTOX-positive area. n=5. (D) The representative immunofluorescence images of MPO (green), CitH3 (red) and DAPI (blue). (E) The quantitative analysis of neutrophils for the MPO-DAPI double-positive area per DAPI positive area, the CitH3-DAPI double-positive area per DAPI positive area, and the MPO-CitH3-DAPI triple-positive area per DAPI positive area, respectively. n=8. (F) Neutrophils pretreated with a 40 μM GSDMD inhibitor (GSDMD-I, Disulfiram) and a 200 μM PAD4 inhibitor (PAD4-I) were incubated for 3 hours in DMEM supplemented with low or high phosphate and calcium buffers without FBS. (G) The representative images of neutrophils. (H) and (I) graphs show the quantification of SYTOX-positive area and calcium phosphate crystal area, respectively (n=5 for each group). (J-L) Neutrophils from HC were incubated with the DMEM supplemented with low or high calcium and phosphate without FBS (J). The protein expression of GSDMD (pro and cleaved p30 (N-terminal fragment)) in neutrophils was detected by immunoblotting using β-actin as a control (K). The graph (L) shows the ratio of GSDMD-p30/β-actin expression. n=3. Data represent mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey’s test (C and H) or Student’s unpaired t test (E, I and L). *P <0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns: not significant. The insets are higher magnification images of the black and yellow boxes. Scale bars: 200 μm (B, D, and G).

Article Snippet: For inhibitory experiments, neutrophils were pretreated with the FGFR1 inhibitor SU5402 (20 μM; Sigma-Aldrich), the GSDMD inhibitor Disulfiram (40 μM; MedChemExpress), the PAD4 inhibitor Cl-amidine (200 μM; Sigma-Aldrich), or the NaPi inhibitor (Phosphonoformic acid, concentrations of 1–5 mM) for 0.5 or 1 hour prior to incubation in high phosphate or calcium phosphate crystal-forming conditions.

Techniques: Derivative Assay, Incubation, Immunofluorescence, Staining, Expressing, Western Blot, Control

Journal: Bioactive Materials

Article Title: 3D-MSCs apoptotic bodies-integrated conductive hydrogel mitigates spinal cord injury via immunoregulation and alleviating neuronal pyroptosis

doi: 10.1016/j.bioactmat.2026.01.043

Figure Lengend Snippet: Composite hydrogel promotes the polarization of BV2 cells to M2 types and alleviates PC12 cell pyroptosis in vitro inflammatory environment. (A) Representative western blots showing protein expression of iNOS and Arg-1 in each group, β-actin was utilized as a loading control. (B) Quantitative analysis of relative expression of iNOS and Arg-1. (C) Representative immunofluorescence images of CD68 positive and iNOS positive BV2 cells (scale bar: 20 μm). (D) Representative immunofluorescence images of CD68 positive and Arg-1 positive BV2 cells (scale bar: 20 μm). (E, F) Quantitative analysis of relative fluorescence intensity of iNOS and Arg-1. (G) Representative western blots showing the expression of NLRP3, Caspase-1, IL-1β, ASC, GSDMD-N, and IL-18 protein associated with pyroptosis, β-actin was utilized as a loading control. (H) PI staining of PC12 cells in each group (scale bar, 100 μm). (I) Quantitative analysis of PI staining of PC12 cells (n = 3). (J) Quantitative analysis of relative expression of NLRP3, Caspase-1, IL-1β, ASC, GSDMD-N and IL-18 (n = 3). The data are presented as the means ± SEMs (n = 3); ∗p < 0.05, indicates significant differences; ns, is not significant. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Antibodies against GSDMD-N (cat. no. DF12275), CD44 (cat. no. BF9213), and Integrin beta1 (cat. no. AF5379) were obtained from Affinity Biosciences in Ohio, USA.

Techniques: In Vitro, Western Blot, Expressing, Control, Immunofluorescence, Fluorescence, Staining, Comparison

Journal: Bioactive Materials

Article Title: 3D-MSCs apoptotic bodies-integrated conductive hydrogel mitigates spinal cord injury via immunoregulation and alleviating neuronal pyroptosis

doi: 10.1016/j.bioactmat.2026.01.043

Figure Lengend Snippet: The composite hydrogel inhibits post-SCI pyroptosis in neurons. (A) Immunofluorescence images of residual neurons existing in the anterior horn of the spinal cord (scale bar, 500 μm and 200 μm). (B) Immunofluorescence image of Caspase-1 expression of neurons 3 days after SCI (scale bar, 20 μm). (C) Quantitative analysis of relative fluorescence intensity of Caspase-1 protein expression in neurons within the specified groups (n = 3). (D) Immunofluorescence image of GSDMD-N expression of neurons 3 days after SCI (scale bar, 20 μm). (E) Quantitative analysis of relative fluorescence intensity of GSDMD-N protein expression in neurons within the specified groups (n = 3). (F) Representative western blots showing the expression of NLRP3, Caspase-1, IL-1β, ASC, GSDMD-N, and IL-18 protein 3 days after SCI, GAPDH was utilized as a loading control. (G) Quantitative analysis of relative expression of NLRP3, Caspase-1, IL-1β, ASC, GSDMD-N and IL-18 (n = 3). The data are presented as the means ± SEMs (n = 3); ∗p < 0.05, indicates significant differences; ns, is not significant. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Antibodies against GSDMD-N (cat. no. DF12275), CD44 (cat. no. BF9213), and Integrin beta1 (cat. no. AF5379) were obtained from Affinity Biosciences in Ohio, USA.

Techniques: Immunofluorescence, Expressing, Fluorescence, Western Blot, Control, Comparison