Journal: Acta Pharmaceutica Sinica. B

Article Title: Clostridium perfringens alpha toxin drives pathological NETosis via immature neutrophil mobilization and functional reprogramming

doi: 10.1016/j.apsb.2025.09.011

Figure Lengend Snippet: Elevated NETosis is observed in mice after challenged with CPA. (A–C) CPA intoxication induces an increase in NETs markers. (A) The levels of plasma MPO-DNA of PBS controls and CPA-treated at different time points were detected using ELISA. (B) The levels of plasma CitH3 of PBS controls and CPA-treated at different time points were detected using ELISA. (C) The levels of plasma cfDNA of PBS controls and CPA-treated at different time points were detected using the Picogreen method. Data are mean ± SD, n = 4. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test (∗∗ P < 0.01; ∗∗∗ P < 0.001 vs. Control. ns, not significant). (D) Representative immunofluorescence images of CitH3 and MPO in neutrophils isolated from peripheral blood of PBS controls and CPA-treated. Scale bar = 10 μm. (E) Protein levels of PAD4 and CitH3 in peripheral blood, spleen and lung from PBS controls and CPA-treated were detected using Western blot ( n = 3). (F) Quantitative analysis of PAD4 and CitH3 in peripheral blood, spleen and lung from mice PBS controls and CPA-treated. Data are mean ± SD, n = 3. Statistical significance was determined using an unpaired two-tailed t- test (∗∗ P < 0.01; ∗∗∗ P < 0.001 vs. PBS-treated. ns, not significant). (G) Representative images demonstrating NETs presence through co-expression of CitH3 and MPO in the spleen and lung subjected to CPA treated. Scale bar = 50 μm. (H) Quantitative analysis of MPO and CitH3 in spleen and lung from mice PBS controls and CPA-treated. Data are mean ± SD, n = 3. Statistical significance was determined using an unpaired two-tailed t- test (∗ P < 0.05; ∗∗∗ P < 0.001 vs. PBS-treated. ns, not significant). (I) Caspase-3 activity was measured in neutrophils using a fluorometric assay. The relative Caspase-3 activity is shown, with minimal activation observed, indicating a lack of apoptosis. Flow cytometric analysis of neutrophils stained with Annexin V-PE and 7AAD. Representative dot plots show the distribution of cells in different quadrants: Q1 (Annexin V − /7AAD + ), Q2 (Annexin V + /7AAD + ), Q3 (Annexin V + /7AAD − ), and Q4 (Annexin V − /7AAD − ). The majority of neutrophils were found in Q4, indicating intact cell membranes and absence of phosphatidylserine exposure, which is inconsistent with apoptosis. Data are mean ± SD, n = 3. Statistical significance was determined using an unpaired two-tailed t- test (∗∗∗ P < 0.001 vs. PBS-treated. ns, not significant).

Article Snippet: Cell apoptosis was measured using an Annexin V-PE/7AAD apoptosis kit (MULTISCIENCES, AT104-100, Hangzhou, China) and Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor® 488 Conjugate) (Cell Signaling Technology, 9669S, Danvers, MA, USA).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Control, Immunofluorescence, Isolation, Western Blot, Two Tailed Test, Expressing, Activity Assay, Activation Assay, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Clostridium perfringens alpha toxin drives pathological NETosis via immature neutrophil mobilization and functional reprogramming

doi: 10.1016/j.apsb.2025.09.011

Figure Lengend Snippet: CPA directly induces NETosis in both murine and human neutrophils in vitro . (A) Representative immunofluorescence images of purified neutrophils from mice stimulated in vitro with PBS, CPA, or PMA (positive control). Cells were stained for CitH3 (red) and MPO (green). Scale bar = 10 μm. (B) Representative immunofluorescence images of purified neutrophils from humans stimulated in vitro with PBS, CPA, or PMA (positive control). Cells were stained for CitH3 (red) and MPO (green). Scale bar = 10 μm. (C) Flow cytometric analysis of intracellular ROS production in mice PBMCs stimulated in vitro with PBS, CPA, or Rosup (positive control). Histograms show DCFH-DA fluorescence within the gated neutrophil population. (D) Flow cytometric analysis of intracellular ROS production in human PBMCs stimulated in vitro . Histograms show DCFH-DA fluorescence within the gated neutrophil population. (E) Relative mRNA expression of NETs-associated genes in stimulated mice PBMCs ( Tlr4 , Padi4 , Hmox1 , Nfe2l2 , and Cybb ) and human PBMCs ( TLR4 , PADI4 , HMOX1 , NFE2L2 , and CYBB ), as determined by qPCR. Data are mean ± SD, n = 3. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test (∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 vs. PBS-treated. ns, not significant). (F) Representative Western blot detecting PAD4 and CitH3 protein levels in lysates from stimulated mice PBMCs and human PBMCs ( n = 3). (G) Densitometric quantification of the protein bands shown in (F). Data are mean ± SD, n = 3. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test (∗ P < 0.05; ∗∗∗ P < 0.001 vs. PBS-treated. ns, not significant). (H) Caspase-3 activity measured by flow cytometry within the gated neutrophil population of stimulated mice PBMCs and human PBMCs. Data are mean ± SD, n = 3. Statistical significance was determined using an unpaired two-tailed t- test ( vs. PBS-treated. ns, not significant). (I) Flow cytometric analysis of apoptosis in stimulated mice PBMCs and human PBMCs using Annexin V and 7-AAD staining. Representative dot plots are shown. The bar graph shows the quantification of the live cell percentage (Annexin V − /7-AAD - ) within the gated neutrophil population. Data are mean ± SD, n = 3. Statistical significance was determined using an unpaired two-tailed t- test (∗∗∗ P < 0.001 vs. PBS-treated. ns, not significant).

Article Snippet: Cell apoptosis was measured using an Annexin V-PE/7AAD apoptosis kit (MULTISCIENCES, AT104-100, Hangzhou, China) and Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor® 488 Conjugate) (Cell Signaling Technology, 9669S, Danvers, MA, USA).

Techniques: In Vitro, Immunofluorescence, Purification, Positive Control, Staining, Fluorescence, Expressing, Western Blot, Activity Assay, Flow Cytometry, Two Tailed Test

Journal: International Journal of Molecular Medicine

Article Title: Exportin 1 inhibitor KPT-330 reverses oxaliplatin resistance via p53 nuclear retention in colorectal cancer

doi: 10.3892/ijmm.2025.5675

Figure Lengend Snippet: Combination of KPT-330 and oxaliplatin induces apoptosis in oxaliplatin-resistant colorectal cancer cells. (A) Apoptosis rate of HCT116/L-OHP and HCT8/L-OHP cells treated with oxaliplatin, KPT-330 or the combination for 48 h, assessed by flow cytometry. The sum of early apoptotic cells (annexin V + /PI - ) in the lower-right quadrant and late apoptotic cells (annexin V + /PI + ) in the upper-right quadrant indicates apoptotic cells, and was used for quantification. (B) Viability of HCT116/L-OHP and HCT8/L-OHP cells treated with oxaliplatin and KPT-330 or the combination with Z-VAD-FMK for 48 h, analyzed using a Cell Counting Kit-8 assay. (C) Apoptosis rate of HCT116/L-OHP and HCT8/L-OHP cells treated with oxaliplatin and KPT-330 or the combination with Z-VAD-FMK for 48 h, analyzed by flow cytometry. The sum of early apoptotic cells (annexin V + /PI - ) in the lower-right quadrant and late apoptotic cells (annexin V + /PI + ) in the upper-right quadrant indicates apoptotic cells, and was used for quantification. (D) Immunoblot analysis of HCT116/L-OHP and HCT8/L-OHP cells treated with oxaliplatin, KPT-330 or the combination for 48 h. ** P<0.01, *** P<0.001, **** P<0.0001. PARP, poly (ADP-ribose) polymerase.

Article Snippet: Apoptosis was detected using the Annexin V-FITC/PI kit [cat. no. AT101C; MultiSciences (Lianke) Biotech Co., Ltd.].

Techniques: Flow Cytometry, Cell Counting, Western Blot