Journal: Frontiers in Immunology
Article Title: Mitochondrial metabolism is rapidly re-activated in mature neutrophils to support stimulation-induced response
doi: 10.3389/fimmu.2025.1572927
Figure Lengend Snippet: Mitochondrial metabolism and neutrophil response in varied calcium availability. (A) Immunoblot of phospho-PDH[ser293], phospho-PDH[ser300], total PDH E1 subunit, and β-actin in differentiated ER-Hoxb8 neutrophils stimulated with ionomycin for indicated duration. Relative signals (normalized by β-actin from the same blot) from 4 different independent experiments were made relative to the unstimulated condition and compiled. Representative images of immunoblots are shown to the right. Significance was determined with RM two-way ANOVA with Geisser-Greenhouse correction and Šídák’s multiple comparisons test. (B) Labeling pattern of glycolysis and TCA intermediates from U 13 C-glucose in primary human neutrophils treated under specified conditions for 1 hour. Data shown are representative of experiments from 3 different human donors. (C) Extracellular DNA release over time of primary human neutrophils cultured under specified conditions. Representative microscopy images at the 4.5-hour time point are shown to the right. Data shown are representative of experiments from 3 different human donors. (D) MPO activity assay from supernatant of primary human neutrophils treated for 4 hours under specified conditions. Data shown are representative of experiments from 3 different human donors. Significance was determined by ordinary one-way ANOVA and Šídák’s multiple comparisons test. (E) Number of neutrophils that migrated through a transwell to C5a after 1.5 hours under specified treatment conditions. Data shown are representative of experiments from 3 different human donors. Significance was determined by unpaired t test. Statistical significance is indicated by the following annotations: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Article Snippet: Primary antibodies: Citrullinated Histone H3 (abcam, ab5103), Histone H3 (Cell Signaling, #9715), phospho-PDH [ser293] (Novus Biologicals, NB110-93479), phospho-PDH [ser300] (Millipore Sigma, ABS194), PDH (Cell Signaling, #2784), β-actin (Cell Signaling, 4967); and secondary antibodies: Goat-anti-rabbit 800 (LI-COR, 925-32211), Goat-antimouse 680 (LI-COR, 925-68070).
Techniques: Western Blot, Labeling, Cell Culture, Microscopy, Activity Assay
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![Mitochondrial metabolism and neutrophil response in varied calcium availability. (A) Immunoblot of <t>phospho-PDH[ser293],</t> phospho-PDH[ser300], total PDH E1 subunit, and β-actin in differentiated ER-Hoxb8 neutrophils stimulated with ionomycin for indicated duration. Relative signals (normalized by β-actin from the same blot) from 4 different independent experiments were made relative to the unstimulated condition and compiled. Representative images of immunoblots are shown to the right. Significance was determined with RM two-way ANOVA with Geisser-Greenhouse correction and Šídák’s multiple comparisons test. (B) Labeling pattern of glycolysis and TCA intermediates from U 13 C-glucose in primary human neutrophils treated under specified conditions for 1 hour. Data shown are representative of experiments from 3 different human donors. (C) Extracellular DNA release over time of primary human neutrophils cultured under specified conditions. Representative microscopy images at the 4.5-hour time point are shown to the right. Data shown are representative of experiments from 3 different human donors. (D) MPO activity assay from supernatant of primary human neutrophils treated for 4 hours under specified conditions. Data shown are representative of experiments from 3 different human donors. Significance was determined by ordinary one-way ANOVA and Šídák’s multiple comparisons test. (E) Number of neutrophils that migrated through a transwell to C5a after 1.5 hours under specified treatment conditions. Data shown are representative of experiments from 3 different human donors. Significance was determined by unpaired t test. Statistical significance is indicated by the following annotations: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6771/pmc12066771/pmc12066771__fimmu-16-1572927-g003.jpg)