Fig 2: Senescent cells accumulate in metaphysis of long bone in young mice after GC treatment.a–c Flow cytometry analysis of the tdTom+ cells in femoral metaphysis. a Schematic diagram illustrating the experimental procedure. Three-week-old p16tdTom mice were treated with methylprednisolone (MPS) at 10 mg/m2/day or vehicle by daily intraperitoneal injection for 2 weeks. Metaphyseal bone tissue from distal femur was digested, and the isolated cells were subjected to flow cytometry analysis (see the detailed description in the “Methods” section). Representative images of tdTom-expressing cells of the femoral metaphysis is shown in (b). Percentages of tdTom+ cells in bone/bone marrow are shown in (c). d–f Three-week-old p16tdTom mice were treated with MPS at 10 mg/m2/day or vehicle by daily intraperitoneal injection for 2 weeks. Representative confocal images from frozen sections of the femur in (d). Red: tdTom+ cells; Blue: nuclear staining by DAPI. Boxed areas are shown at a higher magnification in corresponding panels to the right. Quantified numbers of tdTom+ cells in primary spongiosa and secondary spongiosa per mm2 tissue area (N. tdTom+ cells/ Ar) are shown in (e) and (f), respectively. g–l Three-week-old BALB/c mice were treated with MPS at 10 mg/m2/day or vehicle by daily intraperitoneal injection for 3 weeks. SA-ßGal staining of femoral bone sections was performed. Representative images of SA-ßGal+ cells (blue) in metaphysis are shown in (g). Quantified numbers of SA-ßGal+ cells in primary spongiosa and secondary spongiosa per mm2 tissue area (N. SA-ßGal+ cells/Ar) are shown in (h) and (i), respectively. Immunofluorescence staining of femoral bone sections was performed using antibody against HMGB1. Representative images of HMGB1+ cells (red) are shown in (j). DAPI stains nuclei blue. Quantified fluorescence intensity of HMGB1+ cells in primary spongiosa and secondary spongiosa was shown in (k) and (l), respectively. GP growth plate. Ar tissue area. PS primary spongiosa, SS secondary spongiosa. n = 5–6 mice. Data are represented as mean ± s.e.m. **p < 0.01, as determined by two-tailed Student’s t-tests.

Fig 3: HMGB1 from platelet-mediated neutrophil autophagy promotes NET formation.(A) HMGB1+ platelets were detected in whole blood samples from healthy individuals and patients and analyzed by flow cytometry. (B) The proportion of HMGB1+ platelets was significantly elevated in samples from TBI patients, especially those with coagulopathy. (C–E) Platelets from each group stained for HMGB1 (red) and CD41 (green) and observed by confocal microscopy. (F) Control neutrophils were cultured with platelets (PLT) from each group in the presence of Box A (HMGB1 competitive antagonist). The concentration of citH3-DNA complexes in the culture supernatant was measured by ELISA. (G) The expression of LC3B on neutrophils treated with platelets from each group. (H) Control neutrophils were treated with HMGB1 in the presence of HCQ (autophagy inhibitor) or Box A. The expression of LC3B on neutrophils from each group was detected by Western blotting. (I) Control platelets were incubated with NETs (0.5 µg DNA/mL) or with NETs in the presence of NET inhibitors (DNase I, APC, sivelestat). HMGB1+ platelets were detected by flow cytometry. (J–L) Representative images of platelets incubated with NETs (0.5 µg DNA/mL) or with NETs plus DNase I. Scale bars: 20 µm (C–E and J–L). Data are presented as the mean ± SD. **P < 0.01; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA with Tukey’s multiple-comparison test (B and J) or Brown-Forsythe and Welch’s ANOVA test (F and I).

Fig 4: Overview of immunogenic cell death (ICD) induced by chromomycin A5 (CA5) on melanoma. B16-F10 cells treated with CA5 displayed cell stress and cell death along with release of damage-associated molecular patterns (DAMPs). The phosphorylation of eIF2α, due to endoplasmic reticulum (ER) stress, drives crucial immunogenic events, as externalization of the “eat me” signals calreticulin (CRT) and ERp57, autophagy and apoptosis. Dendritic cells are activated by DAMPs and present tumor antigens to T lymphocytes via MCH in the presence of co-stimulatory molecules (CD80 or CD86). The activation of T lymphocytes by dendritic cells confers antitumor response. AVOs, acidic vesicular organelles; Casp3, caspase 3; PRRs, pattern recognition receptors, HMGB1, high mobility group box 1; ATP, adenosine triphosphate.