Fig. 2: Immunogenic treatments elicit chemokine production independent of cell death. | Cell Death & Differentiation

Fig. 2: Immunogenic treatments elicit chemokine production independent of cell death.

From: Stress-induced inflammation evoked by immunogenic cell death is blunted by the IRE1α kinase inhibitor KIRA6 through HSP60 targeting

Fig. 2: Immunogenic treatments elicit chemokine production independent of cell death.

A Heatmap obtained from RNA-seq data representing the log2(fold changes) of cytokine and chemokines genes upon treatment with CDDP, MTX, or Hyp-PDT compared to time-matched untreated controls. B Chemokine transcription was evaluated at short timepoints after treatment with CDDP, MTX, or Hyp-PDT with a time course analysis performed by qPCR. Data are expressed as log2(fold change) compared to time-matched untreated control. Asterisks are color-matched to the treatment. C Intracellular CXCL8 accumulation and CXCL3 depletion at the indicated timepoints upon treatment with MTX or Hyp-PDT. D Intracellular accumulation of CXCL8 (green) in control (CTRL) condition or 8 h after treatment with CDDP, MTX, or Hyp-PDT. Nuclei were counterstained with DAPI (blue). Scale bar: 10 μm. E CXCL8 secretion was measured by ELISA in conditioned medium at 8 h and 24 h after treatment with CDDP, MTX, or Hyp-PDT. CXCL8 secretion at 24 h after treatment was blocked by Brefeldin A (BFA, 50 ng/ml). F–H Evaluation of the impact of z-VAD-FMK (50 μM) on CXCL8 transcription (F), intracellular protein accumulation (G) and secretion (H) after treatment with Hyp-PDT or MTX was assessed by qPCR, western blot, and ELISA, respectively. Values are represented as log2(fold change) in (F) and fold change over untreated control in (G). I Impact of siRNA mediated DR5 silencing (siDR5) on CXCL8 protein production measured by western blot 24 h after treatment with MTX or Hyp-PDT compared to scrambled siRNA (siCTRL). J Secretion of CXCL8 in conditioned medium of siDR5, siCasp8, and siCTRL A375p cells measured by ELISA 24 h after treatment with MTX. K Cell death was assessed in siDR5, siCasp8, and siCTRL A375p cells 24 h after treatment with MTX or Hyp-PDT. In all western blots β-actin was used as loading control. In all graphs values are presented as mean ± SD of at least n = 3 independent biological replicates. Data are analyzed by one-sample t-test in (B), One-way ANOVA followed by Dunnett’s multiple comparison test in (E, K) and two-tailed Student’s t-test in (F–H), *p < 0.05,**p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.

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