Supplementary Figure 6: Regulation of protein translation and integrated stress response in cDCs.

(a) histograms show fluorescence of OP-puro incorporation in in-vitro cultured DCs, in presence or absence of the translation inhibitor cycloheximide (CHX). (b) in-vivo OP-puro incorporation in splenic cells after IP injection into CD45.1 wild type/CD45.2 XBP1ΔDC BM chimeric mice. Bar graphs represent mean protein synthesis rate +/- S.E.M. (n = 6 mice). (c) analysis of eIF2α phosphorylation in WT and eIF2α^S51A mutant MEFs upon thapsigargin treatment. Histograms and bar graphs depicting geometrical mean fluorescence are shown (n = 1 experiment). (d) phosphorylation status of 4E-BP1 in lung and intestinal cDC1s of Xbp1^fl/fl and XBP1ΔDC mice. Ratio of phosphorylated P-4E-BP1 levels over unphosphorylated 4E-BP1 in lung and intestinal WT versus XBP1ΔDCs. Bar graph shows mean +/- S.E.M. (n = 5 mice). (e) levels of DAP5 protein in lung and intestinal cDC1s of Xbp1^fl/fl and XBP1ΔDC mice. Figures in histograms depict fluorescence of 1 representative sample. Bar graphs show mean fluorescence +/- S.E.M. P = 0,0159, Mann-Whitney test (n = 5 mice). (f) RT-qPCR analysis of Gadd34 RNA prepared from sorted splenic cDC1s of Xbp1^fl/fl or XBP1ΔDC treated with ISRIB or vehicle. Results are normalized to gapdh and b-actin. Bar graphs represent mean +/- S.E.M. (n^{Xbp1fl/fl DMSO} = 11 mice, n^{XBP1fl/flDC ISRIB} = 8 mice, n^{Xbp1ΔDC DMSO} = 11 mice, n^{XBP1ΔDC ISRIB} = 10 mice). Unpaired Student T-test, P^{(Xbp1fl/fl vs XBP1ΔDC)} = 0,0483, P^{(Xbp1fl/fl DMSO vs ISRIB)} = 0,0049, P^{(XBP1ΔDC DMSO vs ISRIB)} = 0,0039. (g) histograms show fluorescence of 4E-BP1 in splenic cDC1s from Xbp1^fl/fl and XBP1ΔDC mice treated with ISRIB or vehicle. Bar graph shows mean +/- S.E.M. of 4E-BP1 expression in intestinal cDC1s (n^DMSO = 4 mice, n^ISRIB = 5 mice). Data are representative of 1 (a, c) or 2 (b, d–g) independent experiments. Source data for panel 6g can be found in Supplementary Table 2. (h) scheme illustrating ISR downstream of PERK. XBP1 deletion in cDC1s activates PERK, likely resulting into the (temporary) phosphorylation of eIF2α and ATF4 activation. ATF4 is a transcriptional regulator of Gadd34, 4ebp1 and several ISR target genes such as Mtfhd2, Sesn2, Chac1, Mars, Asns, Shmt2 and Gpt2. Upon ATF4 mediated GADD34 expression, eIF2α is rapidly dephosphorylated again. However, protein translation inhibition is sustained in a 4E-BP1-dependent manner. 4E-BP1 engages IRES-dependent translation, leading to DAP5 induction. ISRIB blocks the PERK pathway preventing induction of ATF4 and 4E-BP1. However, despite inhibition of 4E-BP1, ISRIB did not trigger cell death.