Extended Data Fig. 2: Tumor cells reinforce protumorigenic polarization in macrophages via an IL-4/IL-13 independent manner.
a, Representative histogram (left) and quantitative plot (right) of BODIPY FL C12 staining in BMDM cultured in DMEM (Ctrl) or YUMM1.7 CM (n = 4 per group). Data are representative of two independent experiments. b, c, Representative histogram (left) and quantitative plot (right) of ARG1 (b) and MRC1 (c) expression in BMDMs cultured in DMEM or CM (n = 3 per group). Data are representative of three independent experiments. d, qPCR analysis of mRNA expression of the indicated genes in BMDMs cultured in DMEM or CM for 18 h (n = 3 per group). Data are representative of three independent experiments. e, Proliferation of CFSE-labeled T cells activated with anti-CD3 and anti-CD28 alone or in co-culture with BMDM Naïve or previously exposed to CM in a ratio 2:1 (n = 6). Data are pooled of two independent experiments. f, qPCR analysis of Arg1 mRNA expression in BMDMs treated with IL-4 and IL-13 (10 ng/ml) in the absence or presence of 0.25μg/ml anti-IL-4 and 0.25μg/ml anti-IL-13 neutralizing antibodies for 18 h (n = 3 per group). g, qPCR analysis of mRNA expression of indicated genes in BMDMs treated with CM in the absence or presence of 0.25μg/ml anti-IL-4 and 0.25μg/ml anti-IL-13 neutralizing antibody for 18 h (n = 3 per group). Data are representative results of three independent experiments. h, Multiplex cytokine array was used to determine the concentration of IL-13 (left) and IL-4 (right) in CM from YUMM1.7 and MEF cells. Stand0 to Stand7 show the increased concentration detected by the standard provided by the kit (n = 4). Data are representative of two independent experiments. i–k, Representative histogram (left) and quantitative plot (right) of BODIPY staining (i), and protein expression of ARG1 (j), and MRC1 (k) in BMDMs stimulated with regular culture medium (Ctrl) or CM from YUMM1.7 (CM) or MEF (CM MEF) (n = 3 per group). Data are representative results of three independent experiments. l, qPCR analysis of indicated genes in BMDMs exposed to CM or Tunicamycin for 18 h (n = 3 per group). Data are representative results of three independent experiments. m, Quantification of BODIPY staining in BMDMs treated with CM in the absence or presence of 50μM STF083010; Ctrl (n = 7), CM (n = 7), CM-STF (n = 6). Data are pooled from three independent experiments. n, Immunoblots of indicated proteins in BMDM transduced with retrovirus expressing scramble or IRE1-targeting gRNAs. o, qPCR analysis of BIP and sXBP1 mRNA expression in BMDMs treated with 1μγ/ml of Tunicamycin, 1μM thapsigargin and CM for 16 h (n = 3 per group). Data are representative results of three independent experiments. Data are mean ± s.e.m. were analyzed by two-tailed, unpaired Student’s t-test or one-way ANOVA with Tukey’s multiple comparison test (e).
