Extended Data Fig. 3: (related to Figure 3): Dying tumor cells activate mTORC1 via ATP/P2X4 in human CRC and PDAC.

a, Relative mRNA expression levels of the indicated genes in the colon tumors from Lgr5^EGFP-DTR(+) mice 24h after injection of DT injection or vehicle determined by qRT-PCR. (n = 10 tumors for controls and n = 9 tumors for DT). b, Relative mRNA expression levels of the indicated genes in Lgr5^EGFP-DTR(+) tumor organoids at 24h after DT treatment determined by qRT-PCR. Expression of vehicle treated organoids was set to 100 arbitrary units (n = 4 biological replicates). c, Immunoblot analysis of Lgr5^EGFP-DTR(+) tumor organoids treated as indicated for 30 min (n = 3). d, Immunoblot analysis of Lgr5^EGFP-DTR(+) tumor organoids treated as indicated for 16h (n = 3). e, Immunoblot analysis of Sting WT and Sting KO Lgr5^EGFP-DTR(+) tumor organoids treated as indicated (n = 4). f, Immunoblot analysis of mouse (CMT93 and CT26, left panel) and human CRC cells (HCT116 and RKO, right panel) treated as indicated (n = 2). g, Immunoblot analysis of Lgr5^EGFP-DTR(+) tumor organoids treated as indicated for 12h (n = 3). h, Left: Immunoblot analysis of ATP (100 µM) treated CT26 cells transfected with control or Rheb siRNA (n = 3). Right: Relative mRNA expression levels of Rheb in siRNA transfected cells determined by qRT-PCR (n = 3 biological replicates). i, Left: Immunoblot analysis of ATP (100 µM) treated CT26 cells transfected with control or a combination of RagA and RagB siRNA (n = 3). Right: Relative mRNA expression levels RagA and RagB in siRNA transfected cells determined by qRT-PCR (n = 3 biological replicates). j, Relative mRNA expression levels of P2x and P2y receptors in mouse colon tumor organoids tissues determined by qRT-PCR (n = 3 biological replicates). k, Immunohistochemistry analysis of P2X4 expression in healthy mouse colon or colon tumors of AOM/DSS treated mice. Images from one representative staining (n = 3, scale bar = 300 µm). l, Reseeding capacity of hCRC tumor organoid line 2 treated as indicated (n = 3, one of three biological replicates). m, Reseeding capacity of doxycycline-inducible hCRC^shP2X4#2 tumor organoids treated as indicated (n = 4, one of three biological replicates). n, P24 qRT-PCR analysis of doxycycline treated hCRC^shP2X4#2 tumor organoids (n = 3 biological replicates). o, Reseeding capacity of human pancreatic tumor organoids treated as indicated (n = 3, one of three biological replicates). p, Reseeding capacity of human pancreatic tumor organoids treated as indicated (n = 3, one of three biological replicates) All data are mean ±SD and analysed by two-tailed Student’s t-test (a,b,h,i,n,o,p) or by 1-way ANOVA with Bonferroni’s multiple comparison (l,m).