Extended Data Fig. 5: Perk signaling is required for ROCK-induced production of Creld2; Related to Fig. 5.
a, mRNA expression of indicated genes in R-PyMT tumor cells following transfection with non-targeting control (NTC) or siRNAs against Atf6 (Atf6 UPR pathway), Ern1 (Ire1α UPR pathway), Eif2ak2 (Pkr ISR pathway), Eif2ak4 (Gcn2 ISR pathway) and Eif2ak3 (Perk pathway) (n = 2/group). Mean values shown. ND = not detected. Representative data from more than 3 independent experiments. b, Bip (Atf6 pathway), Xbp1−spliced (Ire1 pathway) and Atf4 (Perk pathway) (green) in R-PyMT and K-PyMT tumor cells, transfected with either NTC or siRNAs to separately knockdown UPR pathways as indicated. Counter-labels: Phalloidin (F-Actin, red); DAPI (blue). Scales: 50 µm. Repeated twice with similar results. c, Mean distance migrated by PyMT fibroblasts exposed to CM from 4HT-treated K-PyMT tumor cells transfected with NTC or siRNAs against Ern1, Atf6 and Eif2ak3 (n = 6 KD + NTC, 4 KD + Ern1, 4 KD + Atf6, 5 KD + Eif2ak3) in inverse invasion assay. Mean±SEM, one-way ANOVA. d, Atf4 in K-PyMT and R-PyMT tumor cells transfected with NTC or siRNA targeting Perk (Eif2ak3). Mean±SEM (n = 8 KD + NTC, 11 ROCK + NTC, 9 ROCK + siRNA) relative to KD, one-way ANOVA. Representative images shown in e. e, Atf4 (green) in R-PyMT and K-PyMT tumor cells, transfected with NTC or siRNAs to knockdown the ISR mediators, Eif2ak2 (Pkr), Eif2ak4 (Gcn2) and Eif2ak3 (Perk). Counter-labels: Phalloidin (red) and DAPI (blue). Scales: 50 µm. f, Creld2 (green) in R-PyMT tumor cells treated with ISR inhibitors against Pkr and Gcn2. Counter-labels: Phalloidin (red); DAPI (blue). Scales: 50 µm. Mean±SEM (n = 4/group) relative to untreated, one-way ANOVA. See also Statistics Source Data for Extended Data Fig. 5.
