FIGURE 3.

Activation of aPKCι/λ triggers STAT3 activation when PAR3 is silenced. A, NICD1-mMECs infected with lentivirus to express the shRNAs indicated were harvested, and equal amounts of lysate were immunoblotted for PAR3, phosphothreonine 560 aPKC, total aPKCι/λ, and β-tubulin (loading control). B, NICD1-mMECs infected with lentivirus to express the shRNA constructs indicated were harvested, and equal amounts of lysate were immunoblotted for PAR3, aPKCι/λ, phospho-STAT3, total STAT3, and β-tubulin (loading control). C, quantitation of phospho-STAT3/total STAT3 in B (n = 4). n.s., not significant. D and E, NICD1-mMECs or NMuMGs infected with lentivirus to express the constructs indicated were harvested, and equal amounts of lysate were immunoblotted for PAR3, aPKCι/λ, phospho-STAT3, total STAT3, and GAPDH (loading control). Arrowhead, expressed tRFP-tagged aPKCι/λ. F, NICD1-mMECs expressing tRFP-tagged aPKCi-CA or tRFP (control) were lysed and immunoblotted for phospho-T560 aPKC, total aPKCι/λ, phospho-STAT3, total STAT3, and β-tubulin (loading control). Arrowheads, expressed tRFP-tagged aPKCi-CA. G, quantitation of phospho-STAT3/total STAT3 in F (n = 4). H, NICD1-mMECs infected with lentivirus to express the indicated constructs were harvested and immunoblotted for phospho-Thr-560 aPKC, total aPKCι/λ, GP130, phospho-STAT3, total STAT3, and β-tubulin (loading control). I, quantitation of phospho-STAT3/total STAT3 in H (n = 3). J, primary murine mammary cells were harvested and infected with lentivirus to express the constructs indicated and then cultured as mammospheres in non-adherent conditions for 5 days. After culture, the mammospheres were harvested and lysed, and equal amounts of lysate were immunoblotted for phospho-Thr-560 aPKC, total aPKCι/λ, and GAPDH (loading control). Error bars, S.E.