Supplementary Figure 5: Modulation of Ppp6c expression affects sensitivity to type 1 interferons.

(a) Flow cytometry for CD69 expression after IFN-ɣ treatment of T hybridoma cells. 7678 hybridoma cells were infected with lentiviral constructs driving either expression of miR-31 or shRNAs against miR-31 target genes (Lats2, Stk40, Ppp6c, Sh2d1a, luciferase as control). Cells were mixed with uninfected cells, treated with the indicated concentration of IFN-ɣ for 18 hours, and CD69 expression relative to uninfected cells was determined by FACS. (P values from ratio paired two-tailed Students t test: sh-Control vs pre-miR-31= 0.005; sh-control vs sh-Ppp6c = 0.002) (b) Flow cytometry for CD69 expression after IFN-ɣ treatment of Mir31^-/- OT-I T cells infected with lentiviral constructs driving expression of miR-31 or shRNAs targeting Ppp6c or luciferase (control). CD69 expression was measured following treatment with the indicated IFN-ɣ for 18 hours. (P values from ratio paired two-tailed Students t test: sh-Control vs pre-miR-31= 0.005; sh-control vs sh-Ppp6c = 0.003) (c) Western blot for Ppp6c protein expression in Mir31^+/+ and Mir31^-/- OT-I T cells. OT-I T cells from three mice/condition were activated with peptide-pulsed splenocytes for 24 hours and cultured in media containing IL-2 for 15 days; 3x10⁵ cells/lane were lysed and expression of β-actin and Ppp6c was examined by Western blot. Relative expression of Ppp6c normalized to b-actin is indicated. (n=3/group, p value from unpaired two-tailed Students t test=0.0008) (d) Full blot images for Western blot data.. Left panel displays size marker data from SeebluePlus2 pre-stained protein size standards. Middle and right panel display images collected within the linear response range for β-actin (middle) and Ppp6c (right). Data are representative of one (a), three (c), or two (d) experiments. ** p value <0.01, *** p value <0.001)