GSK-3 Governs Inflammation-Induced Nfatc2 Signaling Hubs to Promoter Pancreatic Cancer

GSK-3 governs inflammation-induced NFATc2 signaling hubs to promoter pancreatic cancer progression

Supplementary Figure Legends

Supplementary Figure 1

(A) Representative views of nude mice treated with 10% DMSO or 10 mg/kg AR-A014418 (hereafter designated GSK-3i)

(B) IMIM-PC1 cells were starved and nuclear translocation was stimulated with 0.5 μMionomycin for 30 min. Subsequently, they were treated with 25 and 50 μM GSK-3i and subjected to subcellular fractionation and immunoblotting as indicated. PARP serves as nuclear loading control.

(C) PaTu8988t cells were transfected with HA-tagged wt NFATc2 and NFATc2 SP2 constructs and subjected to co-immunoprecipitation and immunoblotting as indicated.

(D) PaTu8988t cells stably expressing HA-tagged wt NFATc2 were treated with GSK-3i for 24 h and analyzed for expression levels of indicated proteins. Densitometric quantification of NFATc2 and p-NFATc2 expression (n=3), illustrated as p-NFATc2/NFATc2, *p=0.065.

(E) PaTu8988t stably expressing V5-tagged GSK-3b S9A were analyzed for expression levels of indicated proteins.Densitometric quantification of NFATc2 and p-NFATc2 expression (n=3), illustrated as p-NFATc2/NFATc2 ratio: *p=0.005.

(F) Immunohistological analysis of p-NFATc2 (S213/217/221), p-GS (S641/645) expression and ki67 in tissues from nude mice after treatment with 10% DMSO or GSK-3i.

(G) Quantification of ki67 staining in n≥6 xenograft tumors shows significant reduction upon GSK-3i treatment, ***p<0.001.

(H) PaTu8988t cells stably expressing wt NFATc2 and NFATc2 pSP2 following GSK-3 knockdown were tested for HA-NFATc2, GSK-3 and -actin expression.

Supplementary Figure 2

(A) Immunoblot demonstrating successful knockdown of GSK-3 in PaTu8988t cells (related to reporter gene assay shown in Figure 2A).

(B) Luciferase reporter gene assay demonstrating regulation of the cisNFATpromoter in PaTu8988t cells upon transfection with wt NFATc2 and NFATc2 pSP2 or mock cells following treatment with 25 μM GSK-3i. Means ± SD.

(C) PaTu8988t cells with stable expression of HA-tagged wt NFATc2 and NFATc2 pSP2 were subjected to co-immunoprecipitation and immunoblotting as indicated.

(D) Reporter gene assay of Suit-007 cells following transfection of STAT3 siRNA, GSK-3 S9A and cisNFAT. Means ± SD.

Supplementary Figure 3

(A) Expression of p-STAT3 (Y705) in PaTu8988t cells following GSK-3b knockdown.

(B) Expression of p-STAT3 (Y705) and STAT3 in murineKrasG12D;p53-/-cells following treatment with GSK-3i.

(C) PaTu8988t cells upon treatment with IL-6 and GSK-3i were subjected to co-immunoprecipitation as indicated.

(D) PaTu8988t cells with stable expression of wt NFATc2 and NFATc2 SP2 were transfected with STAT3 siRNA and 3H-thymidine incorporation was measured after 72h.

(E) Immunoblot analysis for CDK 6 expression in PaTu8988t cells following GSK-3 inhibition.

Supplementary Figure 4

Immunohistochemistry for CD45 in 3 representative Pdx1-Cre;KrasG12D mice shows increased numbers of infiltrating immune cells upon caerulein treatment whereas GSK-3i markedly reduces this phenotype.

Supplementary Table 1

Results from tissue microarray (TMA) analysis of 217 human pancreatic cancer cases showing both frequency of nuclear NFATc2 expression (A) as well as frequency of pSTAT3 and NFATc2 expression (B).