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. 2015 Dec 15;6:10204. doi: 10.1038/ncomms10204

Figure 4. Epigenetic-dependent sustained JAK1/STAT3 signalling in CAF.

Figure 4

(a) Immunoblot of pJAK1, pSTAT3 and AcSTAT3 in CAF after 7-days treatment in the presence or absence of 5′-Aza inhibitor at 10 and 3 μM final concentration. Immunoblot of JAK1, STAT3 and tubulin as control. (b) Representative images of H&E coloration of paraffin-embedded sections of SCC12 in response to CAF following 5′-Aza inhibitor alone or in combination with PTP inhibitor 1 (n=3, mean±s.d., ***P<0.001). (c) Immunoblot of DNMT1, pSHP-1, SHP-1, pJAK1, pSTAT3 and AcSTAT3 in CAF following 5′-Aza inhibitor alone or in combination with PTP inhibitor 1. Immunoblot of JAK1, STAT3 and tubulin as control. (d) Immunoblot of DNMT3b, SHP-1, pJAK1, pSTAT3 and AcSTAT3 in hDF activated or not by LIF transfected with siRNA control (siLuc) or siRNA targeting DNMT3b (siDNMT3b#1, #2). Immunoblot of JAK1, STAT3 and tubulin as control. (e) Schematic illustration of the PTPN6 gene structure and the regions analysed by bisulfite sequencing. The PTPN6 gene has two alternative promoters. We restricted our analysis to Promoter 1 since Promoter 2 is mostly active in the haematopoietic lineage. Vertical lines in Region 1 and Region 2 indicate the position of the CpG dinucleotides. (f) Bisulfite-sequencing results in two regions of the PTPN6 promoter (R1 and R2) in hDF transfected with control RNAi (siLuc) and RNAi targeting DNMT3b (siDNMT3b#1 and #2) and subsequently long-term control (−) or LIF-activated (+). Each line represents an individual sequence. Open and closed circles denote unmethylated and methylated CpG dinucleotides, respectively. Donut charts summarize the global proportion of methylated CpG with the percentage in the center (n=2, P<0.0001, two-tailed Fisher's exact test). (g) Representative images of H&E coloration of paraffin-embedded sections of SCC12 in response to CAF overexpressing control vector (GFP) or SHP-1 gene (SHP-1_GFP) (n=3, mean±s.d., ***P<0.001). (h) Immunoblot of SHP-1, pJAK1, pSTAT3 and AcSTAT3 in CAF overexpressing control vector (GFP) or SHP-1 gene (SHP-1_GFP). Immunoblot of JAK1, STAT3 and tubulin as control.