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. Author manuscript; available in PMC: 2015 Jun 15.
Published in final edited form as: Nat Commun. 2014 Dec 15;5:5798. doi: 10.1038/ncomms6798

Figure 4. STAT3 transcriptional activity is required to repress Ube2n expression.

Figure 4

(a) Ube2n mRNA was measured in bone marrow-derived macrophages from Stat3-sufficient mice following treatment with IL-6 as indicated, using qPCR. (b and c) Ube2n promoter activity was measured by reporter assays in HEK293T cells transfected with pGL3-Ube2n (Ube2n), pGL3-Ube2n-mut (Ube2n-mut; containing a mutated STATx site), pMX vector (vector), pMX-STAT3C (STAT3C), pMX-STAT3 DN (STAT3 DN) and pTK-Renilla, as indicated. pMX-STAT3 DN and pMX-STAT3C were included in some samples in a 3:1 or 6:1 ratio, as shown. (d) Stat3−/− MEFs were transfected with pMX-STAT3C, pMX-STAT3 DN or both, at the ratios indicated. The expression level of pSTAT3, total STAT3 and Ubc13 was detected by immunoblotting. Ran served as loading control. (e and f) Bone marrow-derived macrophages from Stat3-sufficient mice were cultured in M-CSF- and RANKL-containing medium in the absence or presence of the STAT3 inhibitor PM-73G, as indicated. Ubc13 amounts were detected by immunoblotting (e); osteoclast gene expression was measured by qPCR (f). (d and e) Relative Ubc13 expression vs. STAT3C transfection alone (d) or untreated cells on 1 d (e) was calculated as indicated in Fig. 1g and displayed below bottom panels. (a, b, c and f) Data represent mean values of 3 independent experiments. Error bars indicate SEM. Two-way Anova with Bonferroni multiple comparison used. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 for the indicated comparisons. (d and e) Results represent 3 independent experiments.