Fig 2: USP4 and USP17L22 affect the timing of activation of STAT3. a, b Induction of USP17L22 results in a shift of STAT3 activation to later time points in BJhTERT inducible cells. Expression of Flag-tagged USP17L22 was induced with doxycycline, cells were serum-starved and stimulated with PDGF-BB (20 ng/ml) for the indicated time periods. Expression of total and phosphorylated proteins was determined in total cell lysates using antibodies against PDGFRβ, Flag, STAT3 (pY705), STAT3, PLCγ (pY783), PLCγ, pS473 Akt1/2/3, Akt1/2/3, pThr202/pThr204 Erk1/2, Erk1/2 and β-actin (a). Phosphorylated STAT3 (pSTAT3) versus total STAT3 levels were quantified in three experiments; peak of phosphorylation at 10 min of stimulation with PDGF-BB was set as 1. Standard deviation is shown between repeats; *p < 0.05 (b). c, d Induction of USP4 increases the STAT3 activation in BJhTERT-USP4 inducible cells. BJhTERT-USP4 inducible cells were treated as described and immunoblotting was performed for proteins as in panel a; α-tubulin was used as loading control. Activated pSTAT3 relative to STAT3 protein levels were quantified in three independent experiments as in panel b; *p < 0.05 (d). e CRISPR-Cas9 knockout of USP4 decreases the STAT3 activation in BJhTERT-CRISPR-Cas9-USP4 knockout cells. The control BJhTERT and CRISPR-Cas9-USP4 knockout BJhTERT cells were starved overnight and stimulated with PDGF-BB (20 ng/ml) for the indicated time periods. Immunoblotting was performed for the indicated proteins, as in panels a–d. f USP4 does not deubiquitinate STAT3. After USP4 induction and serum starvation, BJhTERT-USP4 cells were stimulated with PDGF-BB (20 ng/ml) for the indicated time periods, lysates were divided and immunoprecipitated with anti-PDGFRβ (CTβ) or anti-STAT3 antibodies. Eluates were immunoblotted for ubiquitin. Levels of PDGFRβ, Flag-USP4 and α-tubulin were determined by immunoblotting (IB)

Fig 3: (A) Determination of STAT3 activation in U251 cells using a STAT3 assay kit. STAT3 activation was measured in cells cultured for 24 h in the absence (EtOH) or presence of 1α,25-dihydroxyvitamin D3 (1α,25) or tacalcitol (Tac) in the indicated concentrations. The values are presented as mean ± standard deviation of three independent determinations. (B) Effects of 1α,25-dihydroxyvitamin D3 and tacalcitol on anchorage-independent growth of U251 cells; representative images of colonies in wells treated with vehicle (EtOH) or 1 μM of 1α,25-dihydroxyvitamin D3 or tacalcitol. (C) Quantitative analysis of colonies in the presence of vehicle or 0.2–5 μM of 1α,25-dihydroxyvitamin D3 (1α,25) or tacalcitol (Tac) in U251 cells. The number of formed colonies with a diameter ≥40 μm were counted. The values represent mean ± standard deviation of 7–9 data points from three independent experiments. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Fig 4: Activation of STAT3 promotes increased DNA binding and results in acute induction of STAT3 target genes. a, b Transcriptional activity of STAT3 is increased upon induction of DUBs and PDGF-BB stimulation. USP17L22 (a) or USP4 (b) were induced in the BJhTERT tet-inducible cell lines, cells were starved overnight and stimulated with PDGF-BB for 0, 15, 30 and 60 min. 20 µg of nuclear extracts were incubated with STAT3 binding element 5ʹ-TTCCCGGAA-3ʹ in a microplate and binding was quantified by colorimetric ELISA. Positive control was 5 µg of HepG2 cells stimulated with 100 ng/ml IL-6 (as supplied in the kit). The value for negative control was obtained by incubating each of the unstimulated or stimulated for 15, 30 or 60 min nuclear extracts from the uninduced BJhTERT cells with the addition of 20 pmol of unbound oligonucleotide that competed with the binding of activated STAT3 to the plate (as supplied in the kit). Absorbance values were plotted for three independent repeats; standard deviation is shown. The statistical significance was determined by one-tailed t test with unequal variance; *p < 0.05. c–h Induction of USP17L22 promotes acute upregulation of STAT3 target genes. USP17L22 was induced in the BJhTERT tet-inducible cell line which were stimulated with PDGF-BB for 1, 2 or 3 h and the expression of mRNA for STAT3 (c), CSF-1 (d), myc (e), SOCS3 (f), junB (g) and CDKN1A (h) was determined by quantitative PCR

Fig 5: IL-1ß and VEGF-A expression levels are increased in inflammatory relative to alternatively activated macrophages(A) Volcano plot demonstrating RNA-seq transcriptome data displaying expression profiles from mouse bone marrow-derived macrophages (BMDMs) treated with either LPS+IFN-? or IL-4+IL-13 for 24 h. IL-1ß, VEGF-A, HIF-1a, STAT3, and RELA are marked by arrows and red dots. Dashed line, p = 0.05.(B–F) Quantitative RT-PCR of relative (normalized to HPRT) mRNA expression for IL-1ß, VEGF-A, HIF-1a, STAT3, and RELA from the BMDM lysates treated as in (A) (*, p < 0.05 by t test; n = 4 mice total; two males and two females). Data, mean ± SD.