Fig 2: KIAA1199 exerts negative feedback on canonical Wnt signaling. A. Ectopic KIAA1199 expression in HEK293 cells inhibits the transcriptional activity elicited by wild-type CTNNB1 or by a constitutively active form of the protein, CTNNB1-T41A. Cells were transiently transfected with either a TOPflash or a FOPflash reporter vector, together with an empty vector or an expression vector for KIAA1199 or TCF4-DN. TOP/FOP ratios are the mean ± SEM of triplicate experiments. B. Total and active CTNNB1 levels in SW480 cells expressing KIAA1199 (V5) and in empty vector-transfected (Dest) controls. The table shows fold changes (vs. controls) in the expression of both CTNNB1 forms induced by KIAA1199 expression based on quantification of band intensity relative to that of CDH1 or TUBB in the same lane. TUBB was used as loading control for cytoplasmic and nuclear fractions since it was detectable in these fractions of the SW480-V5 and Dest cell extracts under the same extraction conditions. Furthermore, TUBB has been reported to shuttle to the nucleus ([50] and references herein).

Fig 3: Western blot analysis of GAPDHS, PGK2 and LDHC on ejaculated spermatozoa in normal, mature, immature and asthenozoospermia samples. Data were compared by one-way analysis of variance; *P<0.05. AS, asthenozoospermia; GAPDHS, glyceraldehyde-3-phosphate dehydrogenase, testis-specific; IM, immature spermatozoa; M, matured spermatozoa; N, normozoospermia; PGK2, phosphoglycerate kinase 2; LDHC, lactate dehydrogenase C; TUBB, ß-tubulin.

Fig 4: Cytoskeletal changes in NHDF cells after a 3-day s-µg-exposure: Immunofluorescence images of 1g-control cells, RPM-AD and RPM-MCS of vimentin (A–C). Transcriptional and translational vimentin analysis: Quantitative gene expression levels of VIM (D) and intracellular vimentin levels (E). Transcriptional and translational ß-tubulin analysis: Quantitative gene expression levels of TUBB (F) and intracellular ß-tubulin levels (G). Quantitative gene expression levels of ACTB (H). Intracellular pan-actin levels (I). Transcriptional and translational alpha smooth muscle actin analysis: Quantitative gene expression levels of ACTA2 (J) and intracellular alpha smooth muscle actin levels (K). Full-length blots of cropped Western blot images are presented in Supplementary Fig. S2. *p < 0.05 1g vs. RPM; #p < 0.05 AD vs. MCS. Scale bars: 50 µm.

Fig 5: Characterization of mPrOs-ERGM40A. Phenotypic analysis of mPrOs-WT and mPrOs-ERGM40 treated for 96 h with doxycycline (doxy) or left untreated (mock). Scale bars: 200 µm B. ERG expression in mPrOs. cDNA from VCAP cell line was used as positive control. C. Immunoblot with ERG-specific antibody of protein extracts from mPrOs-WT and mPrOs-ERG M40 treated with doxycycline for 96 h or left untreated. Gapdh was used as loading control. D. RT-qPCR analysis of known ERG-targeted genes in mPrOs-ERGM40 after treatment with or without doxycycline for 96 h mPrOs-WT were used as reference. E. Immunofluorescence analysis of ERG, Krt 8 and Krt 5 in mPrOs-WT and mPrOs-ERGM40 treated with doxycycline for 96 h (doxy) or left untreated (mock). DAPI was used for nuclear staining. Scale bars: 50 µm. F. Immunoblot analysis of Krt 8 and Krt 5 expression in mPrOs-WT and mPrOs-ERGM40 treated with doxycycline for 96 h or left untreated. Gapdh was used as loading control. G. RT-qPCR analysis of Krt 5 and Krt 8 expression in mPrOs-ERGM40 treated with doxycycline (doxy) for 96 h or left untreated (mock). H. Immunofluorescence analysis of ERG and Ki67 expression mPrOs-WT and mPrOs-ERGM40 treated with doxycycline for 96 h or left untreated. DAPI was used for nuclear staining. Scale bars: 50 µm. I. Percentage of Ki67+ cells in mPrOs-WT and mPrOs-ERGM40 treated with doxycycline for 96 h or left untreated. Quantification was performed on sections of n = 10 organoids per condition (WT mock = 1.396; WT doxy = 1.181; ERGM40 = 1.380; ERGM40 doxy = 1.345 total cells counted).J. Analysis of cell cycle progression of mPrOs-WT and mPrOs-ERGM40 treated with doxycycline for 96 h or left untreated. Histogram shows the quantification of the FACS analysis.K. Phenotypic analysis of mPrOs-WT and mPrOs-ERGM40 cultured with Egf-free medium for up to two weeks. Doxycycline was maintained throughout the duration of the experiment. Fluorescent images were acquired following 1 h incubation with 5 µM calcein. Scale bars: 200 µm. Statistical analyses were performed on at least n = 3 independent biological replicates. * = p-value <0.05; ** = p-value <0.01; *** = p-value <0.001.