Fig 2: ATXN7L3 positively regulates SMAD7 transcription. (a) The mRNA expression of ATXN7L3 is positively correlated with that of SMAD7 in TCGA database. The relative level of ATXN7L3 was plotted against that of SMAD7. (b) Knockdown of ATXN7L3 downregulated mRNA expression of SMAD7. HCCLM3 cells were transfected with siCtrl or siATXN7L3. After 48 hr, cells were collected for RNA extraction and quantitative real-time PCR were performed. Data represents the mean ± SD of triplicate real-time PCR. (c) Knockdown of ATXN7L3 downregulated protein expression of SMAD7. HCCLM3 cells were transfected with siCtrl or siATXN7L3. After 48 hr, cells were collected for protein extraction and subjected to western blot analysis using indicated antibodies. The results were quantified through Image J software and GAPDH was used as internal control. (d) Ectopic expression of ATXN7L3 upregulated protein expression of SMAD7. HCCLM3 cells were transfected with ATXN7L3-expressing plasmids with different doses or control vector. After 24 hr, cells were collected for protein extraction and subjected to western blot analysis using indicated antibodies. The results were quantified through Image J software and GAPDH was used as internal control.

Fig 3: ATXN7L3 associates with ERα and acts as a coactivator of ERα. (a) ATXN7L3 and ERα interacts with each other in HEK293 cells. HEK-293 cells were co-transfected with plasmids expressing ATXN7L3 and ERα. After 24 hr, cells were treated by E2 (10−7 M) or ethanol vehicle for 4 hr. Then cells were harvested and equal amounts of cell lysates were subjected to immunoprecipitation with normal IgG, or anti-ERα antibodies. The immunoprecipitated proteins were subjected to western blot analysis using indicated antibodies. (b-c) Endogenous ATXN7L3 and ERα interacts with each other in HCCLM3 cells. Upon treatment of 10−7 M E2 or ethanol vehicle for 24 hr, cells were harvested and equal amounts of cell lysates were subjected to immunoprecipitation with normal IgG, anti-ATXN7L3 or anti-ERα antibodies. The immunoprecipitated proteins were subjected to western blot analysis using indicated antibodies. (d) Subcellular localizations of the ATXN7L3 and ERα. HCCLM3 cells were co-transfected with plasmids expressing ATXN7L3 and ERα. After 10−7 M E2 stimulation or ethanol vehicle for 4 hr, HCCLM3 cells were fixed and stained with antibody against ATXN7L3 (green) or ERα (red). DAPI was used to visualize the nucleus (blue). Merged images were shown as indicated. Scale bar, 20 μm. (e) Identification of domains in ERα for ATXN7L3 interaction. ATXN7L3 protein was synthesized by transcription and translation kit in vitro, then was incubated with GST or GST- ERα deletion mutants. Bound proteins were washed and analyzed by western blot. GST and GST- ERα deletion mutants were stained by Coomassie brilliant blue staining. *, position of GST and GST- ERα deletion mutants. (f-h) ATXN7L3 enhances ERα-mediated transactivation. HEK-293 cells (f), HepG2 cells (g) or HCCLM3 (h) were co-transfected with ERE-luc, pRL-TK and ERα, together with the indicated expression plasmids or siRNA in the presence of 10−7 M E2 or ethanol vehicle. After 24 hr, cells were lysed and assayed using the dual-luciferase reporter assay system. The expression of ATXN7L3 was evaluated by western blot in lower panel. Error bars represent mean ± SD of three replicates. The western blot results were quantified through Image J software and GAPDH was used as internal control.

Fig 4: Schematic representation of ATXN7L3 functions on SMAD7 transactivation and suppression of tumor growth in HCC.

Fig 5: Related to Fig 4, USP22 is required for efficient recruitment of Pol II to activator‐induced target genes independent of Ub‐H2B AFlowchart depicting ChIP‐seq subsetting.BMetagene profiles for Pol II (top) and Ub‐H2B (bottom) ChIP‐seq for all genes not directly bound by USP22 and insensitive to USP22 knockdown, termed “Insensitive” genes.C, DChIP‐qPCR for ATXN7L3 and GCN5 at the indicated loci, before and after ER stress, in the presence and absence of shRNA targeting USP22, represented from three independent experiments as mean ± SEM.