Fig 2: KCTD15 homo-and hetero-oligomerizes via BTB-POZ domain.a KCTD15 forms homo-oligomeric complexes via the BTB-POZ domain. Co-IP experiments performed in lysates from HEK293T cells cotransfected with expression vectors encoding for the indicated proteins. IP was performed with anti-Flag conjugated agarose beads. Blots were IB with anti-HA antibody and reblotted with anti-Flag antibody. b KCTD15 forms hetero-oligomeric complexes with KCASH2 via the BTB-POZ domain. Co-IP experiments were performed as above after co-transfection of vectors expressing KCTD15-HA or KCASH2 fragments as depicted in the upper panel. IP was performed with anti-Flag agarose beads, IB with anti-KCTD15 antibody and IP was controlled by reblot with anti-Flag antibody

Fig 3: KCTD15 interacts specifically with KCASH2.a KCTD15 co-immunoprecipitates with KCASH2 but not with KCASH1 and KCASH3. Co-IP assays was performed on total lysates from HEK293T cells transfected with expression vectors encoding for the indicated proteins and immunoprecipitated (IP) with anti-Flag agarose beads. IP samples and a fraction of the total lysate (Total) were separated on SDS-PAGE gels. Blots were immunoblotted (IB) with anti-KCTD15 antibody and reblotted with anti-Flag antibody. b KCASH2 interacts with KCTD15 in vitro. IVT KCTD15 was incubated with GST control or GST-KCASH2. After GST pull-down (see lower panel), the interacting protein was detected by WB with anti-KCTD15 antibody

Fig 4: KCTD15 reduces Gli1 transcriptional activity and HDAC1 protein levels but does not interact with Cul3 and HDAC1.a KCTD15 expression reduces Gli1-responsive Luciferase activity. Assays were performed on lysates from HEK293T cells transfected with 12×-GliRE-Luc alone or with Gli1, KCASH2, KCTD15 and pRL-TK Renilla (as a normalizer). Data are indicated as mean ratios with respect to Renilla luciferase signal. *P < 0.05 Gli1 versus control, **P < 0.01 KCASH2 or KCTD15 versus Gli1. b KCTD15 expression reduces HDAC1 protein levels. HEK293T were co-transfected with HDAC1-HA vector, plus empty or Flag-KCTD15 vectors and protein lysates were analyzed by WB. c HEK293T were co-transfected with Flag-GLI1 vector plus empty or KCTD15. IP was performed with anti-Flag conjugated agarose beads. Blots were IB with anti-acetylated lysine (K518) antibody and reblotted with anti-Flag antibody (upper panel). Total lysates were analysed by Western Blot using anti-KCTD15 and anti-Flag antibodies (lower panel). d, e KCTD15 is not able to interact with Cul3 (d) and HDAC1 (e). Co-IP experiments are performed in lysates from HEK293T cells transfected with expression vectors encoding for the indicated proteins

Fig 5: KCTD15 expression increases KCASH2 protein levels, and reduces Hh-dependent medulloblastoma cells proliferation.a KCASH2 protein levels are increased in DAOY MB cells expressing KCTD15 while Gli1 protein is reduced. DAOY cells were transfected with KCTD15-Flag and protein lysates were immunoblotted with anti-KCASH2 antibody (upper panels) or anti-Gli1, anti-Flag antibodies (lower panels). Anti-Actin and anti-Tubulin antibodies were used as loading controls. b Hh pathway activity is downregulated in KCTD15-transfected MB cells. Q-RT-PCR analysis of endogenous Hh targets mRNA levels are normalized to the control (Ctr). *p < 0.05 versus Ctr. c–g MB cells proliferation is reduced following KCTD15 expression. DAOY cells overexpressing KCTD15 were assayed for their capability to form colonies (c–e). Representative images were shown from three independent experiments (c) and number of colonies (d) are shown in correlation with the empty vector (Ctr). e Percentage of small, medium, and large size colonies is indicated in DAOY Ctr cells (upper panel) and DAOY transfected with KCTD15 (lower panel). f DAOY cells were transfected with indicated vectors for 24 h. Following 6 h incubation with EdU, the cells were fixed and stained with Click-iT kit. Number of EdU positive cells was calculated over total cells and expressed as percentage vs total cells. Results are expressed as the mean ± SD of three independent experiments (*P < 0.05, Student’s t-test). g MTT assay on DAOY cells transfected with empty or KCTD15 vectors and analyzed after 24 h. Results are expressed as the mean ± SD of five independent experiments, each performed in triplicate (*P < 0.05, Student’s t-test)