Fig 2: TTC36 reduces the binding of STK33 to HPD and subsequent STK33-mediated HPD T382 phosphorylation. Immunoblotting analyses were performed with the indicated antibodies a–i. a Immunoprecipitation analyses of LO2 cell lysates were performed with an anti-HPD antibody. b, c Ttc36 shRNA b or Flag-TTC36 c were stably expressed in LO2 cells. Immunoprecipitation analyses with an anti-HPD antibody were performed. d Phos-tag analysis was performed with bacterially purified His-STK33 and His-HPD or His-HPD T382A mutant in the presence or absence of ATP. e Ttc36 shRNA#1 (left panel) or Flag-TTC36 (right panel) were stably expressed in LO2 cells. The quantification numbers represent relative intensity of presented bands. f LO2 cells expressing Flag-HPD or Flag-HPD T382A mutant were treated with the indicated dosages of ML281 for 48 h. The quantification numbers represent relative intensity of presented bands. g LO2 cells expressing Stk33 shRNA#1 or #2 (left panel) with or without reconstituted expression of RNA-resistant(r) STK33 (right panel) were examined by immunoblotting analyses with the indicated antibodies. The quantification numbers represent relative intensity of presented bands. h, i HEK293T cells were transfected with the indicated plasmids. MG-132 (10 μM) was added 12 h before they were harvested with a guanidine–HCl-containing buffer. A Ni-NTA pull-down assay was performed

Fig 3: miR-454 and siSTK33 reduce cell viability and protein expression. (A) Cell viability changes for miR-454-3p mimic or siSTK33-transfected NB cell lines were determined at 96 hours post-transfection relative to control with Quanti-Max assays. (B, C) Quanti-Max viability assays were performed with miR-454-3p mimic or antagonist and their controls in SK-N-DZ- and SK-N-AS-transfected NB cell lines at the indicated time points post transfection. Cell viability was detected in 24-hour intervals for 96 hours. (D, E) Western blot and Quanti-Max assays were performed to evaluate STK33 expression and cell viability changes in cells transfected with siSTK33, with or without overexpression constructs tagged with C-HA and N-Flag. Plots were “% Viability” with respect to the control-treated cells. (F) Cell viability was measured at different time points in SK-N-DZ and SK-N-AS cell lines following transfection with siSTK33, using the Quanti-Max assay and plotted as “% Viability” with respect to the control-treated cells. Data represent mean ± SEM t-test: *P < .05, **P < .01, ***P < .001.

Fig 4: STK33 levels are regulated by growth factors and stress conditions. (A) Relative mRNA levels of STK33 and miR-454-3p in the SK-N-DZ and SK-N-AS treated with FGF2/heparin, insulin, starved, or treated with tunicamycin for 24 hours are shown. miR-454-3p was calculated by the miR-454-3p:U6 ratio, and STK33 was calculated through the relative expression of the STK33:GAPDH ratios from the qPCR reads. (B) Relative protein levels of STK33 in the FGF2/heparin, insulin, starved, or treated with tunicamycin for 24 hours were determined by Western blotting, and the blots are shown. (C, D) Western blot and Quanti-Max assays were performed to evaluate STK33 expression and cell viability in cells transfected with miR-454-3p mimics, with or without overexpression STK33 constructs tagged with C-HA and N-Flag. Plots were “% Viability” with respect to the control-treated cells. Data represent mean ± SEM t-test: *P < .05, **P < .01, ***P < .001. Abbrevation: GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Fig 5: miR-454-3p directly targets STK33 in NB cells. (A) Relative protein expression of STK33 was confirmed in 5 neuroblastoma cell lines by Western blotting for the indicated proteins. (B) Protein expression changes for STK33 were confirmed by transfecting miRNA mimics and antagonists into NB cell lines by Western blotting for the indicated proteins. (C) Based on the miRNA prediction database, a schematic representation of the direct binding sites of miR-454-3p and STK33 is shown. (D) Luciferase activities were performed in SK-N-DZ and HEK-293 cells cotransfected with STK33 3′ UTR (wild type or binding motif mutant) reporter plasmid, and miR-454-3p mimic or miR NC. (E) qRT-PCR analysis of STK33 mRNA expression in SK-N-DZ and SK-N-AS cells transfected with miR-454-3p mimic or miR NC was performed. miR-454-3p levels were calculated by the miR-454-3p:U6 ratio in the pPCR, and STK33 RNA levels were calculated as a relative expression from the STK33:GAPDH ratio from the qPCR signal. Data represent mean ± SEM t-test: *P < .05, **P < .01, ***P < .001. Abbrevation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR: Real-Time Quantitative Reverse Transcription PCR