Fig 1: 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 2: miR-454-3p or siSTK33 induces autophagy in NB cells. (A, B) Relative protein expression levels of STK33, p62, and LC3-II in SK-N-DZ and SK-N-AS cells transfected with miR-454-3p or siSTK33 were determined by Western blotting, and the blots are shown. Data represent mean ± SEM t-test: *P < .05, **P < .01, ***P < .001.
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: Schematic illustration of miR-454-3p inducing autophagy and apoptosis of neuroblastoma via inhibiting the RNA levels of STK33. RNA levels of STK33 are suppressed by miR-454-3p and result in increased autophagy and apoptosis and reduced cell viability according to the markers indicated(Created in BioRender. (2024) https://BioRender.com/a57y202).
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
Supplier Page from Sino Biological, Inc. for Human STK33 Gene ORF cDNA clone expression plasmid, N-Flag tag