Fig 1: MiR-451a hampers the tumorgenesis of osteosarcoma in vivo. (A) The volume of tumor xenografts in mice was detected every three days after seven days after treatment (i.e. injection with HOS or 143B cells expressing agomir NC or miR-451a agomir). (B) The weight of tumors excised from the mice sacrificed on the 28th day for xenografts. The weight of tumor xenografts was measured. (C-D) IHC analysis of cell proliferation markers (Ki67 and PCNA) in tumor xenografts in mice was shown. (E) TUNEL assay was performed to evaluate the apoptosis of paraffin-embedded tissues. (F-G) QPCR and western blot were applied to detect PDPK1 expression in mice tumor xenografts. (H) Western blot analysis of EMT markers in mice tumor xenografts. **P < 0.01.
Fig 2: YTHDC1 modifies the m6A methylation of PDPK1. (A) RNA pulldown followed by q-PCR was performed to determine the binding between miR-451a and PDPK1 in HOS and 143B cells. (B) The levels of candidate mRNAs in transfected HOS and 143B cells with miR-451a mimics or NC mimics were measured. (C) RNA pulldown assay was carried out to validate the binding between miR-451a and YTHDC1 3'UTR. (D) Luciferase reporter assay was conducted to further verify that miR-451a binds to YTHDC1 3'UTR. (E) RIP followed by q-PCR was performed to determine whether m6A modification occurs in the sequence of PDPK1. (F) Luciferase reporter assay was used to determine the YTHDC1-specific methylation site in the sequence of PDPK1. (G) RNA pulldown assay was done to further verify m6A-1588 site as the YTHDC1-specific methylation site in the sequence of PDPK1. (H) Remaining levels of PDPK1 and ß-actin mRNA were detected via q-PCR in the transfected HOS and 143B cells after 50 mM a-amanitin treatment, with 18S rRNA as internal reference. **P < 0.01.
Fig 3: MiR-451a inhibits AKT/mTOR signaling pathway via PDPK1-induced phosphorylation modification. (A) Western blot was applied to measure the expression of AKT/mTOR signaling pathway associated proteins (AKT, p-AKT, mTOR, and p-mTOR) in transfected HOS and 143B cells with miR-451a mimics, taking ß-actin as internal reference. (B) AKT/mTOR signaling pathway associated protein levels were evaluated by western blot analysis. (C) Q-PCR detection of PDPK1 expression level in transfected HOS and 143B cells with miR-451a mimics or mimics NC. (D) Co-IP assay was used to detect the interaction between PDPK1 and AKT proteins in osteosarcoma cells. (E) In vitro phosphorylation experiment was carried out to verify Thr308 as PDPK1-specific phosphorylation site in the sequence of AKT. **P < 0.01.
Fig 4: YTHDC1 regulates the progression of osteosarcoma via PDPK1. (A-C) The proliferative ability of osteosarcoma cells transfected with sh-NC, sh-YTHDC1-1 or sh-YTHDC1-1 + pcDNA3.1-PDPK1 was evaluated by proliferation assays. (D) The apoptosis of transfected osteosarcoma cells in different groups was determined by TUNEL assay. (E-F) The migratory ability of differently transfected osteosarcoma cells was assessed by Transwell migration and wound healing assays. (G) The morphology of transfected osteosarcoma cells was observed via microscopy. (H) Western blot analysis of EMT markers in the transfected osteosarcoma cells. **P < 0.01.
Fig 5: YTHDC1 propels the malignant progression of osteosarcoma. (A) Bioinformatics site SRAMP predicts m6A methylation sites in the sequence of PDPK1. (B) The upregulation efficiency of pcDNA3.1-YTHDC1 was validated via q-PCR. **P < 0.01.
Supplier Page from Abcam for Anti-PDPK1 antibody