Fig 2: Genetic knockdown of CLEC3A with si-CLEC3A decreases the proliferation of OS cells and induces G1 phase arrest. Transfection efficiency of CLEC3A knockdown with si-CLEC3A compared with si-NC in MG63 and SaOS-2 cells detected using (A) reverse transcription-quantitative PCR and (B) western blotting. (C) Cell Counting Kit-8 assays were performed to detect the effect of CLEC3A knockdown with si-CLEC3A on OS cell proliferation. (D) Colony formation assays were performed to examine the effect of CLEC3A knockdown with si-CLEC3A on OS cell colony formation in MG63 and SaOS-2 cells (diameter of dishes=6 cm). (E) Cell cycle distribution was analyzed in the si-NC and si-CLEC3A groups in MG63 and SaOS-2 cells to assess the effect of CLEC3A on the cell cycle. *P<0.05, **P<0.01. OS, osteosarcoma; CLEC3A, C-type lectin domain family 3 member A; si, small interfering RNA; NC, negative control; OD, optical density.

Fig 3: Knockdown of CLEC3A suppresses the AKT1/mTOR/HIF1α pathway. (A) Scatter diagram demonstrating the co-expressing relationship between CLEC3A and AKT1 in mRNA level in patient OS tissues. (B) Expression levels of AKT1, mTOR, and HIF1α in the si-NC- and si-CLEC3A-transfected MG63 and SaOS-2 cells was detected using western blotting. (C) Immunofluorescence was used to analyze the nuclear location of HIF1α in the si-NC- and si-CLEC3A-transfected MG63 cells (magnification, ×200). VEGF, GLUT1 and MCL1 mRNA expression levels were detected by reverse transcription-quantitative PCR in the si-NC- and si-CLEC3A-transfected (D) MG62 and (E) SaOS-2 cells. (F) VEGF, GLUT1, and MCL1 protein expression levels were detected in the si-NC- and si-CLEC3A-transfected cells by western blotting.*P<0.05. OS, osteosarcoma; CLEC3A, C-type lectin domain family 3 member A; NC, negative control; si, small interfering RNA; HIF1, hypoxia-inducible factor-1; VEGF, vascular endothelial growth factor; GLUT1, glucose transporter 1; MCL1, induced myeloid leukemia cell differentiation protein Mcl-1.

Fig 4: Overexpression of CLEC3A increases the proliferation of OS cells and decreases G1 phase arrest. Transfection efficiency of CLEC3A overexpression plasmid compared empty plasmid (NC) in MG63 and SaOS-2 cells detected using (A) reverse transcription-quantitative PCR and (B) western blotting. (C) Cell Counting Kit-8 assays were performed to detect the effect of CLEC3A overexpression on OS cell proliferation. (D) Colony formation assays were performed to examine the effect of CLEC3A overexpression on OS cell colony formation in MG63 and SaOS-2 cells (diameter of dishes=6 cm). (E) Cell cycle distribution was analyzed in the NC group and CLEC3A-overexpressing group in MG63 and SaOS-2 cells to assess the effect of CLEC3A on the cell cycle. *P<0.05, **P<0.01. OS, osteosarcoma; CLEC3A, C-type lectin domain family 3 member A; NC, negative control; OD, optical density.

Fig 5: CLEC3A is highly expressed in OS tissues. (A) Volcano plot demonstrating the differently expressed genes in OS using the Gene Expression Omnibus data profile GSE99671 and CLEC3A is one of the upregulated gene (B) Reverse transcription-quantitative PCR was used to detect CLEC3A mRNA expression levels in clinical normal adjacent bone tissue (n=15) and OS tissue (n=30). (C) Representative micrographs demonstrating CLEC3A expression levels in OS and normal tissues. Black triangles indicate normal bone tissue infiltrated by OS cells. Scale bars, 100 µm. **P<0.01. OS, osteosarcoma; CLEC3A, C-type lectin domain family 3 member A.