Fig 2: LV-si-RBP1 suppressed tumor growth in vivo.a Representative graph showing tumor growth 32 days after subcutaneously implanted OSCC cells. b, c Calculated tumor volume and weight in LV-si-RBP1 and LV-control groups. d, e Expression of RBP1 as measured by qRT-PCR and western blot in LV-si-RBP1 and LV-control groups. f RBP1 expression was measured by immunohistochemistry staining in LV-si-RBP1 and LV-control groups. Scale bar: 100 µm; *P < 0.05, **P < 0.01.

Fig 3: RBP1-induced autophagy and enhanced autophagic flux in OSCC cells.a Western blot analysis showing the expression levels of ATG5, BECN1, LC3-II, and ß- actin in the cell transfected with control, RBP1 plasmid, or si-RBP1. Quantification of the amount of ATG5, BECN1, and LC3-II to the amount of ß- actin for each of the indicated sample. b Electron microscopic examination showing autophagic vacuoles (arrows) in the cytoplasm in the cell transfected with control and RBP1 plasmid. Scale bar: 2 µm. c Immunofluorescent staining showing the number of LC3 dot in the cell transfected with control, RBP1 plasmid, or si-RBP1. Cells were stained by indirect immunofluorescence using anti-LC3 antibody, and the number of LC3 dot per cell was quantified. Scale bar: 20 µm. d RBP1 overexpression enhanced autophagic flux. The number of GFP-LC3 dot in the RBP1-overexpressed cells and control cells treated bafilomycin A1 (BAF). Cells were transfected with GFP-LC3 adenovirus. 24 h after transfection, cells were treated with BAF, then GFP-LC3 dot was examined by fluorescence microscopy. Scale bar: 10 µm; *P < 0.05, **P < 0.01.

Fig 4: The expression of RBP1 in patients with OSCC and cell lines.a, b iTRAQ technical flow chart and mass spectrometry map of a DEP. c, d Volcanic maps and cluster analysis of DEPs. e The expression of RBP1 in 63 pairs of OSCC tissues and normal tissues were analyzed by qRT-PCR. f IHC was used to analyze the expression of RBP1 in 63 pairs of OSCC tissues with different degrees of differentiation and normal tissues. Scale bar: 100 µm. g The expression of RBP1 in OSCC cell lines and HOK cell were analyzed by qRT-PCR. All data were shown as mean ± SD; *P < 0.05, **P < 0.01.

Fig 5: The effects of RBP1 on OSCC growth, migration, and invasion in vitro.a Effective overexpression and elimination of RBP1 mRNA in SCC15 cells were achieved, as determined by qRT-PCR analysis of total RNA preparations of these cells. b After transfection with control plasmid, RBP1 plasmid, or si-RBP1, the image of 1000 cells were severally plated in a six-well plate for 12 days; the average colony number of per well. c At 24, 48, and 72 h after transfection with control plasmid, RBP1 plasmid, or si-RBP1, cell growth was examined by the MTT assay. d The relative proportion of SCC15 cell cycle, after transfection with control plasmid, RBP1 plasmid, or si-RBP1. e The view of wound-healing migration assay; the average rate of SCC15 cells migration at 0, 12, and 24 h after treatment with control plasmid, RBP1 plasmid, or si-RBP1. Scale bar: 100 µm. f The results of the transwell assay of SCC15 cells at 24 h after treatment with control plasmid, RBP1 plasmid, or si-RBP1; the relative ratio of migrated and invasive cells per field was shown. All data were shown as mean ± SD. Scale bar: 100 µm; *P < 0.05, **P < 0.01.