Fig 2: NOP influences the proliferation and apoptosis of HCC cells through autophagy.A CCK-8 assay and colony formation assay showing the proliferation of MHCC-LM3-vector, MHCC-LM3-NOP, Huh7-shNC, Huh7-shNOP-1, and Huh7-shNOP-2 cells. B Apoptosis rates in MHCC-LM3-vector, MHCC-LM3-NOP, Huh7-shNC, Huh7-shNOP-1, and Huh7-shNOP-2 HCC cells. C IB analysis of the indicated proteins associated with apoptosis in MHCC-LM3 and Huh7 cells. D IB analysis of the indicated proteins associated with autophagy in MHCC-LM3 and Huh7 cells. E IF staining showing the GFP-mCherry-LC3B fusion protein in MHCC-LM3-vector, MHCC-LM3-NOP, Huh7-shNC, and Huh7-shNOP cells (GFP: green, mCherry: red; merged: yellow). Scale bars = 20 µm. F Electron microscopy showing the autolysosome (red arrow) and autophagosome (yellow arrow) numbers in MHCC-LM3-vector, MHCC-LM3-NOP, Huh7-shNC, and Huh7-shNOP cells. Scale bars = 2 µm. G Volumes and weights of xenograft tumors formed from MHCC-LM3-vector, MHCC-LM3-NOP, Huh7-shNC, Huh7-shNOP-1, and Huh7-shNOP-2 cells. H IB analysis of the indicated proteins associated with apoptosis and autophagy in MHCC-LM3 cells treated with 3-MA. I CCK-8 assay showing the proliferation of MHCC-LM3-vector and MHCC-LM3-NOP HCC cells treated with 3-MA or DMSO. J Apoptosis rates in MHCC-LM3-vector and MHCC-LM3-NOP HCC cells treated with 3-MA or DMSO. ***P < 0.001, **P < 0.01, *P < 0.05.

Fig 3: E2F1 is a transcription factor of NOP.A Correlations between NOP expression and the expression of candidate transcription factors. B Expression of E2F1 and MAZ in HCC and adjacent tissues. C Correlation between NOP expression and E2F1 or MAZ expression. D IB analysis of the indicated proteins in MHCC-LM3 and Huh7 cells. E CCK-8 assay showing the proliferation of vector + shNC, vector + shNOP, E2F1 + shNC, and E2F1-shNOP HCC cells. F Apoptosis rates in vector + shNC, vector + shNOP, E2F1 + shNC, and E2F1-shNOP HCC cells. G CCK-8 assay showing the proliferation of shNC + vector, shNC + NOP, shE2F1 + vector, and shE2F1 + shNOP HCC cells. H Apoptosis rates in shNC + vector, shNC + NOP, shE2F1 + vector, and shE2F1 + shNOP HCC cells. I IB analysis of the indicated proteins associated with autophagy and apoptosis in MHCC-LM3 and Huh7 cells. J Schematic drawing of the full-length and truncated NOP fragments. K Luciferase assay showing the binding site of NOP and E2F1. L. A specific band of the expected size was detected in the input DNA and the anti-E2F1 antibody (anti-E2F1)-precipitated DNA by ChIP. M Schematic showing the effect of increased expression of NOP via the transcription factor E2F1. ***P < 0.001, *P < 0.05.

Fig 4: NOP is upregulated and predicts poor prognosis in HCC.A Venn diagram of the differentially expressed genes in three HCC datasets. B Boxplots of NOP mRNA expression in HCC (n = 38) and adjacent tissue (n = 19) in the Mas Liver dataset. C Kaplan–Meier analysis based on NOP expression in the TCGA dataset (n = 360). D Boxplots of NOP mRNA expression in normal tissue (N) (n = 35), HCC (T) (n = 52), and adjacent tissue (NT) (n = 52) in the SYSUCC cohort. E IHC staining showing strong, moderate, and weak positive expression and negative expression of NOP in HCC. Scale bar: ×4 = 100 µm; ×40 = 10 µm. F Boxplots of the IHC score of NOP in HCC (n = 288) and adjacent tissue (n = 288).

Fig 5: NOP promotes autophagy by activating the NF-kB signaling pathway.A IB analysis of the indicated proteins associated with the NF-kB pathway in MHCC-LM3 and Huh7 cells. B IB analysis of the indicated proteins associated with the NF-kB pathway in MHCC-LM3 and Huh7 cells treated with JSH-23 or DMSO. C, D IF staining showing LM3 expression in MHCC-LM3-vector and MHCC-LM3-NOP HCC cells treated with JSH-23 or DMSO. Scale bars = 20 µm. E CCK-8 assay showing the proliferation of MHCC-LM3-vector and MHCC-LM3-NOP HCC cells treated with JSH-23 or DMSO. F Apoptosis rates in MHCC-LM3-vector and MHCC-LM3-NOP HCC cells treated with JSH-23 or DMSO. ***P < 0.001, **P < 0.01, *P < 0.05.