Fig. 2

METTL16 is highly expressed in liver CSCs and genetic depletion of METTL16 attenuates liver CSC self-renewal. A, B Histogram plot (A) and the statistical results (B) showing METTL16 abundance in CD133⁻ and CD133⁺ populations in HCC cell lines (n = 5). C, D Histogram plot (C) and the statistical results (D) showing METTL16 abundance in CD133⁻ and CD133⁺ populations in HCC tumors (n = 3). E, F Representative images (E) and the statistical results (F) showing the effects of METTL16 KO on liver CSC maintenance as determined by spheroid formation assay in HepG2 cells (n = 4; mean ± SD). G, H Representative images (G) and the statistical results (H) showing the effects of METTL16 KO on liver CSC maintenance as determined by spheroid formation assay in Hep3B cells (n = 3; mean ± SD). I, J Representative images (I) and the statistical results (J) showing the effects of METTL16 KO on liver CSC frequency as determined by in vitro limiting dilution assay (LDA) in HepG2 cells. K, L Representative images (K) and the statistical results (L) showing the effects of METTL16 KO on liver CSC frequency as determined by in vitro LDA in Hep3B cells. M, N Percentage of liver CSCs in HepG2 (M) and Hep3B (N) cells upon METTL16 KO as determined by flow cytometry (n = 3; mean ± SD). O Table showing the injected cell numbers and the ratios of xenograft tumors implanted with HepG2 cells at the indicated number of days post transplantation. P Quantitative and statistical results showing the effects of METTL16 KO induced by sgMETTL16-2 (left panel) and sgMETTL16-3 (right panel) on liver CSC frequency as determined by in vivo LDA. Q Percentage of liver CSCs in xenograft tumors implanted with HepG2 cells with or without METTL16 KO [n = 3 (left panel); n = 6 (right panel); mean ± SD]. Statistical analyses: paired t test (B, D); un-paired t test (F, H, M, N, Q); extreme limiting dilution analysis (ELDA) (https://bioinf.wehi.edu.au/software/elda/) (J, L, P). *P < 0.05, **P < 0.01, ***P < 0.001