Fig 2: Evaluation of CABE-Ts and CBE-Ts in therapeutically relevant cell contexts.a, Gene-editing outcomes of primary human hepatocytes transfected with mRNAs encoding CBE-T1.46 and BE4 at three sites within the PCSK9 gene (left, P = 0.2882 (NS); right, P = 0.0017 (double asterisk)). Positional edit within the protospacer indicated. b, Evaluation of relative change in PCSK9 secretion between day 9 (collection time point) and day 0 (transfection time point) through ELISA. The targeted PCSK9 site is indicated on the x axis. P values are as follows: Q555X versus untreated, P = 0.001001 (double asterisk); E4 splice versus untreated, P = 0.001295 (double asterisk). c, Relative change in LDL-R present in supernatant between day 9 and day 0 assessed by ELISA. P values are as follows: Q555X versus untreated, P = 0.001116 (double asterisk), E4 splice versus untreated, P = .009481 (double asterisk). d, Gene-editing efficiencies from sgRNA screens in primary human T cells using CBE-Ts. X axis label indicates the targeted gene and target base within sgRNA. e,f, Percent C·G to T·A conversion (e) and surface protein loss (f) achieved by each base editor or control in multiplex-edited primary human T cells. Primary hepatocyte data were generated from n = 3 (‘none’/untreated samples) or 4 (BE4 and CBE-T1.46 samples) independent biological replicates. T cell data were generated from n = 2 independent donors. Where applicable, statistical significance was computed via two-tailed unpaired t tests: NS, P = 0.05; *P < 0.05; **P < 0.01.Source data

Fig 3: Correlation between PCSK9 level and length of hospital stay.

Fig 4: PCSK9 activated EGFR and HER3 by reducing lipid rafts on cell membranes. A) Representative images and quantified results of colony formation for the EV, MβCD‐treated, PCSK9‐overexpressing and LDLR‐knockdown groups. One thousand 231‐GFP cells were cultured in 6‐well plates for 12 days, and five thousand 468‐Clover cells were cultured in 6‐well plates for 14 days. B) Representative images and quantified results of the transwell migration assay for the EV, MβCD‐treated, PCSK9‐overexpressing and LDLR‐knockdown groups in 231‐GFP cells and 468‐Clover cells. Five thousand cells were cultured for 16 h in migration assays. C,D) Quantified results of the colony formation and transwell migration assays. E) Representative images of left lung colonies and metastatic tumors after 5 weeks (n = 5–6). F) Quantified results of left lung colonies. G) Quantified results of metastatic tumors in mediastinal LN. H) Quantified results of metastatic tumors in brain. I) Western blotting depicting the phosphorylated and total protein levels of CAV1, EGFR, HER3, Src, ERK, c‐Jun, cyclin D3, and vimentin in the EV, MβCD‐treated and O‐PCSK9 groups of 231‐GFP cells, and in the DMSO‐treated and MβCD‐treated groups of 4–11 cells with shPCSK9. J) Western blot showed the phosphorylated and total protein levels of EGFR, HER3, LDLR and CAV1 after LDLR was knocked down in 231‐GFP cells. The significance of differences was determined by two‐way ANOVA (C, D) or Student’s t‐test (F).

Fig 5: High expression of PCSK9 reduced cholesterol levels in the cell membrane by decreasing the cell‐surface LDLR. A,B) Representative images (A) and quantified results (B, n ≥ 100 cells) of DiI‐LDL in 231‐GFP and 468‐Clover cells with EV or O‐PCSK9. C) The contents of cholesterol in 231‐GFP, 4–11 and O‐PCSK9 cells using a sensitive high performance liquid chromatography (HPLC)‐based method. D) Quantified results of cholesterol levels by HPLC. E) Western blotting showed the protein levels of LDLR and CAV1 after overexpressing PCSK9 in 231‐GFP and 468‐Clover cells, respectively. F) Coimmunostaining of LDLR and CD44 in MDA‐MB‐231 cells with EV and O‐PCSK9. The white arrow indicates the coordinates (G) used for the intensity profiles of scan line. G) The coordinates for intensity profiles of scan line were shown for colocalization of CD44, LDLR and DAPI in the EV (top) and O‐PCSK9 (bottom) groups of MDA‐MB‐231 cells. (H, n ≥ 50 cells) Ratio of colocalization LDLR and CD44 (left) and fluorescence intensity of LDLR (right). I,J) Representative images (I) and quantified results (J, n ≥ 20 cells) of lipid rafts and filipin III staining in 231‐GFP cells. (K, n ≥ 20 cells) Quantified results of lipid raft and Filipin III staining in 468‐Clover cells. The significance of differences was determined by one‐way ANOVA (D, J), two‐way ANOVA (B, K) or Student’s t‐test (D, H).