Fig 2: iTRAQ technical flow chart and localization of the identified proteins and phosphorylation events in RKO cells, according to PCK1-OE and PCK1-Ctrl. A–D GO analysis of the involved biological processes. E iTRAQ technical flow for assessing the effect of PCK1 over-expression on serine phosphorylation of UBAP2L at 445/454/467/477

Fig 3: IHC analysis of PCK1, P62, ATG5, UBAP2L, and p-UBAP2L Ser454 expression in PCK1-OE and PCK1-KD tumor tissues. All images are at 100 × magnification. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig 4: PCK1-induced autophagy inhibits CRC cell growth via down-regulation of UBAP2L Ser 454 phosphorylation. A IHC analysis p-UBAP2L Ser 454, UBAP2L, ATG5, and P62 expression in the p-UBAP2L Ser 454 (A) tumor and control groups. B–C P62, ATG5, and LC3B protein expression in PCK1-OE RKO (B) and PCK1-OE SW480 (C) cells transfected with Ser 454-Ctrl and Ser 454 (A) for 24, 48, and 72 h. D and E LC3B expression in Ser 454-Ctrl and Ser 454 (A) cells after treatment with rapamycin or BAF for 4 h. Actin was used as a control. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig 5: A single initiating ribosome blocks stress granule assembly. (A) Representative polysome profiles (n = 3) of U-2 OS cells treated with 0.1% DMSO (carrier; black) or 2 µg/mL harringtonine (HT) for 10 min (red) or 30 min (blue) followed by cycloheximide addition. The X-axis represents the distance down the sucrose gradient from 10% to 50%. (B) Average ± SEM is reported for the polysome to monosome ratio from polysome profiles in A from n = 3 replicates (denoted as green, red, and black circles). ANOVA tests were done to determine the significance between the DMSO and HT-treated conditions. (****) P < 0.001. (C) Schematic of experimental time line for D–F (2 µg/mL harringtonine [HT] and 250 µMarsenite [As]). (D) U-2 OS cells were pretreated with DMSO or harringtonine for 30 min followed by live-cell imaging at 40× magnification with or without arsenite for 60 min. Images were taken every minute, and representative images (n = 3 for all times and conditions except the DMSO 20 min time point, which is n = 2) of U-2 OS cells stably expressing GFP-G3BP1 (green) are shown. Scale bar, 10 µm. See also Supplemental Move S1. (E) The average ± SEM of the percentage of cells in a frame positive for stress granules corresponding to images in D is shown. (F) After live-cell imaging (shown in D,E), cells were fixed and stained for polyadenylated RNA [oligo(dT); magenta] and stress granule protein UBAP2L (cyan). GFP-G3BP1 is shown in green (n = 2). Scale bar, 5 µm. See also Supplemental Figure S4. (G) U-2 OS cells were pretreated with DMSO or 50 µM lactimidomycin (LTM) for 30 min followed by live-cell imaging with or without arsenite as done in D. (Left) Representative images. Scale bar, 10 µm. (Right) The average ± SEM of the percentage of cells in a frame with stress granules (n = 3) is shown. See also Supplemental Movies S2–S4 and Supplemental Figure S5.