Fig 1: CX3CL1 promotes the invasion and migration of HCC cells through Src. A total of 50 or 100 nM recombinant human CX3CL1 was added to the medium. CX3CL1 antibody was added to neutralize free CX3CL1 in medium. Bosutinib was used to inhibit Src. (A) Representative images and statistical analysis of Transwell migration assays showed the migratory ability of HCC cells. The number of cells which had invaded was quantified; n=5; scale bar, 200 μm. (B) Representative images and bar graphs of Transwell invasion assays showed the invasive ability of HCC cells after 24 h. The number of cells which had migrated was quantified; n=5; scale bar, 200 μm. Cell migration and invasion were analyzed using ANOVA. (C) Expression of THP-1 cells surface markers MRC1 and CD163 were detected by western blot analysis; n=3. Western blot analysis data were analyzed using a Student's t-test following log transformation. HCC, hepatocellular carcinoma; CX3CL1, C-X3-C motif chemokine ligand 1; MRC1, mannose receptor C-type 1.
Fig 2: Interaction between BMECs and HCC cells enhances tumor growth in the spine. (A) si-CX3CL1 was transfected into BMECs to construct CX3CL1low BMECs. Transwell assays were used to examine recruitment of BMECs co-cultured with or without HMCC97H cells. The number of the recruited cells was quantified. n=3. Cell recruitment were analyzed using ANOVA test. (B) Cell vitality of BMECs was assessed using CCK-8 assay; n=3. Data were analyzed using ANOVA followed by Tukey's test. (C) Tumor size was measured based on the volume. n=5. Scale bar, 5 mm. Data were analyzed using an unpaired t-test. (D) Samples from 2 groups were examined using immunofluorescence. Magnification, ×20. The result was analyzed using an unpaired t-test. Gray bars represent the CX3CL1nor BMECs group and black bars represent the CX3CL1low BMECs group (E) Flow cytometry revealed the percentage and number of F4/80+CD11b+ cells; n=5. (F) Flow cytometry was used for detecting expression of M2 macrophage surface markers, CX3CR1, MRC1 and CD163; n=5. Cells were selected based on higher expression of CD45 for macrophages. Subsequently, CD11b and F4/80 expression were used for further characterization of macrophages. FACS (fluorescent-activated cell sorting) data were analyzed using a Kruskal Wallis test followed by Dunn's non-parametric post hoc test. HCC, hepatocellular carcinoma; BMECs, bone marrow endothelial cells; si, small interfering; CCK-8, Cell Counting kit-8; CX3CL1, C-X3-C motif chemokine ligand 1.
Fig 3: Nitrogen starvation causes similar changes to Cds1 and Mrc1. (a) Cds1-HA2His6, cds1 deletion and rad3 deletion strains without auxotrophic markers were grown in minimal medium with nitrogen overnight, washed and resuspended in minimal medium without nitrogen at 30 °C. The rise in the septation index (G1/S cells) shows the two rounds of DNA replication post-shift. (b) Total protein extracts tested at the indicated hours post-shift and probed for Mrc1, Sty1-T171P + Y173P and Cds1-HA2His6, (P = phosphorylated Cds1 protein on phostag SDS page), (c) Phostag SDS page of Cds1-HA2His6. Twelve mM HU was added at 20 min (1) post-shift or 4 h (2) post-shift. Cells were harvested 2 h later. (d) Total protein extracts analysed at the indicated hours post-shift and probed for H2AX-S129-P and Cdc2. (e) Normal and phostag (PT) SDS page of Cds1-HA2His6, harvested 0 h, 7 h and 24 post-shift. (f) PT-SDS page of Cds1-HA2His6 cells, harvested at 0 h, 1 h, 3 h and 7 h (arrow = N-terminally truncated Cds1 form). (g) SDS page of Sty1-T171P+Y173P in 0.3% glucose or in minimal medium without nitrogen (-N) at 0 h and 24 h. (h) PT-SDS page of Cds1-HA2His6 in 0.3% glucose or in minimal medium without nitrogen (-N) at 0 h and 24 h (3 = phosphorylated form). (i) Survival of the indicated strains in minimal medium without nitrogen.
Fig 4: DBAN suppresses Chk1 phosphorylation. (A,B) cds1-His 6 HA 2 (55 kDa) and chk1-HA 3 (60 kDa) cells were grown in rich medium at 30 °C and treated for 4 h with 10 μM DBAN, 12 μM camptothecin (CPT), 12 mM hydroxyurea (HU) or the combination as indicated. Total protein extracts were loaded on a 6% phostag gel. Full phostag gels are shown. H2AX-S129-P was detected on a 20% acrylamide (37.5:1 acrylamide:bisacrylamide) gel. The Chk1 shift was detected on a 10% (100:1 acrylamide:bisacrylamide) gel. H2AX and Chk1 (normal) panels were cropped. The full images are shown in Figs S3 and S4, respectively. The arrows indicate the smaller Cds1 band, Chk1-S345 phosphorylation and the hyper-phosphorylated forms of Chk1. (C) Model of Chk1 activation by Rad3 at broken replication forks. CPT immobilises Topoisomerase 1 and the Rad9-Rad1-Hus1 ring aids Chk1 phosphorylation. Rad9 is also phosphorylated by Rad341. (D) Drop test of the indicated strains. Chk1-D155E-HA3 is a kinase-dead mutant. DBAN: 10 μM; CPT: 12 μM. (E,F) chk1-HA 3 cells were HU synchronised (3.5 h, 15 mM HU, rich medium) and released into medium without (UT) or with CPT (12 μM), CPT (12 μM) + DBAN (10 μM) or CPT (12 μM) + TCAN (10 μM). Total protein extracts were separated on a 8% (Mrc1) and 10% (Chk1) acrylamide gel. P = Phospho-Chk1-S345 shift band). (G) rad9-HA 3 cells were HU-synchronised and released into rich medium without (UT) or with CPT (12 μM), DBAN (10 μM) or CPT (12 μM) + DBAN (10 μM). P = Phospho-Rad9 shift band. Full images: Mrc1: Fig. S5; Chk1: Fig. S6; Rad9: Fig. S7.
Fig 5: Glucose starvation transiently activates Cds1. (a) Cds1-HA2His6 (56 kDa), Chk1-HA3,(58 kDa), Mrc1 (180 kDa) and Cdc2 (35 kDa) in total protein extracts prepared from growing (G) and stationary cultures (NG) after 24 h of growth in rich medium with 3% glucose at 30 °C. (b) Septation index (G1/S cells) after the 3–0.3% glucose down-shift. (c) Cds1-HA2His6, Cdc2 and H2AX-S129-P in total protein extracts at 3% glucose and post-shift to 0.3% glucose. (d) In vitro kinase activity of immunoprecipitated Cds1-HA2His6 on myelin basic protein post-shift (top: phosphorylated myelin basic protein, bottom: precipitated Cds1 protein, asterisk: unspecific band). (e) Phos-tag SDS page of total protein extracts (P = phosphorylated forms 2–5, arrow = hyper-phosphorylated form 5). (f) Isoelectric focusing of Cds1-HA2His6 (P = more negatively charged species, arrows = changes in low glucose medium) (1–3) to more positively charged forms of Cds1 in 3% glucose medium. (g) H2AX-S129-P and Cdc2 in total protein extracts post-shift in wild type (WT) and selected deletion mutants.
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