Fig 1.
Experimental design and differentially expressed genes (DEG) analyses.
A. ID8 syngeneic mouse EOC line was obtained through spontaneous transformation of normal ovarian surface epithelial cells from C57BL6 mice by repetitive passage in vitro. The ID8-luc cell line (expressing both GFP and luciferase) was established as described previously. The procedures used to establish ID8-P1 and -P2 cell lines and the cells used for RNA-seq are illustrated in the figure. B. Scatter plot of gene expression for ID8-P0 and ID8-P2 cell lines. The red and green dots represent up- and down-regulated DEGs, respectively. Several genes mentioned in the current work are marked in the figure.
Fig 2.
ZIP4 expression regulated SOX9 expression.
A. In PE04-ZIP4-knockout cell lines (K36 and K37 [28]), SOX9 expression was blocked. B. In PE01-ZIP4-overexpression clones, LPA-induced up-regulation of SOX9.
Fig 3.
Altered functions and pathways in ID8-PW2 vs. -P0 cells.
GO functions and pathways significantly enriched in altered genes are grouped based on four major cellular processes. A. Cell proliferation and apoptosis-resistance. B. Cytoskelton/cell adhesion. C. Metabolism (glucose and amino acids). D. Lipid metabolism. Q values (p-values corrected by FDR multiple test adjustment) for corresponding functions and pathways are shown as red dots.
Fig 4.
DSP and PKP1 were up-regulated in in vivo passaged EOC cells.
A. DSP protein was upregulated in different P1 cell lines vs. P0 cells: PW, Peritoneal wall metastases-derived P1 cells; OM, omentum metastases-derived P1 cells; Liver, liver metastases-derived P1 cells; Kidney, kidney metastases-derived P1 cells; ME, mesentery metastases-derived P1 cells; P2, PW-P1 cells in vivo passage one more time; SKOV3ip1, i.p injected SKOV3 cells. B. PKP1 protein was upregulated in different P1 cell lines vs. P0 cells. C. Knock-down (KD) DSP clones (a and b) in PW-P1 cells by shRNA. The first lane were from PW-P1 transfected with control (ctrl) shRNA. D. Representative data showing that DSP was involved in anoikis-resistance. Cell survival was analyzed in cell suspension conditions in the presence of 2% FBS. E. The cell aggregation in P1 cells was inhibited by KD Dsp. Cells were cultured under suspension conditions for 48 hr.
Fig 5.
Focal adhesion kinase (FAK) and paxilin (Pxn) activation was regulated by stresses.
Phosphorylation of FAK and PXN was mainly upregulated when cells were cultured in suspension under starvation. Western blot analysis showing that ID8-P0, ID8-PW1, or ID8-PE2 cells were cultured in FBS, lipid depleted (charcoal-treated) FBS (Fat-cm), or FBS starved condition in attached or detached conditions.
Fig 6.
Gene regulation confirmed by Q-PCR in mouse and human EOC cell lines.
A-B. Mouse ID8 P0/P1 cells (A) or human EOC cells (B) were seeded into 6-well plates in attached or low-attached plates in suspension, RNAs were extracted with the RNeasy mini kit (Qiagen, Valencia, CA) and reverse transcribed by M-MLV reverse transcriptase. Quantitative real-time PCR was performed on a Light Cycler 480 (Roche, Indianapolis, IN) with a SYBR Green I Master Mix (Roche, Indianapolis, IN). mRNA abundance was normalized to GAPDH.
Fig 7.
PKM and PDK1 were up-regulated in P1 vs. P0 cells and the PKM2 inhibitors inhibited cellular functions.
A and B. PKM2 and PDK1 in P0 and different P1 cells (ME, mesentery, PW, peritoneal wall; OM, omentum) as well as human EOC cells (SKOV3 and HEY). C and D. Pretreatment (1 hr) of PKM inhibitors, shikonin (SHK) and menadione (MD) dose-dependently reduced anoikis- resistance. % of cell survival are shown in the Y-axis. E. ID8-P1 and PE04 formed spheroids was disrupted by SHK (1.25 μM).
Fig 8.
DEGs up-regulated in ID8-PW2 associated with hypoxia pathway.
The gene expression heatmap (four samples) of 67 DEGs associated with GO:0001666~response to hypoxia and/or mmu04066:HIF-1 signaling pathway.
Table 1.
Confirmed hypoxic responsive genes up-regulated in ID8-P1 or–PW2 cells.
Fig 9.
ID8-P1 cells had higher ALDH activity.
Aldefluor assays were performed as detailed in Methods. Both ID8-P0 (upper row) and-P1 (lower row) were stained with ALDEFLUOR kit from StemCell Technologies. ALDH negative (left column) in the presence of the inhibitor DEAB; ALDH+ positive (right column) in the absence of the inhibitor DEAB.
Table 2.
Genes related to CSC markers.