Article Figures & Data
Figures
- Figure 1.
Bioinformatics analysis of CHKα expression in PDAC cell lines and tissue samples. A, TEA of the expression data of PDAC lines in the CCLE. PDAC lines are highly represented among the highest CHKα-expressing cells (probe 204233_s_at). B, box plot showing CHKα expression (probe 204266_s_at) in the PDAC subgroups defined by Collisson and colleagues and statistical analysis of the comparison (bottom).
- Figure 2.
CHKα is overexpressed in cultured PDAC cells, and it is important to sustain cell proliferation. A, CHKα expression by western blotting in PDAC cell lines and nontransformed HPDE pancreatic cells; quantification was performed by densitometry. B, CHKα expression in a panel of nonpancreatic cells. C, CHKα expression in Suit2 028 cells infected with 5 different shRNA lentiviruses (Sh-1 to Sh-5) or a scrambled (Nt) sequence. D, growth of control and CHKα-silenced Suit2 028 cells in vitro (left) and in vivo as subcutaneous xenografts (right).
- Figure 3.
CHKα expression in pancreatic tissue samples. A, representative images of CHKα immunostaining in normal pancreas (top left), chronic pancreatitis (top middle), PanIN (low grade, top right; high grade, bottom left), and PDAC (bottom, middle and right). Bottom, middle and right, nuclear and cytoplasmic CHKα, respectively. B, Kaplan–Meier survival curves of a cohort of 74 patients (left) with PDAC showing that nuclear CHKα staining correlates with better outcome. The same analysis, restricted to patients with moderately differentiated tumors, is shown at right.
- Figure 4.
CHKα levels are associated with the apoptotic response to MN58b. A, treatment of IMIM-PC-2 PDAC cells with MN58b reduces phosphocholine (PCho) synthesis. B, colony formation using four PDAC cell lines treated with increasing MN58b concentrations (top). Inverse correlation between levels of CHKα and MN58b IC50 (bottom). C, apoptosis, measured by Annexin V staining and flow cytometry, in three PDAC cell lines treated with MN58b for 24 or 48 hours. D, synergistic effects of MN58b combined with gemcitabine (Gem), oxaliplatin (Oxa), and 5-FU on PDAC cell lines. Cho, choline.
- Figure 5.
Generation and characterization of MN58b-resistant IMIM-PC-2 cells: overexpression of ABCB transporters. A, generation of MN58b-resistant IMIM-PC-2 cells (left). Growth curves of parental versus IMIM-PC-2-R cells (right). B, choline (Cho) uptake in parental and IMIM-PC-2-R cells. C, in vitro choline kinase activity in parental versus IMIM-PC-2-R cells. D, validation of ABCB1 and ABCB4 overexpression in parental IMIM-PC-2-R cells by qRT-PCR (left) and western blotting (ABCB1; right). PCho, phosphocholine.
- Figure 6.
Pharmacologic modulation of ABCB transporter activity restores sensitivity of IMIM-PC-2-R cells to MN58b. A, calcein uptake in parental and IMIM-PC-2-R cells. B, changes in calcein uptake in parental and IMIM-PC-2-R cells treated with variable concentrations of verapamil or zosuquidar. C, representative images of calcein uptake in parental and IMIM-PC-2-R cells treated with verapamil or zosuquidar.
Additional Files
Supplementary Data
- Supplementary Tables 1-5 - Supplementary Table 1. Metrics of the RNA-Seq experiment; Supplementary Table 2. Clinical and pathological characteristics of patients whose tumors were analyzed for CHKA expression; Supplementary Table 3. IC50 of MN58b for the PDAC cell lines used in the study; Supplementary Table 4. IC50 for gemcitabine, oxaliplatin, and 5-FU in control (ShNt, non-targetting) and CHKA-knockdown (Sh-1 and Sh-2) cells; Supplementary Table 5. Effect of modulation of ABCB transporter activity on the sensitivity (IC50) to treatment with MN58b of IMIM-PC-2 and IMIM-PC-2R cells.
- Supplementary Figure 1 - Analysis of antibody specifity
- Supplementary Figure 2 - CHKA expression in tissues and cell lines
- Supplementary Figure 3 - CHKA expression in HPNE cells and pancreas cancer cells
- Supplementary Figure 4 - Detailed results of combination treatments
- Supplementary Figure 5 - RNA-Seq analysis of IMIM-PC-2 and IMIM-PC-2-R cells
- Supplementary Figure 6 - Acute treatment with MN58b: effect on ABCB gene expression
Volume 15, Issue 2
