Figure 6.
Decreased Susceptibility of EGFP(+) ZL55 Cells to Asbestos Toxicity and NF2 Expression; Equal Sensitivity of EGFP(+) and EGFP(−) ZL55 Cells to Calretinin Downregulation
(A) Increased resistance of ZL55-SO-P2 cells exposed to 5 μg/cm2 of crocidolite for 96 hr determined by an MTT assay compared with ZL55-SO cells (n = 3 independent experiments, one-way ANOVA; ∗∗∗p < 0.0005).
(B–D) Downregulation of calretinin in ZL55-SO and ZL55-SO-P2 cells equally decreases the viability in both cell populations for three different shRNA targeting CALB2 (B) (n = 3 independent experiments; one-way ANOVA; n.s., not significant). Overexpression of merlin (NF2) in ZL55-SO (C) and ZL55-SO-P2 cells (D) shows strong proliferation-inhibiting effects on the non-sorted ZL55-SO cells, but almost no effect on ZL55-SO-P2 cells. The inset in (D) shows strong upregulation of full-length NF2 (Mr = ∼68 kDa) using LV-NF2. All error bars represent SD.
(E) Live-cell imaging of ZL55-SO 3 hr after upregulation of NF2; note the presence of EGFP(+) and EGFP(−) cells in the ZL55-SO cell population. The morphology of green and non-green cells is mostly epithelioid.
(F) Twenty-seven hours post infection, the upregulation of NF2 induced strong apoptosis/necrosis in non-green cells (white arrows) demonstrated by cell shrinkage, blebbing, and cell debris. EGFP(+) cells do not show striking morphological changes.
(G) Western blot analysis showing strong downregulation of CR (Mr = 31 kDa) by shRNA CALB2 no. 5 in ZL55-SO and ZL55-SO-P2 cells. At approximately 35 kDa, the nitrocellulose membrane was cut and the upper part was used for GAPDH detection (lower panel). Control infection with shRNA GFP has no effect on CR expression. For the normalization the signal for GAPDH (Mr ∼38 kDa) was used. See also Figure S4.