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. 2017 Aug 18;6:e27301. doi: 10.7554/eLife.27301

Figure 2. LRP16 mediates acquired resistance to etoposide in CRCs.

(A) Expression levels of LRP16 were analyzed in 11 CRC cell lines with Western blotting and RT–qPCR. (B) Cell growth assay. SW480 cells were transfected with the control vector or an LRP16-expressing plasmid for 48 hr, and then treated with etoposide, 5-fluorouracil, or oxaliplatin for a further 72 hr. Cell viability was determined with a CCK-8 assay. (C) SW480 cells transfected with an LRP16-expressing plasmid or the control vector were treated for 36 hr with the indicated concentrations of etoposide and/or BAY 11–7082. Cell lysates were separated with SDS-PAGE and analyzed with Western blotting using the indicated antibody. β-Actin was used as the loading control. (D) SW480 cells transfected with an LRP16-expressing plasmid or the control vector were treated with etoposide (50 μM), 5-fluorouracil (100 μM), or oxaliplatin (2 μg/ml) for 3 hr, and the cell lysates were analyzed with Western blotting using the indicated antibodies. (E) SW480 cells transfected with increasing doses of an LRP16-expressing plasmid or the control vector were treated with etoposide (50 μM), and then immunoblotted with the indicated antibodies. (F) SW480 cells transfected with an LRP16-expressing plasmid or the control vector were pretreated with or without BAY 11–7082 and then treated with etoposide for indicated concentrations, and subjected to a cell viability analysis. (G) SW480 cells transfected with the indicated plasmids and treated with etoposide for indicated concentrations, and subjected to a cell viability analysis. (H) SW620 cells were transfected with control or LRP16-directed shRNAs, and the resulting stable cells were treated with etoposide and subjected to a cell viability analysis. Data are representative of at least three independent experiments.

Figure 2.

Figure 2—figure supplement 1. The cytotoxic and cytostatic effects of chemotherapeutic drugs on CRC cell lines.

Figure 2—figure supplement 1.

(A) Whole cell lysates from 11 different CRC cell lines were immunoblotted with the indicated antibody. β-Actin was used as the loading control. Correlation analysis gray value using ImageJ software for the protein expression level of LRP16 and phosphorylation-p65 by R programming. (B) RKO, LS180, HCT116, LoVo, SW480, and CACO2 cell lines were treated with the indicated concentrations of etoposide, 5-fluorouracil, or oxaliplatin for 3 days and cell viability was measured with a CCK8 assay (upper). RKO, LS180, HCT116, LoVo, SW480, and CACO2 cell lines were treated with etoposide, 5-fluorouracil, or oxaliplatin for the indicated times and then cell viability was measured with a CCK8 assay (bottom). Data are representative of at least three independent experiments.
Figure 2—figure supplement 2. LRP16 plays a critical protective role for the survival of CRC cells.

Figure 2—figure supplement 2.

(A) SW480 cells transfected with the control vector or an LRP16-expressing plasmid were treated with the indicated concentrations of etoposide, 5-fluorouracil, or oxaliplatin and then subjected to a clonogenic cell survival assay for 14 days. (B) Transwell invasion assays of SW480 cells transfected with control vector or LRP16-expressing plasmid were treated with the indicated concentrations of etoposide, 5-fluorouracil, or oxaliplatin. (C) Whole cell lysates from 11 different colon cancer cell lines treated with etoposide (50 μM) or 5-fluorouracil (100 μM) for 2 hr were immunoblotted with the indicated antibody. β-Actin was used as a loading control. Data are representative of at least three independent experiments.
Figure 2—figure supplement 3. The protective roles of LRP16 for CRC cell survival rely on PAR-dependent NF-κB activation in response to DNA damage.

Figure 2—figure supplement 3.

(A) SW480 cells transfected with increasing doses of LRP16 or the control for 48 hr were then treated with increasing amounts of etoposide for 14 days and then subjected to a clonogenic cell survival assay. (B) SW480 cells transfected with increasing doses of an LRP16-expressing plasmid or the control vector were treated with the indicated concentrations of etoposide and/or BAY 11–7082 for 12 days and subjected to a clonogenic cell survival assay. Quantification of the clonogenic cells from four independent experiments is shown. (C) SW620 cells were transfected with control or LRP16-directed siRNAs, and the resulting stable cells were treated with etoposide and subjected to clonogenic cell survival assay, and cell apoptosis analysis. (D) SW480 cells were transfected with the indicated plasmids (control vector, LRP16, LRP16_D160A, and LRP16_I161A) for 48 hr, and then were treated the indicated concentrations of etoposide and/or PARP inhibitors (PJ-34 or 3-AB) subjected to clonogenic cell survival and cell viability assays. One representative experiment of three was shown.