Fig 2: HSP47 promotes AKT activation and decreases PHLPP1 expression in colorectal cancer (CRC) cells exposed to 5-FU. Representative immunoblotting images of (A) HCT116/HSP47_st, (B) RKO/si-HSP47_st, and their corresponding control cell lines treated with 50 μM 5-FU. The immunoblotting images of (C, D) phospho-Akt (S473) and (E, F) PHLPP1 were quantified in Image J software (n = 3). Data represent mean ± SEM. *P < 0.05.

Fig 3: HSP47 interacts with PHLPP1 and decreases its protein stability. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of PHLPP1 mRNA expression levels in (A) HCT116/HSP47_st, (B) RKO/si-HSP47_st, and their corresponding control cell lines treated with 50 μM 5-FU (n = 3). (C) Representative Western blot images of RKO/si-HSP47_st and its control cell line treated with 100 μg/mL cycloheximide (CHX). Cells were collected at the indicated time points and subjected to Western blot analysis. The immunoblotting images were quantified in Image J software (n = 3). (D) RKO cells were double stained with the mouse monoclonal antibody against PHLPP1 (green) and rabbit monoclonal antibody against HSP47 (red), then subjected to confocal immunofluorescence analysis. Colocalization between PHLPP1 and HSP47 is indicated by arrows. (E) Co-immunoprecipitation of HSP47 with PHLPP1 in parental RKO cells. Scale bar = 5 μm. Data represent mean ± SEM. *P < 0.05.

Fig 4: HSP47 expression promotes drug resistance in colorectal cancer (CRC) cells exposed to chemotherapy. (A) Representative image of Western blot analysis of HSP47 protein expression in various human CRC cell lines. (B) HCT116, (C) RKO, and (D) CCL228 CRC cell lines were transiently transduced with HSP47-expression vector and then exposed to 5-FU. MTS cell viability assays of (E) CCL228 cells exposed to various concentrations of 5-FU after HSP47 transient knockdown. MTS assays of (F) HSP47-overexpressing (HCT116/HSP47_st) and (G) HSP47-knockdown (RKO/si-HSP47_st) stable cell lines exposed to 5-FU. HSP47 mRNA and protein expression in (H) RKO and (I) HCT116 resistant cell lines were determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis, respectively. Data represent mean ± SEM, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 5: HSP47 inhibits apoptosis in colorectal cancer (CRC) cells exposed to chemotherapy. (A) HCT116/HSP47_st cells were visualized by fluorescence microscopy (200×) 48 h after 50 µM 5-FU treatment. TUNEL-positive nuclei are shown in green, and total nuclei stained with Hoechst 33258 are shown in blue. (B) Quantitative analysis (percentage of TUNEL positive cells vs. total) was performed for randomly selected fields (n = 10). Representative Western blot images of caspase-3 and -7 cleavage of (C) HCT116/HSP47_st, (D) RKO/si-HSP47_st, and their corresponding control cell lines after treatment with 50 µM 5-FU. Data represent mean ± SEM. **P < 0.01; ***P < 0.001.