Fig. 1

CRC patient specimens and cells express HSP70-2 mRNA and protein. a RT-PCR analysis shows HSP70-2 mRNA expression in stage I-IV, grades WD, MD and PD and CRC cells (COLO205 and HCT 116). ANCT specimens failed to express HSP70-2 mRNA. Testis was used a positive control and β-actin was used as a loading control. b Western blotting reveals HSP70-2 protein expression, COLO205 and HCT116 cells. c IIF analysis depicts predominantly cytoplasmic localization; co-localization reveals HSP70-2 protein in endoplasmic reticulum, golgi bodies, mitochondria and plasma membrane (yellowish-orange staining). No co-localization was seen with nuclear envelope. Co-localization of HSP70-2 in endoplasmic reticulum, Golgi bodies and mitochondria in both COLO205 and HCT116 CRC cells was observed and quantified (see Methods) Histogram depicts average percentage co-localization in n = 20 cells from five examined areas (regions of interest). Original magnification x630, objective x63. Scale bar: 10 μm. d Flow cytometric analysis demonstrates the displacement shift of fluorescence (green color peak: depicts cells fixed with PFA prior to first antibody incubation and blue color peak: depicts cells incubated first with primary antibody followed by PFA fixation) in COLO205 and HCT116 cells as compared to control IgG stained (black color peak) and unstained (red color peak) cells. WD: well differentiated, MD: moderately differentiated and PD: poorly differentiated