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Fig. 5 | Journal of Experimental & Clinical Cancer Research

Fig. 5

From: Triple blockade of EGFR, MEK and PD-L1 has antitumor activity in colorectal cancer models with constitutive activation of MAPK signaling and PD-L1 overexpression

Fig. 5

Expression of PD-L1 and EGFR by immunofluorescence analysis. a. Immunofluorescence analysis of the localization of EGFR and PD-L1 in HCT116 and HCT116-MR cells after exposure to different time of TGFα (5 min, 20 min and 1 h). Cells were stained with anti-EGFR antibody followed by secondary antibody labeled with Alexa Fluor 488 and anti-PD-L1 antibody followed by secondary antibody Alexa Fluor 532. DAPI was included to stain the nucleus. b. Cytofluorimetric analysis of HCT116 MR cells. Cells are stimulated with TGFα for 5 min, 20 min and 1 h alone or in combination with MEKi (BAY86–9766 0,5 μM) and then we investigated the presence of EGFR and PD-L1 on the surface of cells. c. An upregulation of PD-L1 is observed in the nucleus of HCT116 MR cells. Green color from Alexa Fluor 488 for PD-L1 and blue color from DAPI for visualization of nuclei. d. HCT116 and HCT116-MR were lysed to obtain cytosolic and nuclear fragments that were analyzed by Western blot analysis. Tubulin and Histone H3 antibodies were used as loading and purity control of the cytosolic and nuclear fractions, respectively

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