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. 2016 Jul 8;66(1):124–136. doi: 10.1136/gutjnl-2016-312078

Figure 6.

Figure 6

Mitogen-activated protein kinase(MAPK) inactivation in vivo downregulates programmed cell death-ligand 1 (PD-L1) expression on tumour cells. (A) Experimental design is shown. (B) Changes in tumour size (%) post-MAPK kinase (MEK) inhibitor (MEKi) treatment. Data represent mean±SEM, n=4–6. Statistical difference between two groups was analysed by two-way analysis of variance (ANOVA). (C) By flow cytometry, the percentages of CD45EpCAM+PD-L1+ tumour cells and CD45+CD11b+PD-L1+ myeloid cells in subcutaneous 65 671 tumours and iKras*3 tumours extracted from vehicle or MEKi-treated mice were measured. Data represent mean±SEM; each point indicates one tumour (n=4–6). The statistical difference was determined by two-tailed Student’s t-test. (D) Western blotting showing PD-L1 and phosph-ERK1/2 levels in subcutaneous iKras*3 tumours post-vehicle or MEKi treatment. Data represent mean±SEM, n=3. (E) Co-immunofluorescent staining showing E-cadherin (green), PD-L1 (red), phosph-ERK1/2 (magenta) and DAPI (blue) in vehicle or MEKi-treated subcutaneous 65 671 tumours. Scale bar 50 µm. (F) Co-immunofluorescent staining showing GFP (green), PD-L1 (red), phosph-ERK1/2 (magenta) and DAPI (blue) in vehicle or MEKi-treated subcutaneous iKras*3 tumours. Magenta arrows indicate GFP+PD-L1+phosph-ERK1/2+ tumour cells; green arrows indicate GFP+PD-L1phosph-ERK1/2 tumour cells. Scale bar 50 µm. (G) Experimental design and graph showing the changes in tumour size (%) post-MEKi and/or anti-PD-1 treatment. Data represent mean±SEM, n=4–6. Statistical difference between two groups was analysed by two-way ANOVA. DAPI, 4',6-diamidino-2-phenolindole.

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