Vectashield antifade mounting medium with dapi

For autophagy assay, transfected HCT116 cells expressing fusion protein GFP-LC3 (autophagy marker) were seeded in 6-well plates with coverslips for 24 h. The next day, drugs were added, cells were monitored at 8 and 12 h of treatment and fixed using 3.7% paraformaldehyde (PFA) at room temperature for 30 min and permeabilized with 0.5% Triton X-100 for 3 min. Then, coverslips were rinsed with PBS and were mounted with Vectashield (Vector Laboratories VECTASHIELD^® Antifade Mounting Medium with DAPI) and fluorescent images were acquired utilizing the Leica TCS SP8 inverted microscope. All images were processed with Leica Application Suite X.

For autophagy assay, HCT116 and SW480 cells were seeded in six-well plates with coverslips for 24 h. The next day, cells expressing GFP-LC3 (autophagy marker) were treated with 2% FBS and transfected with the mimics of miR-106a, anti-miR, miR-1, and scramble. The cells were monitored at 24 h of treatment. As a positive control were included cells treated only with lipofectamine. Later, they were fixed using 3.7% paraformaldehyde (PFA) at room temperature for 30 min and permeabilized with 0.5% Triton X-100 for 3 min. Then, coverslips were rinsed with PBS and mounted with Vectashield (Vector Laboratories VECTASHIELD® Antifade Mounting Medium with DAPI) and fluorescent images were acquired using the Leica TCS SP8 inverted microscope (objective 40 ×). All images were processed with Leica Application Suite X.