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. 2021 Sep 17;18(6):1274–1296. doi: 10.1080/15548627.2021.1973338

Figure 3.

Figure 3.

Knockdown of TSSC4 increased autophagy. TSSC4 knockdown by siRNA in U87 cells was demonstrated in Figure 1E. (A–C) Endogenous autophagy. (D–E) Autophagy induced by starvation of glucose and glutamine (No GP). (A, D) Autophagy was measured by western blotting the protein levels of the autophagy marker protein LC3-II. The lysosomal inhibitor chloroquine (CQ; 20 µM) was used to measure autophagic flux (the same hereafter). Cells were pre-treated with the autophagy inhibitor 3-methyladenine (3-MA) (2 mM) for 1 hour (H) before treatment with CQ for 24 h (the same hereafter). (B, C, E) Autophagy was measured by counting the numbers of LC3 puncta per cells. Cells stably expressing mRFP-LC3B were treated with and without CQ and 3-MA for 24 h. Then, numbers of mRFP-LC3 puncta (representing autophagosomes/autolysosomes) per cell were counted under fluorescence microscope visualization. (B) Examples of images showing mRFP-LC3 puncta. (C, E) Numbers of mRFP-LC3 puncta per cell. ACTB was used as the loading control for western blot.