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. 2021 Aug 19;11:711043. doi: 10.3389/fonc.2021.711043

Figure 4.

Figure 4

GZ17-6.02 and pemetrexed interact to alter cell signaling, increase autophagosome formation, and kill via toxic autophagy A549 NSCLC cells that express a mutant K-RAS protein. (A) A549 cells were treated with vehicle control, GZ17-6.02 (2 μM final curcumin), pemetrexed (500 nM), or the drugs combined for 6 h. Cells were fixed in place and immunostaining was performed to determine protein expression and phosphorylation (n = 3 ± SD) *p < 0.05 less than vehicle; **p < 0.05 less than GZ17-6.02 alone; # p < 0.05 greater than vehicle control; ## p < 0.05 greater than GZ17-6.02 alone. (B) A549 cells were transfected with siRNA molecules to knock down protein levels or with plasmids to express activated forms of mTOR or STAT3 and then subsequently treated with vehicle or [pemetrexed (500 nM) + GZ17-6.02 (2 μM curcumin final)] in combination for 4 h and 8 h. The number of intense staining GFP+ and RFP+ punctae were determined randomly in at least 50 cells, and the mean number of punctae per cell was determined (n = 3 ± SD). p < 0.05 greater than the corresponding values after 4 h; *p < 0.05 less than the corresponding values in siSCR/CMV-transfected cells. (C) A549 cells were transfected to knock down Beclin1 or ATG5 expression. Subsequently cells were treated with vehicle, pemetrexed (500 nM), GZ17-6.02 (2 μM curcumin final), or the drugs in combination for 24 h. Cell viability was determined by trypan blue exclusion (n = 3 ± SD). *p < 0.05 less than the corresponding values in siSCR cells.