Figure 2 Troponin C1, slow skeletal and cardiac type (TNNC1) overexpression could enhance the sensitivity of A549 cells to gemcitabine (GEM). (A) Detection of the knockout effect of TNNC1. (B) TNNC1 lost model was successfully established in A549/GemR cells by transfecting TNNC1 short interfering ribonucleic acid (siRNA-TNNC1) and negative control RNA (siRNA-NC) respectively by reverse tracscription-quantitative polymerase chain reaction (RT-qPCR) (*** P<0.01). (C) Stable TNNC1-overexpressed cell line was successfully established in A549 cells by infecting with TNNC1 overexpression lentivirus (Lv-TNNC1) or negative control lentivirus (Lv-NC) respectively through RT-qPCR assay (*** P<0.01). (D) Western blot assay was used to evaluate the expression of autophagy-related proteins in A549 and A549/GemR model cells. (E) A549 cells were incubated with increasing concentrations of GEM (0–20 μM) for 24 h; meanwhile A549/GemR cells were incubated with increasing concentrations of GEM (0–80 μM) for 24 h. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). A549 were incubated with 5 μM GEM, whereas A549/GemR were incubated with 40 μM GEM. Then cell viability was determined at 24, 48, and 72 h by MTT assay. The results were shown as mean±SD of four independent experiments. (** P<0.01, *** P<0.001, ## P<0.01, ### P<0.001). (F) Flow cytometry with annexin V-fluorescein isothiocyanate/propidium iodide double staining detected apoptosis in A549 and A549/GemR model cells after GEM treatment respectively with 5 μM and 40 μM for 24 h. (G) LC3 puncta accumulation in A549 and A549/GemR model cells after GEM treatment with 5 μM and 40 μM for 24 h, respectively. The original magnification was ×200.