Fig. 2: NOX4 is responsible for TGF-β downregulation-induced ROS generation.

a A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of NOX1, NOX4, 5-lipoxygenase, and GAPDH were detected by western blot analysis. b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or transfected with pCMV6-myr-Akt or infected with adenovirus expressing shTGF-β1 and subsequently transfected with pCMV6-myr-Akt. After 48 h, NOX4, phospho-Akt, Akt, and GAPDH were detected by western blot analysis. c Expression levels of NOX4 mRNA in A375 and HPAC cells were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). d A375 cells were infected with adenovirus expressing shTGF-β1. After 6 h, infected cells were treated with NAC (10 mM) for 42 h, and then the expression of NOX4 and GAPDH was detected by western blot analysis. e A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with p38 inhibitor (SB203580, 10 μM) or JNK inhibitor (SP600125, 10 μM) or both for 42 h. Then the expression of NOX4 and GAPDH was detected by western blot analysis. f A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by NAC (10 mM) or adenovirus expressing shTGF-β1 followed by p38 inhibitor (SB203580, 10 μM) or adenovirus expressing shTGF-β1 followed by transfection with pCMV6-myr-Akt or adenovirus expressing shTGF-β1 followed by transfection with siRNA of Smad4 based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, the expression levels of NOX4 mRNA were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (*p < 0.05; **p < 0.01). g A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of Smad4. After 48 h, NOX4 and GAPDH were detected by western blot analysis. h A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right). i A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by treatment with NOX4 inhibitor (GKT137831, 140 nM). After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right). j A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 or NOX1 based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right)