Fig 2: Arsenic-induced autophagy was in part alleviated by RNA interference targeting aquaporin 3 (AQP3). (a) Compared with control siRNA, AQP3 siRNA significantly reduced AQP3 mRNA expression in keratinocytes treated with arsenic at a concentration of 5 µM for 6 h. Signal of AQP3 expression was measured in duplicate by quantitative real-time PCR. *P < 0.05. ****P < 0.0001, compared with the arsenic 5 µM 6 h group. (b) Consistent with quantitative real-time PCR findings, AQP3 siRNA reduces immunofluorescence of AQP3 in arsenic-treated keratinocytes. (c) Keratinocytes treated with arsenic shows arsenic-induced autophagy, as detected by an autophagy kit (ab139484, Abcam). AQP3 siRNA partially reduced arsenic-induced autophagy.

Fig 3: Pretreatment with AgNO3, an aquaporin 3 (AQP3) inhibitor, suppresses arsenic-induced autophagy. (a) The EC50 of which AgNO3 inhibits AQP3 activity is > 50 µM. (b) Keratinocytes were pretreated with AgNO3 at a concentration of 0.1, 1, or 10 µM for 15 min. The autophagy was measured by an autophagy assay kit (ab139484, Abcam). Compared with those without pretreatment of AgNO3, keratinocytes with AgNO3 pretreatment had reduced arsenic-induced autophagy. (c) Without pretreatment of AgNO3, arsenic at a concentration of 5 µM induced autophagy. After pretreatment of AgNO3, the percentages of keratinocytes with autophagy decreased. The inhibition effect of AgNO3 on arsenic-induced autophagy showed dose-dependent effect. NS, no statistical significance. *P < 0.05. **P < 0.01. ****P < 0.0001.

Fig 4: Detection of the autophagic flux in U937 cells treated with TBT-OCOCF3 (1 µM) alone or with wortmannin (WMN, 0.5 µM) after double staining with a fluorescent green dye that selectively labels autophagic vacuoles and with the fluorescent DNA-binding dye DAPI (Autophagy Assay kit, Abcam). As a positive control for autophagy, cells were treated with rapamycin 0.5 µM (Rap) for 18 h. (a) Autophagy fold change with respect to the control cells measured by green fluorescence emission using a fluorescence microplate reader after 6 and 18 h of treatment. The results are from three independent experiments performed in duplicate (mean + SD). * p < 0.01 vs. all groups. (b) Representative images of cells subjected to different experimental conditions after 6 h of treatment. In the samples treated with TBT-OCOCF3, typical features of apoptosis, especially evident following cotreatment with WMN, can be detected (white arrows). Fluorescent green autophagic vacuoles can be clearly detected only in the cells treated with rapamycin or, occasionally, in the control cells. Objective 40×, scale bar = 10 μm.