Gtx101395

We treated keratinocytes with IL-33 (100 ng/mL) and IL-17 (100 ng/mL) for 2, 6, and 24 h, respectively. The cells were detached from the cultured dishes by trypsin. The cells were fixed and holed by 1% Saponin for 30 min in room air. We stained the cells with 1:500 Rabbit (Genetex, GTX101395, San Antonio, TX, USA) overnight at 4 °C. Then, the cells were bound by Goat anti-rabbit IgG-568 antibody (secondary antibody) for 1 h in room air. We then measured the expressions of ACE2 by flow cytometry (BD Biosciences, San Jose, CA, USA).
The following cytokines and antibodies were used: IL-33: PeproTech #200-33, 100 ng/mL; IL-17: PeproTech #200-17, 100 ng/mL; ACE2: Rabbit, Genetex, GTX101395; Isotype: Rabbit IgG; secondary antibody: Goat anti-rabbit IgG-568.
In order to validate that IL-33 indeed enhances the expression of ACE2 in keratinocytes, we used an immunofluorescent examination to measure the expression of ACE2 in keratinocytes treated with IL-33. Furthermore, we were interested in the mechanism through which IL-33 enhances the expression of ACE2. Therefore, in order to test this hypothesis, inhibitors of ERK (PD98059) or STAT3 (STA21) were added to the keratinocytes treated with IL-33.

Keratinocytes were cultured with keratinocyte SFM (serum-free medium). KCs, attached on 16 mm cover glasses, were cultured in 12-well plates. Cytokines and protein inhibitors were added to the 12-well plate for 24 h. Then, the cells were fixed and holed by 0.1% triton X for 1 h in room air. We stained the keratinocytes with 1:500 rabbit anti-ACE2 (Genetex GTX101395) overnight at 4 °C, followed by Goat anti-rabbit IgG-568 antibody (secondary antibody) binding for 1 h in room air. Immunostained samples were analyzed using the feature of “Analyze Particles” in ImageJ.
The following cytokines and protein inhibitors were used: IL-33: PeproTech #200-17, 100 ng/mL; PD98059 (ERK 1/2 inhibitor): Sigma P215, 50 uM STA21 (STAT3 inhibitor): CAYMAN 14996, 250 nM. The following antibodies were used for immunofluorescent staining: Isotype IgG: Rabbit IgG, ACE2: Rabbit, Genetex GTX101395. 4,6-Diamidino-2-phenylindole (DAPI) (Fluka) was used for the staining of the nuclei.