A1r ti2 e

To generate the 35S::SMXL6,7,8-GFP (OE-SMXL6,7,8) construct, the full-length coding sequences of SMXL6,7,8 were individually cloned into the KpnI-SalI sites of the pCAMBIA1300-GFP vector via the in-fusion method (Clontech). The primers are shown in Table S1. To generate OE-SMXL6, OE-SMXL7, and OE-SMXL8 transgenic plants, the corresponding constructs were introduced into the Agrobacterium strain GV3101 and then into the wild type via the floral dip method [39 (link)]. Homozygous OE-SMXL6-4, OE-SMXL7-1, and OE-SMXL8-2 were introduced into dwa1-1 to obtain OE-SMXL6/dwa1-1, OE-SMXL7/dwa1-1, and OE-SMXL8/dwa1-1 lines via genetic crossing, respectively.
For the fluorescence observation of the OE-SMXL6/7/8 and OE-SMXL6/7/8/dwa1 transgenic lines, these seeds were sown in the same ½ MS medium plate and subjected to a 16-hour-light/8-hour-dark photoperiod and 60% relative humidity at 22 °C in a greenhouse. Fluorescence observation was performed after 5 days. The seedlings of two transgenic lines, such as OE-SMXL6 and OE-SMXL6/dwa1, were placed under the same slide for observing using the layer scanning method with a confocal laser scanning microscope (Nikon, A1R + Ti2-E, Tokyo, Japan). Then, multiple-layer fluorescent images were superimposed into a single image using a stacking technique in Photoshop. Finally, the overall fluorescence in the multiple roots of the two lines were counted.

The coding regions of SMXL6,7,8 and DWA1 were cloned next to the N-terminal part of YFP (nYFP) in the pEarleygate202-YN vector and the C-terminal part of YFP (cYFP) in the pEarleygate201-YC vector. Different constructs (including empty vectors) were transformed into the Agrobacterium strain GV3101 and then infiltrated into Nicotiana benthamiana leaves for transient expression. Fluorescence was observed and photographed in leaf epidermal cells 2 days after infiltration using a Confocal laser scanning microscope (Nikon, A1R+Ti2-E, Tokyo, Japan).

The cryosections were washed with PBS and then blocked with 5% horse serum in PBS for 1 h and incubated overnight at 4 °C in a solution of the following primary antibody: rabbit anti-AQP5 antibody (1:200, Merck Millipore, Billerica, MA, USA). It was then washed with PBS and reacted with a secondary antibody, Alexa Flour 488 Donkey anti-rabbit IgG (1:800, Invitrogen), for 60 min at room temperature. It was re-washed with PBS followed by nuclear staining with DAPI (1:10,000, Invitrogen) for 3 min at room temperature; then, it was washed with PBS and encapsulated using a VECTASHIELD Vibrance Antifade Mounting Medium (Vector Laboratories). After drying, the stained sections were photographed cross-sectionally using a confocal laser microscope A1R/Ti-2E (Nikon, Tokyo, Japan).

In brief, HaCaT cells were treated with LBP and PM2.5, and then washed three times with PBS and fixed with 4% paraformaldehyde at 25°C for 15 min. After cell fixation, incubated with primary antibody overnight at 4°C, and then washed three times with PBST and incubated with fluorescent secondary antibody for 1 h. After that, with DAPI counterstained-cell nucleus, the samples were observed by a confocal microscope (Nikon, A1R+ Ti2-E).

HaCaT cells (1 × 10⁵ cells/well) were seeded in 6-well plates for 24 h. Then cells were pretreated with or without LBP (or 3-MA) for 2 h, and subsequently treated with or without PM2.5 (100 μg/ml) for another 24 h. Apoptosis was detected by a confocal microscope (Nikon, A1R+ Ti2-E), using an Annexin V- FITC/PI Apoptosis Kit.