Sa00013 4

OCT-embedded liver sections held in an ultralow-temperature refrigerator were cut to a thickness of 6 µm each and were left at room temperature for half an hour, rinsed with TBST to remove OTC on the surface and then fixed with cold acetone (−20 °C). An appropriate amount of goat blocking serum was used to block the liver sections for an hour. Primary antibodies (anti-CK18, ab181597, 1:200, Abcam; anti-ALB, ab207327, 1:500, Abcam; anti-AFP, ab213328, 1:200, Abcam) were used to bind specific proteins on the sections at 4 °C overnight. The liver slices were then subsequently incubated with the secondary antibody (SA00013-4, 1:500, Proteintech) for an hour at 37 °C. The nuclei were stained with DAPI for 4 min. After adding antifade mounting medium (Beyotime, China), the staining of the liver slices was observed under a fluorescence microscope (Olympus, Japan).

Cells were seeded on coverslips and then transfected with pCMV6-Entry, pCMV6-ZDHHC22, or pCMV6-ZDHHC22-(C111A). After 48 hours, cells were fixed with 4% paraformaldehyde for 20 min and then permeabilized with 0.5% Triton X-100 for 10 min. After blocking with blocking buffer, cells were incubated with primary antibodies overnight at 4°C and then incubated with secondary antibodies for 1 hour at 37°C. DAPI (Roche, Palo Alto, CA, USA) was used for DNA counterstaining. Photomicrographs were acquired with a confocal laser scanning microscope (Leica, Hilden, Germany). The following antibodies were used for immunofluorescence: anti-Flag (1:400, #14793, Cell Signaling Technology), anti-mTOR (1:200, #2983, Cell Signaling Technology), anti-phospho-AKT(S473) (1:200, #4060T, Cell Signaling Technology), anti-PHLPP2 (1:40, ab71973, Abcam), ER-Tracker Red (1:2000, C1041, Beyotime), CoraLite488 (1:200, SA00013-1, Proteintech), and CoraLite594 (1:200, SA00013-4, Proteintech).

Immunofluorescence analysis was applied to analysis the expression of EMT-related genes. In briefly, transfected cells were fixed and incubated with the anti-E-cadherin (1:200; Abcam, ab1416, USA) or anti-Vimentin (1:200; Abcam, ab8978, USA) primary antibodies overnight at 4°C. Fluorescence-conjugated secondary antibodies were added for another incubation after washing with PBS for three times at room temperature for 1 h (1:200; ProteintechGroup, SA00013-2 for E-cadherin and SA00013-4 for Vimentin, USA). The images were captured after staining with DAPI solution.

Lung sections or cells treated with 4% paraformaldehyde for 15 min and 0.2% triton for 10 min were blocked with 5% goat serum for 60 min at room temperature. Then, they were incubated with primary antibodies at 4 °C for overnight and stained with FITC- (A0562, beyotime) and Coralite594-conjugated secondary antibody (SA00013-4, Proteintech) at room temperature for 1 h, after which nuclear were stained with DAPI (F6057, sigma). Pictures were captured with confocal microscopy (LSM880, Carl Zeiss) or fluorescence microscopy (Imager D2, Carl Zeiss). Primary antibodies used here were as follows: anti-NOX4 (ab154244, abcam), anti-COX IV (200147, ZENBIO), anti-LC3 II/I (A5179, bimake), anti-SOD2 (A5377, bimake), anti-collagen I (ABM40379, Abbkine), anti-CPT1a (15184-1-AP, proteintech) and anti-PPARα (Abp55667, Abbkine).

This experiment was grouped into control, US, HN-Ti₃C₂, and HN-Ti₃C₂+US groups. hBMSCs seeded on glass coverslips in 24-well plates were incubated for 14 or 21 days under the corresponding conditions according to the groups. The US and HN-Ti₃C₂+US groups were treated with US (1.0 MHz, 0.2 W/cm², 50% duty cycle) every two days for 10 min each time, for a total of 4 times. Cells were washed and fixed with 4% paraformaldehyde for 30 min before permeabilized with 0.5%Triton x-100 for 15 min. After sealing with blocking solution for 30 min, alkaline phosphatase (ALP; rabbit source, DF6225) and OPN (Affinity, rabbit source, AF0227) antibodies diluted in the antibody diluent were added to the corresponding wells and incubated for 12 h at 4 °C. After discarding the antibody, the wells were washed three times with 0.1% PBST and then anti-rabbit antibody (red fluorescence) (Proteintech, SA00013-4) was added to each well and incubated in the dark for 1 h at room temperature. Anti-rabbit antibody was discarded, and the wells were washed again with 0.1% PBST before staining the cytoskeleton and nucleus. The cytoskeleton was stained with green fluorescence-labeled phalloidin solution (Yeasen, 40735ES75), and the nuclei were stained with DAPI solution (Beyotime, P0131). Fluorescence staining images were captured using a fluorescence microscope.