Normal goat serum blocking solution

HKMT was purchased from InvivoGen (San Diego, CA, USA). A small interfering RNA (siRNA; BioneerInc, Daejeon, Korea) against NOX4 based on the target region of the NOX4 gene (sense:5′-UCAGACAAAUGUAGACAC-3′ and antisense: 5′-AGUGUCUACAUUUGUCUG-3′; siNOX4) and scrambled siRNA (sense:5′-GGTCAAGACACTATTAACA-3′ and antisense:5′-GGATTCCTAGTGTATTTCA-3′; SiCon) were used in experiments. The mesothelial cells were treated with 10 ng/mL HKMT for the 1 h after transfection with SiCon or SiNOX4.
To assess the effect of NOX4 on HKMT-induced autophagosome formation in Met5A, LC3 puncta was measured using immunofluorescence staining. In brief, Met5Acells were fixed with 4% paraformaldehyde solution and then blocked with 2.5% normal goat serum blocking solution (Vector Laboratories, Burlingame, CA, USA) for 1 h. The samples were treated with the antibody anti-LC3 (Cell signaling Technology, USA) overnight at 4 °C, followed by treatment with secondary antibody Alex Fluor 488 donkey anti-rabbit IgG (Gibco, Grand Island, NY, USA) for 1 h at 37℃.Finally, cells were rinsed with PBS and examined under a fluorescent microscope (Olympus FV500; Olympus, Tokyo, Japan). The number of LC3 puncta was counted in four separate fields.

Immunostaining was performed as previously described [8 (link)]. Briefly, cells were fixed in 4% PFA for 15 min at room temperature, blocked with 5% Normal Goat Serum Blocking Solution (Vector Laboratories, Burlingame, CA, USA, S-1000). The following primary antibodies were used: mouse anti-cTnT (Thermo Fisher Scientific, MS-295-P1); mouse anti-sarcomeric α-actinin (Sigma Aldrich, 111M4845). Cells were then incubated with secondary antibodies conjugated with Alexa Fluor 488, followed by DAPI (Thermo Fisher Scientific, D1306) counterstaining. The numbers of cells immunopositive for cTnT and α-actinin were counted in all fields per well from at least three independent experiments. The measurements and calculations were conducted in a blinded manner.