P limk1

NP cells were treated with 10 ng/mL leptin (Sigma-Aldrich, Oakville, ON, Canada) for 5 min, 10 min, 30 min and 24 h, respectively. Untreated cells were used as controls. Cell lysates were subject to SDS-PAGE and transferred to PVDF membranes (Millipore, Boston, MA, USA). Primary antibodies against the following proteins were used: LIMK1, p-LIMK1, cofilin-2, p-cofilin-2 (Abcam, Cambridge, UK) and GAPDH (Abcam, Cambridge, UK). HRP-conjugated secondary antibodies were used. Signal was detected using ECL kit (Millipore, Boston, MA, USA).

Tissues fixed with 4% paraformaldehyde were embedded in paraffin and cut into 5-μm thick slices. For H&E staining, the tissue slices were dewaxed in xylene, rehydrated with decreasing concentrations of ethanol, and washed with PBS. The slices were stained with hematoxylin for 30 s with agitation and rinsed with water. Then, the slices were stained with eosin for 10–30 s with agitation and rinsed with water. After staining, the slices were dehydrated, mounted, and covered with coverslips. Immunohistochemical (IHC) staining was performed according to a published protocol (Liu et al., 2017 (link)). Samples were incubated with antibodies (Cell Signaling Technology, Beverly, MA, United States, except as noted) against Ki-67, SphK2 (Proteintech, Wuhan, China), p-PAK1, p-LIMK1 (Abcam, Burlingame, CA, United States), and p-Cofilin1. TUNEL staining was performed according to the manufacturer’s protocol (Beyotime Biotechnology, Nantong, China). The Ki-67-positive cells, TUNEL-positive cells, and the integrated optical density (IOD) of SphK2, p-PAK1, p-LIMK1, and p-Cofilin1 staining were analyzed using ImageJ software.