Hematoxylin and eosin h e

Tert-Butylhydroquinone (TBHQ), streptozocin (STZ), insulin and HClO were provided by (Sigma, St. Louis, MO, USA).
Fetal bovine serum (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) were provided by Gibco BRL (Gibico, Grand Island, NY, USA). The bicinchoninic acid (BCA) protein assay, hematoxylin and eosin (HE) and Hoechst staining kits were provided by Beyotime (Beijing, China). Antibodies targeting p-AKT, AMPKα2, glycogen synthase kinase-3 β (GSK3 β), and glucose transport-4 (GLUT4) were provided by Abcam (Cambridge, MA, USA). Anti-phosphatidylinositol 3-kinase (PI3K) and anti-AKT antibodies were provided by Sangon (Shanghai, China). Antibodies directed against p-PI3K were provided by Cell Signaling Technology (Danvers, MA, USA). Antibodies targeting β-actin were provided by Beyotime (Beijing, China). PRKAA2 siRNA and riboFECT™ CP transfection kit were provided by Guangzhou RiboBio (Guangzhou, China).

Harvested tumor samples were fixed, paraffin embedded, and sectioned into 4-µm sections onto slides. For IHC analysis, sections were stained with Ki-67 antibody (1:500; CST Biological Reagents Co., Ltd., Shanghai, China) at 4 °C overnight, followed by incubation in avidin-biotin-peroxidase complex reagent (Vector Laboratories, Burlingame, CA, USA). For histopathology analysis, sections were stained with hematoxylin and eosin (HE) (Beyotime). Images were captured using a BX51 microscope (Olympus).

Briefly, the rat lung specimens were collected, fixed in 4% buffered paraformaldehyde for 24 h, embedded in paraffin, and sectioned to 5 µm-thick slices by freezing microtome. Next, the tissue sections were deparaffinized, hydrated, and stained with hematoxylin and eosin (H&E) (Beyotime, Shanghai, China) and the Modified Masson’s Trichrome stain kit (Solarbio, Beijing, China). The histological examination was performed by 2 independent pathologists blinded to the study protocol. The wet/dry (W/D) weight ratio was determined to assess the degree of pulmonary edema following PQ administration. In brief, the wet weight was measured after excising the lung tissues, and the lung was then placed in an oven until the weight remained constant, at which time it was weighed again to measure the dried weight. The W/D weight ratio was calculated by dividing the dry weight by the wet weight. A hydroxyproline (HYP) detection kit (Jiancheng, Nanjing, China) was used to measure the HYP content of rat lungs to assess the lung collagen deposition.