Actinonin

Actinomycin D, LPS, Rotenone, Antimycin, Oligomycin, Etomoxir and antibodies against β-actin were purchased from Sigma-Aldrich, USA, and Chloramphenicol were obtained from Wako Pure Chemicals, Japan. Actinonin was purchased from Enzo Life Sciences, Germany. Polyclonal antibodies against mouse p32 were raised in our laboratory. Antibodies against p38, phospho-p38, Erk1/2, phospho-Erk1/2, NFκB p65, phospho-NFκB p65, IκB, phospho-IκB, ATF4 were purchased from Cell Signaling Technology, USA. Anti-COX1 antibodies and anti-B23 antibodies were from Thermo Fisher scientific, USA. Total OHPHOS rodent antibodies cocktail were from Abcam, USA.

The inhibitor for multiple MMP proteases (Actinonin) (Chemical formula C₁₉H₃₅N₃O₅, Enzo, Farmingdale, NY), MMP9 specific inhibitor (Chemical formula C₁₆H₁₇F₂N₃O₃S, Sigma-Aldrich), and MMP13 specific inhibitor (Chemical formula C₂₂H₂₀F₂N₄O₂, Sigma-Aldrich) were used to perform MMP inhibition assay. Actinonin can inhibit the activity of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP12, and MMP13. hANOs were treated with Actinonin (25 μM), MMP9 inhibitor (100 μM), and MMP13 inhibitor (10 μM) twice a day, respectively, from Day 10 (when the organoids started differentiation) to Day 24. MMP activity was measured in the supernatant of hANOs collected on Day 17 and Day 24. The dosages of Actinonin, MMP9 specific inhibitor and MMP13 specific inhibitor were modified based on the manufacturer’s manual and published articles^{57 (link)–59 (link)}.