Fig. 8: Caspase-2 inhibitors prevent sterol regulatory element-binding protein 2 (SREBP2) activation via the blockage of Casp2-dependent S1P proteolytic activation.
From: Genuine selective caspase-2 inhibition with new irreversible small peptidomimetics
a Schematic representation of cDNAs encoding HA-tagged Casp2, Flag-tagged PIDD1, 6His tagged RAIDD, Myc-tagged S1P, V5-tagged SREBP2. b, c Effects of Casp2 inhibitors on Casp2-dependent S1P and SREBP2 activation. HEK 293 cells were transfected with indicated plasmids in order to overexpress SREBP2, S1P, RAIDD, PIDD1, and Casp-2. After 5 h, cells were incubated in DMEM/F12 medium supplemented with DMSO (Veh.), LJ2a, LJ3a, or Δ2ME-TRP601 (10 μM) for 16 h. b Membrane fraction (Memb. Fraction) and nuclear extract (Nucl. Extracts) were prepared and subjected to WB to detect full-length (FL) and cleaved (Cl.) S1P (through Myc tag) and SREBP2 (through V5 tag). IRE1, Inositol-requiring transmembrane kinase/endoribonuclease 1. c Whole cell extracts (WCL) were prepared and subjected to WB to detect full-length (FL) and cleaved (Cl.) SREBP2. d Effects of Casp2 inhibitors on the expression of transfected Casp2, RAIDD, PIDD1 components. HEK 293 cells were transfected with the indicated plasmids. After 5 h, cells were incubated in DMEM/F12 medium supplemented with DMSO (Veh.), LJ3a, or Δ2Me-TRP601 (10 µM) for 16 h. Then, whole cell extracts (WCL) were prepared and subjected to WB analysis to detect Casp2 (through HA tag), RAIDD (through 6HIS tag), and PIDD1, PIDD1-C, PIDD1-CC (through Flag tag). FL full length, Cl. cleaved, N.S. non-specific. Full-length uncropped original WB is shown in Supplementary Fig. S5.
