Figure 2. SDHB loss activates a succinate-Ca²⁺-calpain-Cdk5 cascade.

(A) [Ca²⁺]_i activity reported by time-lapse live-cell imaging of cells loaded with Fluo-4 AM, images acquired pre or post stimulation with ionomycin (10 μM). Representative pseudo-colored images are shown with time course of fluorescence intensity quantification as %[Ca²⁺]_i, scale bar, 80 μm.

(B) Liquid chromatography-mass spectrometry (LC-MS) quantitation of succinate in WT and KO cell extracts.

(C) Quantitation of fluorescence intensity and photomicrographs showing effects of ionomycin on %[Ca²⁺]_i in WT hPheo1 cells treated with dimethyl succinate (DMS, 2 mM) or controls.

(D) Time-lapse measurement of [Ca²⁺]_i in SDHB KO cells pretreated with SUCNR1 antagonist, NF-56-EJ40, with representative images, scale bar, 50 μm

(E) Schematic workflow of calpain sensor, and representative pseudocolor FRET map and normalized FRET index of WT and SDHB KO hPheo1.

(F) Immunoblots of Cdk5, p25/p35 in WT and SDHB KO cells with quantitation.

(G) 3D surface plot of immunostained cells comparing relative levels of p35/25 between WT and KO hPheo1, scale bar, 80 μm.

(H and I) Representative confocal 3D photomicrographs and quantitation showing p35/25 expression in WT hPheo1 treated with increasing concentrations of succinate as indicated.

(J) Quantitative immunoblotting of Cdk5/p35/p25 levels in tumor lysates derived from WT (n = 4) and KO (n = 6) xenografts. Values are means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001; n.s., non-significant compared by t test and/or one-way ANOVA.

(K) Schematic illustrating the mechanism of action mediated via succinate accumulation in chromaffin cells.