Fig 1: Fh1- and Sdhb-deficient cells accumulate fumarate and succinate respectively and display decreased oxygen consumption rate.(A) Western blot confirming CRISPR/Cas9 mediated KO of Sdhb (left panel) and Fh (right panel). GAPDH was used as a loading control. (B) Functional validation of succinate (left panel) and fumarate (right panel) levels by LC-MS in isogenic cell lines (n = 3). (C) Seahorse assay showing altered OCR in Fh1-KO (top panel) and Sdhb-KO (bottom panel). (D) Proliferation rates of mutant vs. parental cells (n = 3). Error bars represent means ± SEM. *** P < 0.001, ** P < 0.01, * P < 0.05.
Fig 2: Gene expression profile of engineered cell lines by RNA-seq.(A) The volcano plot displays the significant up-regulated (pink) and down-regulated (blue) genes in the Sdhb-KO (left panel) and Fh1-KO (right panel) cells compared to the parental cells. Top 20 DGE’s are labelled. (B) Reactome over-representation analysis showing top 20 over-represented biological Reactome pathways for DGE’s associated with Sdhb-KO (left panel) and Fh1-KO (right panel). (C) Venn diagram showing upregulated genes and downregulated genes shared between Sdhb-KO and Fh1-KO. (D) Venn diagram showing the hypermethylated CpG island shared between Sdhb-KO and Fh1-KO. (E) Logistic regression volcano plots. Each point represents a differentially methylated CpG site, with red points exhibiting FDR adjusted p-Value < 0.05.
Fig 3: SOD2-overexpressing cells showed an increase in amino-acid synthesis pathways and a partial Krebs Cycle blockage through a decrease in succinate dehydrogenase. Metabolomic analysis of LNCaP-Mock (MOCK) and LNCaP-SOD2 (SOD2). (A) Summary graph and list of the main metabolic pathways altered in SOD2 cells, showing impact and enrichment. (B) Relative uptake of 13C by succinate and malate, indicating accumulation of recently formed succinate (m + 4) and a decrease in fumarate. (C) Protein levels of succinate dehydrogenase (SDH) in MOCK and SOD2 cells, values of densitometric quantification relative to B-ACTIN are shown. (D) Levels of lactate loaded with 1-, 2-, or 3-13C in its structure (n = 3 each group). For all panels, values are expressed as mean ± SEM; t-test was used as statistical analysis; * = p-value < 0.05, ** = p-value < 0.01.
Fig 4: Sdhb deficiency confers sensitivity to combined PARP-inhibitor and low-dose TMZ in vivo.(A) Experimental schema of in vivo treatments. RENCA Sdhb-KO cells were injected subcutaneously into the flank of BALB/c mice. Seven days after injection, tumors were measured and mice were randomized into four-arm treatment groups. Treatment was initiated when tumors reached an average size of 40–100 mm3. (B) Mice carrying flank tumors of Sdhb-KO cells were treated with no treatment (n = 9), TMZ alone (3 mg/kg) (n = 10), BGB-290 (3 mg/kg/dose, 2x/day) (n = 10), or TMZ (3 mg/kg) and BGB-290 (3 mg/kg/dose, 2x/day) (n = 9). Mice were treated in 5-day cycles for a total of 4 cycles. Data are represented as mean ± SEM. (C) Mean body weight of mice during Sdhb-KO flank tumor experiment. Data are represented as mean ± SEM. P values were calculated using two-way ANOVA. *** P < 0.001, ** P < 0.01, * P < 0.05.
Fig 5: Suppression of expression of NDUFAF5 and assembly of complex I. Human 143B cells were transfected three times with either negative control siRNA or siRNA specific for NDUFAF5, denoted by a and b, respectively, and samples of mitoplasts and inner membranes were made 48 h after each transfection. Parts A and B, mitoplasts and inner mitochondrial membrane proteins, respectively, fractionated, in A by SDS-PAGE and, in B, by BN-PAGE. In part A, samples taken from three duplicate gels were Western-blotted with antibodies against subunits NDUFS2, NDUFS7, and ND1 of complex I, and one of them, employed as a loading control, was stained with Coomassie Blue dye, after Western blot analysis; in part B, membranes were probed with antibodies against the peripheral arm subunit NDUFS2, and membrane arm subunit NDUFB8, and probed a second time with an antibody against complex II subunit SDHB as a loading control, shown for the 192 h sample. CI-980 kDa, mature complex I; CI-200 kDa, -370 kDa, -550 kDa, sub-complexes of complex I; CII-130 kDa, complex II. The control cell results (denoted by the letter a in A and B) have been presented previously in another context (19).
Supplier Page from MilliporeSigma for Anti-SDHB antibody produced in rabbit