Journal: Frontiers in Endocrinology

Article Title: Succinate Mediates Tumorigenic Effects via Succinate Receptor 1: Potential for New Targeted Treatment Strategies in Succinate Dehydrogenase Deficient Paragangliomas

doi: 10.3389/fendo.2021.589451

Figure Lengend Snippet: Structure and purity of SUCNR1 inhibitors.

Article Snippet: Slides were incubated with rabbit anti-SUCNR1 antibody (ab140795 Abcam, Cambridge, UK) in blocking solution in a humidified chamber for 1 h at 37°C.

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Succinate Mediates Tumorigenic Effects via Succinate Receptor 1: Potential for New Targeted Treatment Strategies in Succinate Dehydrogenase Deficient Paragangliomas

doi: 10.3389/fendo.2021.589451

Figure Lengend Snippet: (A) Box and whisker Tukey plots showing the expression of SUCNR1 in PPGLs of three published series. Data from GSE19422 and GSE51081 ( , ), showing two different probes for SUCNR1 . SDHx (n = 19: 10 SDHB , 3 SDHC , 6 SDHD ), cluster 2 (n = 37: 3 FGFR1 , 7 HRAS , 3 MAX , 5 NF1 , 16 RET , and 3 TMEM127 ). (B) Data from the TCGA project , SDHx (n = 20: 17 SDHB , 3 SDHD ) and cluster 2 (n = 61: 2 FGFR1 , 17 HRAS , 2 MAX , 22 NF1 , 17 RET , and 1 TMEM127 ); (C) Data from E-MTAB-733 , SDHx (n = 23: 1 SDHA , 16 SDHB , 1 SDHB+SDHA , 2 SDHC , 3 SDHD ) and cluster 2 (n = 67: 2 FGFR1 , 6 HRAS , 2 MAX , 36 NF1 , 17 RET , 1 TMEM127 , and three tumors with mutations in two different drivers: MAX + HRAS , NF1 + FGFR1 , and RET + SDHA ). One-tailed Mann-Whitney test was applied to test for significant differences. (D) SUCNR1 protein expression determined by immunohistochemical staining in human PPGL tissue and normal adrenal medulla. Paraffin embedded PPGL samples from patients with mutations in succinate dehydrogenase B ( SDHB , n = 2), succinate dehydrogenase D ( SDHD , n = 4), the von-Hippel-Lindau gene ( VHL , n = 2) as well as one sample of normal adrenal medulla (NAM) were used. SDHD PGLs were from the head and neck area (HNP).

Article Snippet: Slides were incubated with rabbit anti-SUCNR1 antibody (ab140795 Abcam, Cambridge, UK) in blocking solution in a humidified chamber for 1 h at 37°C.

Techniques: Whisker Assay, Expressing, One-tailed Test, MANN-WHITNEY, Immunohistochemical staining, Staining

Journal: Frontiers in Endocrinology

Article Title: Succinate Mediates Tumorigenic Effects via Succinate Receptor 1: Potential for New Targeted Treatment Strategies in Succinate Dehydrogenase Deficient Paragangliomas

doi: 10.3389/fendo.2021.589451

Figure Lengend Snippet: (A) Relative SUCNR1 mRNA expression in hPheo1 treated with succinate or oxygen deprivation (n = 3). Three-way ANOVA showed significant differences for oxygen (p = 0.033) and treatment (p = 0.014), after verifying there was no interaction between oxygen and treatment. Dunnett’s post-hoc test was performed for the treatment main effect. The * above the 10 mM succinate bars reflects the significant difference from 0 mM, p = 0.022. (B) Relative expression of SDHB mRNA in hPheo1 parental cells (Ctr), SDHB knockout ( SDHB KO23 ), and SDHB knockout cells re-expressing SDHB ( SDHB KO23Rec ). Representative Western blot for SDHB, SDHA, and GAPDH in hPheo1-Ctr, - SDHB KO23 , - SDHB KO23Rec (right). SDHB protein expression was diminished in hPheo1- SDHB KO23 and normalized in - SDHB KO23Rec with re-constitution of human SDHB-FLAG. (C) Basal oxygen consumption rate of hPheo1-Ctr, - SDHB KO23 , - SDHB KO23Rec as determined by Seahorse XF analyzer. Basal respiration measured as oxygen consumption rate was significantly decreased in SDHB KO23 and normalized with SDHB re-constitution (n = 2). (D) Succinate-to-fumarate ratios in cell extracts (left) of hPheo1-Ctr, - SDHB KO23 , - SDHB KO23Rec and conditioned media (right) (n = 3, each). Data are shown as mean ± SEM. (E) Relative mRNA expression of SUCNR1 and PTSG2 in hPheo1-Ctr, - SDHB KO23 , and - SDHB KO23Rec . ANOVA with Dunnet’s post hoc test for difference from Ctr was performed for delta Cts. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (n = 3).

Article Snippet: Slides were incubated with rabbit anti-SUCNR1 antibody (ab140795 Abcam, Cambridge, UK) in blocking solution in a humidified chamber for 1 h at 37°C.

Techniques: Expressing, Knock-Out, Western Blot

Journal: Frontiers in Endocrinology

Article Title: Succinate Mediates Tumorigenic Effects via Succinate Receptor 1: Potential for New Targeted Treatment Strategies in Succinate Dehydrogenase Deficient Paragangliomas

doi: 10.3389/fendo.2021.589451

Figure Lengend Snippet: PC12 cells transfected with a fusion protein of mCherry and SUCNR1 or EGFP . (A) Confocal microscopy confirmed punctate mCherry signal in accordance with cell surface location typical for G-protein coupled receptors, while EGFP was equally distributed throughout the cells. (B) Western blot for mCherry and GFP confirmed successful transfection. (C) Cell viability of transfected cells was determined by XTT assay after 24 and 48 h of exposure to the indicated succinate concentrations. ANOVA with post-hoc Dunnet’s test for difference from 0 mM succinate treatment was performed. *p < 0.05 (n = 4). (D) Representative Western blot showing increased ERK phosphorylation in SUCNR1 transfected cells after 5 min exposure to 2 mM or 10 mM succinate, while no difference in phospho-ERK could be determined in EGFP transfected cells (n = 3). Mean optical density ratios of phospho-ERK to ERK ± SEM of three independent experiments are shown as bar graph. Three-way ANOVA of the log transformed pERK/ERK ratios revealed significant interaction between cell type and succinate concentration (p = 0.045). ANOVA for the effect of treatment in PC12-SUCNR1 was significant at p = 0.006, with Dunnet’s post-hoc test indicating a significant difference in phosphorylation at 10 mM succinate compared to control (p = 0.023). In PC12-EGFP cells, succinate had no effect on ERK phosphorylation (p = 0.454). (E) PC12-SUCNR1 cells were treated with candidate inhibitors (A – C) in presence and absence of succinate. The bars show the relative viability of cells treated with the drug or vehicle and succinate relative to drug or vehicle alone (n = 2). Data are shown as mean ± SEM.

Article Snippet: Slides were incubated with rabbit anti-SUCNR1 antibody (ab140795 Abcam, Cambridge, UK) in blocking solution in a humidified chamber for 1 h at 37°C.

Techniques: Transfection, Confocal Microscopy, Western Blot, XTT Assay, Transformation Assay, Concentration Assay

Journal: Nature Communications

Article Title: Succinate and its G-protein-coupled receptor stimulates osteoclastogenesis

doi: 10.1038/ncomms15621

Figure Lengend Snippet: As described in the method, ficoll-processed non-adherent bone marrow cells were seeded at 20,000 per well in 96-well plate and stimulated with the indicated concentration of cytokines (M-CSF, 30 ng ml −1 from day 1; RANKL, 50 ng ml −1 from day 3); fresh cytokines were added every other day. ( a ) Western blotting of SUCNR1 in various cell lineages from bone. Representative TRAP staining images and analysis of ( b , c ) WT mice bone marrow in vitro OC cultures treated with 1 mM succinate or control in the presence of SUCNR1 antagonist 4c at indicated concentrations. Scale bar, 200 μm. ( d , e ) Succinate effects in the OC cultures derived from bone marrow of WT and SUCNR1 KO mice, scale bar 250 μm. Data show mean±s.e.m. of triplicates. ** P <0.01, *** P <0.001 according to Bonferroni post hoc test after ANOVA, n =5.

Article Snippet: Antibodies against p50, p65, β actin, DAPI (4′,6-diamidino-2-phenylindole) and Histone H3 were purchased from Cell Signalling (Danvers, MA, USA), SUCNR1 was from Novus Biologicals (Littleton, CO, USA).

Techniques: Concentration Assay, Western Blot, Staining, In Vitro, Derivative Assay

Journal: Nature Communications

Article Title: Succinate and its G-protein-coupled receptor stimulates osteoclastogenesis

doi: 10.1038/ncomms15621

Figure Lengend Snippet: ( a ) Representative TRAP staining images (scale bar, 250 μm) and ( b ) analysis of OC cell culture on day 6 derived from WT and MKR mouse bone marrow cells. PBS or metformin (200 mg kg −1 ) were administrated daily to the mice for 14 days before bone marrow cells were harvested for OC cultures; succinate: 1 mM succinate was added in OC cell cultures; data show mean±s.e.m. of triplicated wells; * P <0.05, ** P <0.01 according to a two-tailed t -test post ANOVA, n =4. ( c ) Western blotting of OCs culture derived 12-week-old MKR mice. Suc: 1 mM, Met: 500 μM. Levels of p50 and p65 of nuclear proteins from bone marrow OC cells. ( d ) Indirect IF of P65, P50 and DAPI (4′,6-diamidino-2-phenylindole) in OCs derived from WT and SUCNR1 KO mouse bone marrow cells stimulated with 30 ng ml −1 MCSF and 30 ng ml −1 RANKL on day 5.

Article Snippet: Antibodies against p50, p65, β actin, DAPI (4′,6-diamidino-2-phenylindole) and Histone H3 were purchased from Cell Signalling (Danvers, MA, USA), SUCNR1 was from Novus Biologicals (Littleton, CO, USA).

Techniques: Staining, Cell Culture, Derivative Assay, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: Succinate and its G-protein-coupled receptor stimulates osteoclastogenesis

doi: 10.1038/ncomms15621

Figure Lengend Snippet: Under hyperglycaemic condition, BMSCs accumulate and release succinate into the bone microenvironment. Succinate binds to its G-protein coupled receptor SUCNR1 to stimulate OC differentiation and bone resorption. Targeting succinate action either through the activation or expression of SUCNR1 or reduction of succinate level, effectively blocks osteoclastogenesis activated by succinate in hyperglycaemic conditions.

Article Snippet: Antibodies against p50, p65, β actin, DAPI (4′,6-diamidino-2-phenylindole) and Histone H3 were purchased from Cell Signalling (Danvers, MA, USA), SUCNR1 was from Novus Biologicals (Littleton, CO, USA).

Techniques: Activation Assay, Expressing