Journal: Current Biology

Article Title: Injury primes mutation-bearing astrocytes for dedifferentiation in later life

doi: 10.1016/j.cub.2023.02.013

Figure Lengend Snippet:

Article Snippet: For human astrocytes, p53 downregulation was induced by lentiviral infection with shp53 or vector control. shp53 pLKO.1 puro was a gift from Bob Weinberg (Addgene plasmid # 19119; http://n2t.net/addgene:19119 ; RRID:Addgene_19119). pLKO.1 - TRC control was a gift from David Root (Addgene plasmid # 10879; http://n2t.net/addgene:10879 ; RRID:Addgene_10879).

Techniques: Virus, Plasmid Preparation, Recombinant, Imaging, cDNA Synthesis, In Vivo, Control, Software

Journal: Communications Biology

Article Title: SIRT5-mediated desuccinylation of MTHFD2 enhances chemoresistance in breast cancer cells by reducing therapy-induced senescence

doi: 10.1038/s42003-025-08878-z

Figure Lengend Snippet: A Volcano plot of changes in MTHFD2, ME2, and IDH2 succinylation modification sites in U2OS cells treated with 20 μM Etoposide. B In vitro MTHFD2 succinylation assay. MTHFD2 proteins were incubated with different concentrations of succinyl-CoA. Protein succinylation levels were analyzed by western blotting. C Possible succinylation sites of MTHFD2 identified by LC-MS. D Measurement of MTHFD2 succinylation in HEK293T cells transfected with Flag-tagged wide-type (WT) or mutant (K44R, K50R, K88R, K93R, K139R, K164R, K220R, and K286R) MTHFD2. MTHFD2-Flag was immunoprecipitated and its succinylation was examined with a pan-succinylated lysine antibody. E Identification of succinylated peptides around MTHFD2 K44 as determined by mass spectrometry. F Succinylation levels of ectopically expressed MTHFD2 in HEK293T cells were measured. Cells were treated with 5 mM NAM for 3 h before harvesting. MTHFD2-Flag was immunoprecipitated from cell lysates and its succinylation was examined with a pan-succinylated lysine antibody. G SIRT5, instead of other sirtuins, decreases MTHFD2 succinylation level. HEK293T cells were cotransfected with Flag-tagged MTHFD2 and Myc-tagged SIRT1, SIRT2, SIRT3, SIRT4, SIRT5 or SIRT6. Precipitated SHMT2 proteins or ectopically expressed SIRT3, SIRT4, or SIRT5 were determined. Relative succinylation level was quantified. H Endogenous interaction of SIRT5 and MTHFD2 in HEK293T cells. I The effect of knockdown of SIRT5 on succinylation of MTHFD2 in MDA-MB-231 cells. J SIRT5 decreases the MTHFD2 succinylation level. Myc-SIRT5 plasmid was transfected into HEK293T cells. The MTHFD2 succinylation level was detected by western blotting. K SIRT5 mainly desuccinylates MTHFD2 in mitochondria. Flag-tagged MTHFD2 and Flag-tagged SIRT5 or mitochondrial transit peptide defective SIRT5Δ50 were cotransfected into HEK293T cells. Succinylation of precipitated MTHFD2 was determined. Relative succinylation level was quantified. L WT SIRT5 but not its catalytic-dead H158Y mutant decreases the MTHFD2 succinylation level. WT SIRT4 and H158Y mutants were transfected into HEK293T cells. The MTHFD2 succinylation level was detected by western blotting. M The K44 residue of MTHFD2(marked red) is evolutionarily conserved. N In vitro succinylation of MTHFD2 decreased its activity. MTHFD2 proteins were purified and incubated with or without 100 μM succinyl-CoA for 30 min at 37 °C before western blotting and MTHFD2 enzymatic assays were performed. Unpaired two-tailed t-test was used for statistical analyses. O MTHFD2 enzyme activity was determined after SIRT5 knockdown. MTHFD2 enzymatic activity was quantified. P K44E mutation decreased MTHFD2 activity. Different MTHFD2 plasmids were transfected into HEK293T cells, and MTHFD2 proteins were purified from these cells and subjected to MTHFD2 enzymatic activity analyses. Q NADPH levels in MDA-MB-231 cells overexpressing Different MTHFD2 plasmids in the absence of MTHFD2 siRNA are shown. R NADP + /NADPH ratio in MDA-MB-231 cells overexpressing Different MTHFD2 plasmids in the absence of MTHFD2 are shown. S NADPH levels in MCF-7 cells overexpressing Different MTHFD2 plasmids in the absence of MTHFD2 siRNA are shown.

Article Snippet: The shRNA sequences for MTHFD2 and SIRT5 were cloned into pLKO.1 TRC (Addgene plasmid 10879) and verified by DNA sequencing.

Techniques: Modification, In Vitro, Incubation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Transfection, Mutagenesis, Immunoprecipitation, Mass Spectrometry, Knockdown, Plasmid Preparation, Residue, Activity Assay, Purification, Two Tailed Test

Journal: Communications Biology

Article Title: SIRT5-mediated desuccinylation of MTHFD2 enhances chemoresistance in breast cancer cells by reducing therapy-induced senescence

doi: 10.1038/s42003-025-08878-z

Figure Lengend Snippet: A HFF cells (P30) that express FLAG-MTHFD2 and FLAG-MTHFD2 K44E were transfected with siMTHFD2, the ROS level was determined by flow cytometry analysis. B Effect of ectopic expression of WT MTHFD2 and MTHFD2 K44E mutant on cellular senescence. HFF cells (P30) were transfected with siMTHFD2 or siControl to knock down the endogenous MTHFD2, and then express FLAG-MTHFD2 and FLAG-MTHFD2 K44E. The cellular senescence was indicated by SA-β-gal staining. C HFF cells (P30) that were transfected with siMTHFD2 to knock down the endogenous MTHFD2, and express FLAG-MTHFD2 K44E, and add or not add the 2 mM ROS scavenger(NAC), the ROS level was determined by immunofluorescence. D Senescence-associated β-galactosidase activity was assessed in serially passaged HFF cells (P50) following treatment with NAC (2 mM). E HFF cells (P18) transfected with SIRT5 siRNA or control siRNA, the relative ROS level was determined by flow cytometry analysis. F Effect of SIRT5 silencing (siSIRT5) and siControl on HFF (P18) cellular senescence. G HFF (P18) were cultured in medium and then transfected with siSIRT5 and siControl, respectively. The cellular senescence was indicated by SA-β-gal staining. H HFF cells (P30) were transfected with siRNA to knock down the endogenous SIRT5, and then expressed FLAG-MTHFD2 and FLAG-MTHFD2 K44E. The cellular senescence was indicated by SA-β-gal staining.

Article Snippet: The shRNA sequences for MTHFD2 and SIRT5 were cloned into pLKO.1 TRC (Addgene plasmid 10879) and verified by DNA sequencing.

Techniques: Transfection, Flow Cytometry, Expressing, Mutagenesis, Knockdown, Staining, Immunofluorescence, Activity Assay, Control, Cell Culture

Journal: Communications Biology

Article Title: SIRT5-mediated desuccinylation of MTHFD2 enhances chemoresistance in breast cancer cells by reducing therapy-induced senescence

doi: 10.1038/s42003-025-08878-z

Figure Lengend Snippet: A Immunofluorescence of MTHFD2 co-localization with SIRT5. B Effect of ectopic expression of WT MTHFD2, MTHFD2 K44E and Gln132Lys/Asp155Ala-MTHFD2 mutant on DNA damage. MCF-7 cells were transfected with siSIRT5 or sicontrol to knock down the endogenous SIRT5, and then express FLAG-MTHFD2, FLAG-MTHFD2 K44E and Gln132Lys/Asp155Ala-MTHFD2. The γH2AX protein expression was detected by immunofluorescence. C MDA-MB-231 cells overexpressing Myc-SIRT5 or Myc-vector control in the presence of 3xFlag-MTHFD2-WT or 3xFlag-MTHFD2-K44E, cell proliferation was analyzed by EdU-488 experiment. D RNA-sequencing expression (level 3) profiles and corresponding clinical information for BRCA were downloaded from the TCGA dataset ( https://portal.gdc.com ). The two-gene correlation map is realized by the R software package ggstatsplot. Spearman’s correlation analysis to describe the correlation between quantitative variables without a normal distribution. P -values less than 0.05 were considered statistically significant (* p < 0.05). E MDA-MB-231 cells overexpressing Myc-SIRT5 or Myc-vector control in the presence of 3xFlag-MTHFD2-WT or 3xFlag-MTHFD2-K44E, and treated with 40 μM Olaparib, cell proliferation was assayed by crystal violet staining. F Average images (left) and weights (right) of xenograft tumors (4 weeks, means ± S.D., n = 5) generated by control or MTHFD2-silenced MDA-MB-231 cells overexpressing 3xFlag-MTHFD2-WT or 3xFlag-MTHFD2-K44E as indicated. One week later, when the tumors reached a volume of 100 mm 3 , Olaparib (5 mg/kg) was intraperitoneally injected once a day for 2 weeks. For all graphs, data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical analysis was performed with a paired t test.

Article Snippet: The shRNA sequences for MTHFD2 and SIRT5 were cloned into pLKO.1 TRC (Addgene plasmid 10879) and verified by DNA sequencing.

Techniques: Immunofluorescence, Expressing, Mutagenesis, Transfection, Knockdown, Plasmid Preparation, Control, RNA Sequencing, Software, Staining, Generated, Injection

Journal: Communications Biology

Article Title: SIRT5-mediated desuccinylation of MTHFD2 enhances chemoresistance in breast cancer cells by reducing therapy-induced senescence

doi: 10.1038/s42003-025-08878-z

Figure Lengend Snippet: A MDA-MB-231 cells were transfected with control or MTHFD2 shRNA#1, and treated with 5 μM Olaparib for 7 days, and cellular senescence was indicated by SA-β-gal staining. B MDA-MB-231 cells were transfected with control or MTHFD2 shRNA#1, and treated with 5 μM Olaparib for 7 days. The p16 INK4a expression was detected by immunofluorescence. C MDA-MB-231 cells were transfected with control or MTHFD2 shRNA#1 in the presence of 3xFlag-MTHFD2-WT, 3xFlag-MTHFD2-K44E, and 3xFlag- Gln132Lys/Asp155Ala-MTHFD2, and treated with 5 μM Olaparib. The cellular senescence was indicated by SA-β-gal staining. D MDA-MB-231 cells were transfected with control or SIRT5 shRNA#2, and treated with 5 μM Olaparib for 7 days, and cellular senescence was indicated by SA-β-gal staining. E MDA-MB-231 cells were transfected with control or SIRT5 shRNA#2, and treated with 5 μM Olaparib for 7 days. The cell proliferation was assayed by crystal violet staining. F Effect of ectopic expression of WT MTHFD2, MTHFD2 K44E, and Gln132Lys/Asp155Ala-MTHFD2 mutant on TIS. MDA-MB-231 cells were transfected with shSIRT5#2 or shControl to knock out the endogenous SIRT5 and express FLAG-MTHFD2, FLAG-MTHFD2 K44E and Gln132Lys/Asp155Ala-MTHFD2, and then treated with 5 μM Olaparib for 7days. The cellular senescence was indicated by SA-β-gal staining. G MDA-MB-231 cells were transfected with control or SIRT5 shRNA#2 and overexpression WT MTHFD2, MTHFD2 K44E and Gln132Lys/Asp155Ala-MTHFD2 mutant, after a 6-day treatment with 5 μM Olaparib, cells were exposed to 2.5 μM Navitoclax combined with 5 μM Olaparib for 2 days. The cell proliferation was assayed by crystal violet staining. H BALB/c nude mice were orthotopically injected with MDA-MB-231 breast cancer cells and treated daily with either vehicle or Olaparib and Navitoclax. 2 × 10 6 MDA-MB-231 cells were subcutaneously implanted into the subcutaneous. When the tumor volume reached 60 mm 3 , Olaparib (5mg/kg) treatment started and was maintained daily (i.p.) for 20 days. Navitoclax (25 mg/kg) treatments started 7 days after Olaparib treatment, and Navitoclax via oral gavage (o.g.) for 2 weeks. I Average images (up) and weights (down) of xenograft tumors (4 weeks, means ± S.D., n = 5) generated by SIRT5-silenced MDA-MB-231 cells and overexpressing 3xFlag-MTHFD2-WT or 3xFlag-MTHFD2-K44E as indicated. For all graphs, data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical analysis was performed with a paired t test.

Article Snippet: The shRNA sequences for MTHFD2 and SIRT5 were cloned into pLKO.1 TRC (Addgene plasmid 10879) and verified by DNA sequencing.

Techniques: Transfection, Control, shRNA, Staining, Expressing, Immunofluorescence, Mutagenesis, Knock-Out, Over Expression, Injection, Generated