Journal: The Journal of Neuroscience

Article Title: Nitric Oxide Mediates Selective Degeneration of Hypothalamic Orexin Neurons through Dysfunction of Protein Disulfide Isomerase

doi: 10.1523/JNEUROSCI.0595-13.2013

Figure Lengend Snippet: nNOS is involved in SD-induced degeneration of orexin neurons. A, Representative photomicrograph showing immunofluorescence of orexin-A (green) and nNOS (red). B, Representative photomicrographs of immunofluorescence of c-Fos (green) and nNOS (red) in LHA of mice with or without 12 h SD. Arrowheads indicate double-positive cells. C, Representative photomicrographs showing NADPH-d staining in the hypothalamus of mice with or without 12 h SD. D, Left panels, Representative photomicrographs of orexin-IR neurons in the hypothalamus of WT mice or nNOS knock-out mice with or without 12 h SD for 7 d. The effect of 12 h SD for 7 d on the number of orexin-IR neurons in WT and nNOS knock-out mice is shown in the right graph (n = 5 or 6). **p < 0.01 (main effect of SD: F(1,18) = 5.361, p = 0.0326; main effect of nNOS deletion: F(1,18) = 0.604, p = 0.447; interaction between SD and nNOS deletion: F(1,18) = 7.995, p = 0.0112). n.s., Not significant. E, Effect of 12 h SD for 7 d on the percentage of aggregate-containing cells in orexin-IR cells in WT mice and nNOS knock-out mice (n = 5 or 6). ***p < 0.001 (main effect of SD: F(1,18) = 12.86, p = 0.0021; main effect of nNOS deletion: F(1,18) = 33.16, p < 0.0001; interaction between SD and nNOS deletion: F(1,18) = 20.07, p = 0.0003). n.s., Not significant. F, Representative immunoblot of biotin switch assay showing the level of SNO-PDI in the hypothalamus of WT mice and nNOS knock-out mice with or without 12 h SD.

Article Snippet: The blots were washed with Tris-buffered saline containing 0.1% Tween 20 and blocked with Blocking One (Nacalai) at 22–25°C for 1 h. The membrane was incubated with mouse anti-KDEL antibody [for detection of Ig binding protein (BiP), 1:2000; SPA-827, Stressgen, Enzo Life Sciences], rabbit anti-PDI antibody (1:2000; ADI-SPA-890, Enzo Life Sciences), rabbit anti-nNOS antibody (1:1000; Cell Signaling Technology), mouse anti-iNOS antibody (1:1000; 610328, BD Biosciences), and mouse anti-β-actin antibody (1:1000; Sigma-Aldrich) overnight at 4°C.

Techniques: Immunofluorescence, Staining, Knock-Out, Western Blot, Biotin Switch Assay

Journal: The Journal of Neuroscience

Article Title: Nitric Oxide Mediates Selective Degeneration of Hypothalamic Orexin Neurons through Dysfunction of Protein Disulfide Isomerase

doi: 10.1523/JNEUROSCI.0595-13.2013

Figure Lengend Snippet: Proposed mechanisms of NO-mediated selective degeneration of orexin neurons. SD causes NO production by activation of nNOS. NO production from nearby nNOS-expressing neurons causes S-nitrosation and inactivation of PDI in orexin neurons. PDI dysfunction promotes formation of orexin-A-IR aggregates that may promote cell death via increased ER stress.

Article Snippet: The blots were washed with Tris-buffered saline containing 0.1% Tween 20 and blocked with Blocking One (Nacalai) at 22–25°C for 1 h. The membrane was incubated with mouse anti-KDEL antibody [for detection of Ig binding protein (BiP), 1:2000; SPA-827, Stressgen, Enzo Life Sciences], rabbit anti-PDI antibody (1:2000; ADI-SPA-890, Enzo Life Sciences), rabbit anti-nNOS antibody (1:1000; Cell Signaling Technology), mouse anti-iNOS antibody (1:1000; 610328, BD Biosciences), and mouse anti-β-actin antibody (1:1000; Sigma-Aldrich) overnight at 4°C.

Techniques: Activation Assay, Expressing

Journal: Nature Communications

Article Title: Hydrogen peroxide positively regulates brassinosteroid signaling through oxidation of the BRASSINAZOLE-RESISTANT1 transcription factor

doi: 10.1038/s41467-018-03463-x

Figure Lengend Snippet: H 2 O 2 induced the oxidation of BZR1 in vitro and in vivo. a , b The flowcharts show the procedures for quantifying redox modification of target proteins by BIAM-labeling assay ( a ) and biotin-switch assay ( b ). c Analysis of the oxidative modification of BZR1 by BIAM-labeling assay. MBP and MBP-BZR1 protein were pretreated with different concentrations of H 2 O 2 , and then directly labeled by BIAM at room temperature for 30 min. The labeled proteins were subjected to separation by SDS-PAGE, and detected by gel blot analysis with HRP-conjugated streptavidin and anti-MBP antibodies. d Analysis of the oxidative modification of BZR1 by the biotin switch assay. MBP-BZR1 proteins pretreated with different concentrations of H 2 O 2 were first incubated with NEM to irreversibly block the free thiols, then treated with DTT to reduce the pre-existing oxidized cysteines in MBP-BZR1. The newly exposed free thiol groups were then relabeled with BIAM. The BIAM-tagged proteins in the samples were then captured with streptavidin beads and detected by gel blot using anti-MBP antibody. e Mass spectrometry analysis of the tryptic fragments of of MBP-BZR1 protein treated as in d . Cys-63 charged with BIAM was identified as an H 2 O 2 -sensitive residue. f , g Effects of mutating various cysteine residues on the H 2 O 2 -induced cysteine oxidation in MBP-BZR1, analyzed as in d . h , i BL and H 2 O 2 treatment induced ( h ), but overexpression of CAT2 ( i ) decreased the oxidative modification of BZR1 proteins in plants. Total proteins from p35S:BZR1-YFP transgenic plants treated with or without 100 nM BL and 1 mM H 2 O 2 for 3 h, or from the p35S:BZR1-YFP/p35S:CAT2 transgenic plants were sequentially treated with NEM, DTT, and BIAM, and then analyzed for biotin label

Article Snippet: Proteins labeled with BIAM were detected with HRP-conjugated streptavidin (Cell Signaling, Cat: 7075S, 1:5000 dilution).

Techniques: In Vitro, In Vivo, Modification, Labeling, Biotin Switch Assay, SDS Page, Western Blot, Incubation, Blocking Assay, Mass Spectrometry, Residue, Over Expression, Transgenic Assay

Journal: Cell

Article Title: Extracellular matrix remodeling regulates glucose metabolism through TXNIP destabilization

doi: 10.1016/j.cell.2018.08.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ZFP36/tristetraprolin ( for immunoblots ) , Cell Signaling , Cat# 71632.

Techniques: Western Blot, Immunohistochemistry, Recombinant, Binding Assay, Transfection, Clinical Proteomics, Membrane, Protein Extraction, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Luciferase, cDNA Synthesis, Colorimetric Assay, Activity Assay, Microarray, Generated, Control, shRNA, CRISPR, Software, In Vivo Imaging, Microscopy

Journal: Circulation: Arrhythmia and Electrophysiology

Article Title: Caveolin-1 Modulates Cardiac Gap Junction Homeostasis and Arrhythmogenecity by Regulating cSrc Tyrosine Kinase

doi: 10.1161/circep.113.001394

Figure Lengend Snippet: Figure 4. Renin–angiotensin system activation induces caveolin-1 (Cav1) S-nitrosation, resulting in Cav1–cSrc dissociation. A, Cav1 S-nitrosation (SNO) was assessed using biotin-switch assay in the cardiomyocytes isolated from wild-type (WT; n=4) and angioten- sin-converting enzyme (ACE8/8; n=4) left ventricle, which showed the level of Cav1 SNO was significantly (P=0.03) higher in ACE8/8 than in WT LV myocytes. B, Coimmunoprecipitation experiments revealed that the interaction between cSrc and Cav1 was reduced in ACE8/8 (n=4) compared with WT (n=4), LV myocytes. C, Human embryonic kidney (HEK) cells cotransfected with mouse cSrc and Cav1 cDNA were subjected to NO donor (S-nitroso-N- acetyl-dl-penicillamine [SNAP], 20 μmol/L; 10 minutes) treatment, where Cav1 SNO was increased, resulting in reduced interaction between cSrc and Cav1 (P=0.03; n=4 in each group). IB indicates immunoblot; and IP, immunoprecipitation.

Article Snippet: For Western blots, total protein lysates were prepared from the LV of 6 week-old WT control, ACE8/8 with and without 2 week treatment of mitochondria-targeted antioxidant (2- (2,2,6,6-Tetra-methylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)-triphenylphosphonium chloride (MitoTEMPO, see below), as well as from adult (2-4 month) Cav1-/- mice with and without 4 weeks treatment of cSrc inhibitor 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine (PP1, see below); in some cases, protein lysates were prepared from the LV cardiomyocytes isolated from ACE8/8 animals using described methods.1 Total protein lysates were fractionated on 8-15% SDS-PAGE and transferred to PVDF membranes, incubated in 5% skim milk in PBS containing 0.1% Tween 20 (blocking buffer) for 1 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies (rabbit monoclonal anti-cSrc, p-cSrc at Tyr416, Cx43, C-terminal Src kinase [CSK] and Tyr14 p-Cav1 antibodies from Cell Signaling, mouse monoclonal anti-Cav1 and Cav3 antibodies from BD Biosciences, rabbit monoclonal anti-eNOS, p-eNOS and nNOS antibodies from Santa Cruz).

Techniques: Activation Assay, Biotin Switch Assay, Isolation, Western Blot, Immunoprecipitation

Journal: Circulation: Arrhythmia and Electrophysiology

Article Title: Caveolin-1 Modulates Cardiac Gap Junction Homeostasis and Arrhythmogenecity by Regulating cSrc Tyrosine Kinase

doi: 10.1161/circep.113.001394

Figure Lengend Snippet: Figure 7. Schematics illustrating molecu- lar mechanisms linking renin–angiotensin system (RAS) activation to gap junction remodeling and ventricular arrhythmias. On RAS activation, angiotensin II (AngII) binds to the AT1 receptor, which elevates the level of mitochondrial reactive oxi- dative species (mitoROS). Increased mitoROS triggers the redistribution of endothelial NO synthase (eNOS) and increases the binding between eNOS and caveolin-1 (Cav1), resulting in increased Cav1 S-nitrosation (SNO) at C156. Increased Cav1 SNO reduces the interac- tion between Cav1 and cSrc, resulting in Cav1–cSrc dissociation and subsequent phosphorylation/activation of cSrc. Phosphorylated cSrc then competes with and displaces connexin 43 (Cx43) from ZO-1 at the intercalated disc, leading to degradation of Cx43, conduction block, and increased propensity of ventricular arrhythmias. VF indicates ventricular fibril- lation; and VT, ventricular tachycardia.

Article Snippet: For Western blots, total protein lysates were prepared from the LV of 6 week-old WT control, ACE8/8 with and without 2 week treatment of mitochondria-targeted antioxidant (2- (2,2,6,6-Tetra-methylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)-triphenylphosphonium chloride (MitoTEMPO, see below), as well as from adult (2-4 month) Cav1-/- mice with and without 4 weeks treatment of cSrc inhibitor 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine (PP1, see below); in some cases, protein lysates were prepared from the LV cardiomyocytes isolated from ACE8/8 animals using described methods.1 Total protein lysates were fractionated on 8-15% SDS-PAGE and transferred to PVDF membranes, incubated in 5% skim milk in PBS containing 0.1% Tween 20 (blocking buffer) for 1 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies (rabbit monoclonal anti-cSrc, p-cSrc at Tyr416, Cx43, C-terminal Src kinase [CSK] and Tyr14 p-Cav1 antibodies from Cell Signaling, mouse monoclonal anti-Cav1 and Cav3 antibodies from BD Biosciences, rabbit monoclonal anti-eNOS, p-eNOS and nNOS antibodies from Santa Cruz).

Techniques: Activation Assay, Binding Assay, Phospho-proteomics, Blocking Assay

Journal: Nature Communications

Article Title: Hydrogen sulfide coordinates glucose metabolism switch through destabilizing tetrameric pyruvate kinase M2

doi: 10.1038/s41467-024-51875-9

Figure Lengend Snippet: a Upper: MDA-MB-231 cell lysates were treated with 100 μM NaHS for 30 min at 37 °C and subjected with or without 1 mM DTT for 10 min. The biotin switch assay was then applied to precipitate sulfhydrated proteins. The biotin-labeled protein was analyzed by immunoblotting with anti-PKM2 antibody to detect sulfhydration of PKM2. Bottom: The SSH-labeled PKM2 was normalized with the level of total PKM2. Data are presented as the means ± SEM ( n = 3 biological replicates). One-way ANOVA followed by Tukey’s multiple comparisons test was used for the statistical analysis (*** p < 0.001; **** p < 0.0001). Immunoblotting experiments were repeated at least 3 times with similar results. b The PK activity on recombinant PKM2 in the presence of 100 μM NaHS for 30 min on ice and subject to 4 mM DTT for another 10 min on ice. Pyruvate kinase activities were then assayed by measuring the amount of pyruvate production. Data are presented as the means ± SD ( n = 3 technical replicates). The results are consistent across two biological replicates. c Upper: Glycerol gradient ultracentrifugation profiles of purified recombinant PKM2 and the effects of FBP and H 2 S on PKM2 oligomerization. Recombinant PKM2 (10 μg) was exposed to 100 μM FBP, 100 μM NaHS, or both. After centrifugation, fractions were collected and analyzed by immunoblotting with PKM2 antibody. The distributions of PKM2 tetramer and monomer/dimer were as indicated. Bottom: Quantitative analysis of PKM2 protein level. The histogram represents normalized means ± SEM ( n = 3 biological replicates in FBP+NaHS group; n = 4 biological replicates in NaHS group; n = 5 biological replicates in control and FBP group). The two-tailed student’s t test was used for the statistical analysis (* p < 0.05, group of FBP compared to the other three groups). Immunoblotting experiments were repeated at least 3 times with similar results. d Left: MDA-MB-231 cells were exposed to 1 μM NaHS for 1 h. Subcellular localization of PKM2 was detected by immunocytochemistry. Nuclei were counterstained with DAPI. The representative images are shown. Scale bars: 10 μm. Right: The percentage of nuclear PKM2 is shown in a violin plot with individual points. Horizontal black dotted lines display the median and the percentage of cells with >20% nuclear localization of PKM2 were indicated ( n = 65–70 individual cells, data were combined from three independent experiments). The two-tailed student’s t test was used for the statistical analysis (**** p < 0.0001). e MDA-MB-231 cells were treated with 1 μM NaHS for 24 h. The relative expression of genes regulated by PKM2 was measured by qRT-PCR. Data are presented as the means ± SD ( n = 3 biological replicates in CCND1 group and n = 5 biological replicates in all the other groups). The two-tailed student’s t test was used for the statistical analysis (* p < 0.05; ** p < 0.01; *** p < 0.001). f Cell proliferation assays were performed in MDA-MB-231 cells with CBS and CTH knockdown by siRNA. Data are presented as the means ± SD ( n = 3 biological replicates). Two-way ANOVA followed by Tukey’s multiple comparisons test was used for the statistical analysis (* p < 0.05, ** p < 0.01, siCBS&CTH-1 or siCBS&CTH-2 compared to the siCon). g Western blot analysis of CBS, CTH, 3MST, and GAPDH expression in MCF-10A, MDA-MB-231(MB-231), MCF-7, and HCC-1395 cells. The samples derive from the same experiment but different gels for CTH and 3MST, and another for CBS and GAPDH were processed in parallel. GAPDH is the internal control. Immunoblotting experiments were repeated at least 3 times with similar results. h Upper: MCF-10A, MDA-MB-231, MCF-7, and HCC-1395 cells were lysed and subjected to the biotin switch assay to precipitate sulfhydrated proteins. The biotin-labeled protein was analyzed by immunoblotting with anti-PKM2 antibody to detect sulfhydration of PKM2. Bottom: The SSH-labeled PKM2 was normalized with the level of total PKM2. Data are presented as the means ± SEM ( n = 4 biological replicates in the group of MCF-7 and HCC-1395; n = 6 biological replicates in the group of MCF-10A and MB-231). The One-way ANOVA followed by Tukey’s multiple comparisons test was used for the statistical analysis (* p < 0.05). Immunoblotting experiments were repeated at least 3 times with similar results. i Upper: MDA-MB-231 cells were under normal (20% O 2 ) or hypoxia incubator (1% O 2 ) for 48 h with or without 0.25 mM AOAA. Cells were then lysed and subjected to the biotin switch assay to precipitate sulfhydrated proteins. The biotin-labeled protein was analyzed by immunoblotting with anti-PKM2 antibody to detect sulfhydration of PKM2. Bottom: The SSH-labeled PKM2 was normalized with the level of total PKM2. Data are presented as the means ± SEM ( n = 4 biological replicates). One-way ANOVA followed by Tukey’s multiple comparisons test was used for the statistical analysis (** p < 0.01). Immunoblotting experiments were repeated at least 3 times with similar results. Source data are provided as a Source Data file.

Article Snippet: PKM2 (D78A4) XP® Rabbit mAb (#4053) (1:1000 for immunoblotting; 1:100 for immunostaining), HA-Tag (C29F4) Rabbit mAb (#3724) (1:1000), and V5-Tag (D3H8Q) Rabbit mAb (#13202) (1:1000 for immunoblotting; 1:100 for immunostaining) were from Cell Signaling Technology (CST).

Techniques: Biotin Switch Assay, Labeling, Western Blot, Activity Assay, Recombinant, Purification, Centrifugation, Control, Two Tailed Test, Immunocytochemistry, Expressing, Quantitative RT-PCR, Knockdown

Journal: Nature Communications

Article Title: Hydrogen sulfide coordinates glucose metabolism switch through destabilizing tetrameric pyruvate kinase M2

doi: 10.1038/s41467-024-51875-9

Figure Lengend Snippet: a The PKM2 monomer shows the positions of ten cysteines in the folded structure. b Purified recombinant PKM2 was treated with 100 μM NaHS and then subjected to trypsin digestion. LC-MS/MS was performed. A side-by-side comparison of the thiol (-SH) and persulfide (-SSH) detected by MS on the same cysteine residues was shown. Dots with a solid orange color are depicted as positive detection by LC-MS/MS; the rest are depicted in hollow circles. c MDA-MB-231 cells were treated with 10 mM MMTS in the cell culture medium for 20 min to block free thiols. Cells were then lyzed and endogenous PKM2 was immunoprecipitated, treated with IAM to block sulfhydrated cysteine, and then subjected for LC-MS/MS analysis. A side-by-side comparison of the thiol (-SH) and persulfide (-SSH) detected by MS on the same cysteine residues is shown. Dots with a solid orange color are depicted as positive detection by LC-MS/MS; the rest are depicted in hollow circles. d Gel filtration analysis of PKM2 recombinant proteins (wild-type or C326S mutant) in the absence of FBP. The molecular mass markers, tetramer, dimer, and monomer are as indicated. e Upper: Glycerol gradient ultracentrifugation profiles of MDA-MB-231 cells transfected with V5-tagged PKM2 or PKM2 C326S mutant. After centrifugation, fractions were collected and analyzed by immunoblotting. Bottom: Quantitative analysis of PKM2 protein level. The histogram represents normalized means ± SEM ( n = 4 biological replicates). The one-tailed student’s t test was used for the statistical analysis (* p < 0.05; ** p < 0.01). Immunoblotting experiments were repeated at least 3 times with similar results. Source data are provided as a Source Data file.

Article Snippet: PKM2 (D78A4) XP® Rabbit mAb (#4053) (1:1000 for immunoblotting; 1:100 for immunostaining), HA-Tag (C29F4) Rabbit mAb (#3724) (1:1000), and V5-Tag (D3H8Q) Rabbit mAb (#13202) (1:1000 for immunoblotting; 1:100 for immunostaining) were from Cell Signaling Technology (CST).

Techniques: Purification, Recombinant, Liquid Chromatography with Mass Spectroscopy, Comparison, Cell Culture, Blocking Assay, Immunoprecipitation, Filtration, Mutagenesis, Transfection, Centrifugation, Western Blot, One-tailed Test

Journal: Nature Communications

Article Title: Hydrogen sulfide coordinates glucose metabolism switch through destabilizing tetrameric pyruvate kinase M2

doi: 10.1038/s41467-024-51875-9

Figure Lengend Snippet: a Crystal structure of the PKM2 C326S mutant. b Structural overlay of the C326S mutant on the T-state conformation (in complex with Phe, PDB: 4FXJ) . The effector loop and α14-α15 regions are indicated by asterisk and star respectively. c Structural overlay of PKM2 C326S on the R-state conformation (in complex with FBP and Ser, PDB: 4B2D) . PKM2 C326S is in blue, T-state in pink, and R-state in green. d Hydrogen bonding of residues 326 to Q310 in PKM2 C326S and the R-state conformation. e , f Zoom-in view of the effector loop region at the C-C interface. Side chains of W482 and W515 are shown in the stick presentation. Protomers are indicated in parentheses. g , h Zoom-in view of the α14-α15 region at the C-C interface. Hydrogen bonds are shown as dashed lines.

Article Snippet: PKM2 (D78A4) XP® Rabbit mAb (#4053) (1:1000 for immunoblotting; 1:100 for immunostaining), HA-Tag (C29F4) Rabbit mAb (#3724) (1:1000), and V5-Tag (D3H8Q) Rabbit mAb (#13202) (1:1000 for immunoblotting; 1:100 for immunostaining) were from Cell Signaling Technology (CST).

Techniques: Mutagenesis

Journal: Nature Communications

Article Title: Hydrogen sulfide coordinates glucose metabolism switch through destabilizing tetrameric pyruvate kinase M2

doi: 10.1038/s41467-024-51875-9

Figure Lengend Snippet: a Upper Left: Western blot analysis of the expression of V5-tagged PKM2 (vector, wildtype, or C326S mutant) in MDA-MB-231 cells. The samples derive from the same experiment but different gels for V5 and GAPDH, and another for PKM2 were processed in parallel. Upper Right: MDA-MB-231 cells with stable expression were lysed and subjected to the biotin switch assay to precipitate sulfhydrated proteins. The biotin-labeled protein was analyzed by immunoblotting with anti-PKM2 antibody to detect sulfhydration of PKM2. Bottom: The PKM2-V5 was normalized with GAPDH and the SSH-labeled PKM2 was normalized with the level of total PKM2. Data are presented as the means ± SEM ( n = 3 biological replicates). The two-tailed student’s t test (Left) or one-way ANOVA followed by Tukey’s multiple comparisons test (Right) was used for the statistical analysis (* p < 0.05; ** p < 0.01; *** p < 0.001). Immunoblotting experiments were repeated at least 3 times with similar results. b Pyruvate kinase activities were assayed by measuring the amount of pyruvate production (nmol) in MDA-MB-231 cells expressing vector control, PKM2 wt , or PKM2 C326S . Data are presented as means ± SD ( n = 4 biological replicates). One-way ANOVA followed by Tukey’s multiple comparisons test was used for the statistical analysis (* p < 0.05). c The oxygen consumption rate (OCR) curves in MDA-MB-231 expressing vector alone, PKM2 wt , or PKM2 C326S . Cells were treated with oligomycin, FCCP, and rotenone/antimycin A, respectively Data are presented as means ± SD ( n = 3 biological replicates). The one-tailed student’s t test was used for the statistical analysis (* p < 0.05 C326S compared to the vector or PKM2 group). d The level of basal OCR and maximal OCR normalized to the cell numbers in MDA-MB-231 expressing vector alone, PKM2 wt or PKM2 C326S Data are presented as means ± SEM ( n = 3 biological replicates). One-way ANOVA followed by Tukey’s multiple comparisons test was used for the statistical analysis (* p < 0.05; ns: not significant). e The ECAR curves in MDA-MB-231 expressing vector alone, PKM2 wt or PKM2 C326S . Cells were treated with glucose, oligomycin, and 2-DG, respectively. Data are presented as means ± SD ( n = 3 biological replicates). f Glycolysis normalized to the cell numbers in MDA-MB-231 cells expressing vector alone, PKM2 wt or PKM2 C326S . Data are presented as means ± SEM ( n = 3 biological replicates). One-way ANOVA followed by Tukey’s multiple comparisons test was used for the statistical analysis (ns: not significant). g The relative expression of PKM2-responded genes was measured by qRT-PCR from MDA-MB-231 cells with PKM2 wt or PKM2 C326S . Data are presented as normalized means ± SD ( n = 3 biological replicates). The one-tailed Student t test was used for the statistical analysis (* p < 0.05; ** p < 0.01; **** p < 0.0001). h Metabolite analysis was conducted in MDA-MB-231 cells with PKM2 wt or PKM2 C326S expression. The level of glycolysis intermediates, including Glucose, Glucose-6-phosphate, Fructose-1,6-Bisphosphate, Ribose-5-phosphate, Glyceraldehyde-3-phosphate, Phosphoenolpyruvate, Pyruvate, and Lactate was determined by mass spectrometry. Data are presented as means ± SD ( n = 4 in the group of Glucose-6-phosphate; n = 6 in all the other groups; data were combined from two independent experiments). The two-tailed student’s t test was used for the statistical analysis (* p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001). i Left: MDA-MB-231 cells expressing PKM2 wt or PKM2 C326S were exposed to 1 μM NaHS for 1 h. Subcellular localization of PKM2 was detected by immunocytochemistry. Nuclei were counterstained with DAPI. The representative images are shown. Scale bars: 10 μm. Right: The percentage of nuclear PKM2 is shown in a violin plot with individual points. Horizontal black dotted lines display the median and the percentage of cells with >20% nuclear localization of PKM2 were indicated ( n = 41–47 individual cells, data were combined from three independent experiments). One-way ANOVA followed by Tukey’s multiple comparisons test was used for the statistical analysis (**** p < 0.0001). Source data are provided as a Source Data file.

Article Snippet: PKM2 (D78A4) XP® Rabbit mAb (#4053) (1:1000 for immunoblotting; 1:100 for immunostaining), HA-Tag (C29F4) Rabbit mAb (#3724) (1:1000), and V5-Tag (D3H8Q) Rabbit mAb (#13202) (1:1000 for immunoblotting; 1:100 for immunostaining) were from Cell Signaling Technology (CST).

Techniques: Western Blot, Expressing, Plasmid Preparation, Mutagenesis, Biotin Switch Assay, Labeling, Two Tailed Test, Control, One-tailed Test, Quantitative RT-PCR, Mass Spectrometry, Immunocytochemistry

Journal: Nature Communications

Article Title: Hydrogen sulfide coordinates glucose metabolism switch through destabilizing tetrameric pyruvate kinase M2

doi: 10.1038/s41467-024-51875-9

Figure Lengend Snippet: a , b The cell cycle of MDA-MB-231 cells expressing vector only, PKM2 wt , or PKM2 C326S was examined by the PI staining method and measured by flow cytometry. a The representative image is shown. b The percentage of cells at different cell cycle phases was analyzed. Data are presented as means ± SD ( n = 4 biological replicates). One-way ANOVA followed by Tukey’s multiple comparisons test was used for the statistical analysis (* p < 0.05; ** p < 0.01). c , d Immunostaining of actin filaments (Red), α-tubulin (Green), and DNA counterstaining with DAPI (Blue) of MDA-MB-231 cells expressing vector only, PKM2 wt , or PKM2 C326S . c The representative images are shown. Scale Bar: 50 μm. d The percentage of poly-nuclei cells (n ≧ 2) was analyzed. Data are presented as means ± SD ( n = 3 biological replicates). One-way ANOVA followed by Tukey’s multiple comparisons test was used for the statistical analysis (* P < 0.05; ** P < 0.01; *** P < 0.001). e , f Real-time live imaging of MDA-MB-231 cells expressing PKM2 wt or PKM2 C326S expression was performed to monitor cytokinesis failure. e Snapshots of a representative cell expressing PKM2 wt or PKM2 C326S undergoing division are shown. The red arrows point to cells undergoing mitotic division. Time stamps denote h:m of elapsed time. See also Movies and . f Mitotic events with cytokinesis failure were recorded by time-lapse live cell microscopy. Results are expressed as means ± SD ( n = 3 biological replicates). The one-tailed student t test was used for the statistical analysis (* p < 0.05). Source data are provided as a Source Data file.

Article Snippet: PKM2 (D78A4) XP® Rabbit mAb (#4053) (1:1000 for immunoblotting; 1:100 for immunostaining), HA-Tag (C29F4) Rabbit mAb (#3724) (1:1000), and V5-Tag (D3H8Q) Rabbit mAb (#13202) (1:1000 for immunoblotting; 1:100 for immunostaining) were from Cell Signaling Technology (CST).

Techniques: Expressing, Plasmid Preparation, Staining, Flow Cytometry, Immunostaining, Imaging, Microscopy, One-tailed Test

Journal: Nature Communications

Article Title: Hydrogen sulfide coordinates glucose metabolism switch through destabilizing tetrameric pyruvate kinase M2

doi: 10.1038/s41467-024-51875-9

Figure Lengend Snippet: a Comparison of cell proliferation rate between MDA-MB-231 cells expressing vector, PKM2 wt , or PKM2 C326S . Cell proliferation assay was performed and measured by MTS reagents. Data are the means ± SD ( n = 3 biological replicates). Two-way ANOVA followed by Tukey’s multiple comparisons test was used for the statistical analysis (# p < 0.05, C326S compared to the PKM2 or the vector group). b – e MDA-MB-231 cells expressing vector, PKM2 wt or PKM2 C326S were injected into the mouse mammary fat pad. b The tumor volumes were monitored every week. Data are means ± SD ( n = 3 in the group of Vector; n = 9 in the group of PKM2; n = 10 in the group of C326S); data were combined from two independent experiments. Two-way ANOVA followed by Tukey’s multiple comparisons test was used for the statistical analysis (** p < 0.01, *** p < 0.001, C326S compared to the vector group; ## p < 0.01, ### p < 0.001, C326S compared to the PKM2 group). c The mean of mouse weight ± SD ( n = 3 in the group of Ve c tor; n = 9 in the group of PKM2; n = 10 in the group of C326S; data were combined from two independent experiments). d Representative bioluminescence images (BLI) at week 7 after implantation of respective cancer cells are shown. e The kinetics of individual tumors was monitored by IVIS. Data are means ± SD (n = n = 3 in the group of Vector; n = 9 in the group of PKM2; n = 10 in the group of C326S; data were combined from two independent experiments). One-way ANOVA followed by Tukey’s multiple comparisons test was used for the statistical analysis (* p < 0.05; **** p < 0.0001). f A schematic illustration revealing H 2 S modulates glucose metabolism switch through destabilizing PKM2 oligomerization. Left: H 2 S promotes dissociation of PKM2 tetramer to monomer/dimer through protein sulfhydration. Inhibition of PKM2 activity results in the accumulation of metabolic intermediates required for the biosynthesis. Meanwhile, the PKM2 monomers or dimers translocate into the nucleus to facilitate multiple gene expressions to promote cancer progression. Right: Blockade of PKM2 sulfhydration at cysteine 326 by mutation stabilizes PKM2 tetramer to maintain high PK activity, resulting in high energy generation, low biosynthesis, and inhibition of tumor growth. Source data are provided as a Source Data file.

Article Snippet: PKM2 (D78A4) XP® Rabbit mAb (#4053) (1:1000 for immunoblotting; 1:100 for immunostaining), HA-Tag (C29F4) Rabbit mAb (#3724) (1:1000), and V5-Tag (D3H8Q) Rabbit mAb (#13202) (1:1000 for immunoblotting; 1:100 for immunostaining) were from Cell Signaling Technology (CST).

Techniques: Comparison, Expressing, Plasmid Preparation, Proliferation Assay, Injection, Inhibition, Activity Assay, Mutagenesis