Journal: bioRxiv

Article Title: Proteomic Investigation of Neurotrophic trans -Banglene Reveals Potential Link to Iron Homeostasis

doi: 10.1101/2024.09.04.611284

Figure Lengend Snippet: Western blot analysis and quantification. β-actin or β-tubulin was used as a loading reference. Each treatment contains 0.6% DMSO: Control - no additive, NGF (10 ng/mL), t -BG (30 μM), t -BG (30 μM) + NGF (10 ng/mL), (–) t -BG (15 μM). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (A). Protein loading: (a) Ferritin (10 μg) (b) Catalase (10 μg) (c) Ndufs1 (30 μg)

Article Snippet: ×10 TBST (24.23 g Tris base, pH 7.4, 87.66 g NaCl, 10 mL tween 20 in 1 L of water), ×10 transfer buffer without HPLC grade methanol (30.3 tris base, 144 g of glycine in 1 L of water), ×1 transfer buffer with HPLC grade methanol (100 mL of 10 transfer buffer without HPLC grade methanol, 800 mL of distilled water, 200 mL methanol), nonfat dry milk, BSA, PKC Pan Polyclonal Antibody (Invitrogen™ PA5120593), Anti-NF-kB p65 antibody (ab16502), Recombinant Anti-c-fos 1 antibody [EPR20769] (ab214672), Anti-Ferritin Light Chain antibody (ab69090), Ndufs1 Rabbit, Polyclonal, Novus Biologicals™ (NBP15652020), Anti-Catalase antibody - Peroxisome Marker (ab52477), ERK1/ERK2 Rabbit anti-Human, Mouse, Rat, Polyclonal (Invitrogen™ 44-654G), AKT Pan Rabbit anti-Human, Mouse, Rat, Polyclonal, (Invitrogen™ PA5-77855), beta Actin Polyclonal Antibody (Invitrogen™ PA116889), beta Tubulin Polyclonal Antibody, (Invitrogen™ PA516863), Phospho-AKT1 (Thr308) Recombinant Polyclonal Antibody (B18HCLC) (Invitrogen™ 710122), Anti-MEF2C antibody [EPR19089-202] - ChIP Grade (ab211493), Goat Anti-Rabbit IgG (H+L), HRP, Secondary Antibody (Promega PRW4011), and 1-Step™ Ultra TMB-Blotting Solution (Invitrogen™ 37574) were used.

Techniques: Western Blot, Control

Journal: Scientific Reports

Article Title: Comprehensive analysis of disulfidptosis-related genes reveals the effect of disulfidptosis in ulcerative colitis

doi: 10.1038/s41598-024-66533-9

Figure Lengend Snippet: The primer sequence used in this study.

Article Snippet: The primary antibodies and concentrations used in this study were anti-vinculin (1:5000, Sigma-Aldrich), anti-SLC7A11 (1:1000, Abmart), anti-LRPPRC (1:5000, Proteintech), anti-NDUFS1 (1:5000, Proteintech), and anti-CD2AP (1:5000, Sigma-Aldrich).

Techniques: Sequencing

Journal: Scientific Reports

Article Title: Comprehensive analysis of disulfidptosis-related genes reveals the effect of disulfidptosis in ulcerative colitis

doi: 10.1038/s41598-024-66533-9

Figure Lengend Snippet: Potential mechanisms of UC disulfidptosis. In UC intestinal mucosa, the expression of SLC7A11 is elevated and its mediated cystine entry into the cell is significantly increased. At the same time, glucose deficiency caused by increased consumption and impaired absorption and mitochondrial dysfunction triggered by decreased expression of NDUFS1 and LRPPRC can lead to a decrease in NADPH synthesis, which is insufficient to completely reduce the excess cystine, and the large amount of accumulated cystine will trigger disulfide stress, leading to the increase in the formation of disulfide bonds between actin backbone proteins, and ultimately contributing to the intestinal mucosal disulfidptosis. In the figure, red dotted arrow showed the expression level of SLC7A11 was upregulated and cytine was increased in the UC intestinal mucosa. Orange solid arrows indicated that the glucose and NADPH was reduced, which were in relative deficiency in the intestinal mucosa of UC patients. Green solid arrow showed the expression level of NDUFS1 and LRPPRC were downregulated.

Article Snippet: The primary antibodies and concentrations used in this study were anti-vinculin (1:5000, Sigma-Aldrich), anti-SLC7A11 (1:1000, Abmart), anti-LRPPRC (1:5000, Proteintech), anti-NDUFS1 (1:5000, Proteintech), and anti-CD2AP (1:5000, Sigma-Aldrich).

Techniques: Expressing

Journal: Nature Metabolism

Article Title: Weak neuronal glycolysis sustains cognition and organismal fitness

doi: 10.1038/s42255-024-01049-0

Figure Lengend Snippet: a , H 2 O 2 production. Data are mean ± s.e.m. P values are indicated; n = 5 biologically independent cell culture preparations; two-way ANOVA followed by Tukey’s test. NS, not significant. b , Mitochondrial ROS production. Data are mean ± s.e.m. P values are indicated; n = 10 (WT, Pfkfb3 ) or n = 9 ( mCat , Pfkfb3 - mCat ) biologically independent cell culture preparations; one-way ANOVA followed by Tukey’s test. c , In vivo AAV intravenous transduction strategy to express GFP in the neurons of Pfkfb3 lox/+ or CamkIIα-Pfkfb3 mice. HP, hippocampus. Created with https://www.biorender.com (Extended Data Fig. ). d , Mitochondrial ROS production in neurons isolated from mice transduced with AAV expressing GFP under the neuron-specific hSyn promoter. Data are mean ± s.e.m. P values are indicated; n = 5 (WT) or n = 4 ( CamkIIα-Pfkfb3 , CamkIIα-mCat , CamkIIα-Pfkfb3-mCat ) mice; one-way ANOVA followed by Bonferroni correction (Extended Data Fig. ). e , Mitochondrial CI activity. Data are mean ± s.e.m. P values are indicated; n = 6 (WT), n = 8 ( Pfkfb3 ), n = 3 ( mCat ) or n = 9 ( Pfkfb3 - mCat ) biologically independent cell culture preparations; one-way ANOVA followed by Bonferroni correction. f , Mitochondrial complex II–III activity. Data are mean ± s.e.m. n = 6 (WT, Pfkfb3 ), n = 3 ( mCat ) or n = 7 ( Pfkfb3 - mCat ) biologically independent cell culture preparations; one-way ANOVA followed by Bonferroni correction. g , In-gel activity of CI (IGA-CI) and BNGE followed by immunoblotting for CI subunits NDUFS1 and NDUFA9, complex (C)III subunit UQCRC2, complex IV subunit MTCO1, complex V subunit ATPβ or complex II subunit SDHA in primary neurons (Extended Data Fig. ). h , Deactive mitochondrial CI activity. Data are mean ± s.e.m. P values are indicated; n = 6 (WT, Pfkfb3 ) or n = 4 ( mCat , Pfkfb3 - mCat ) biologically independent cell culture preparations; one-way ANOVA followed by Bonferroni correction. i , OCR analysis and calculated parameters. Data are mean ± s.e.m. P values are indicated; n = 4 (WT, Pfkfb3 ) or n = 3 ( mCat , Pfkfb3 - mCat ) biologically independent cell culture preparations; one-way ANOVA followed by Bonferroni correction. Ant, antimycin; olig, oligomycin; rot, rotenone. j , Mitochondrial membrane potential (∆ ψ m ). Data are mean ± s.e.m. P values are indicated; n = 5 (WT), n = 8 ( Pfkfb3 ), n = 3 ( mCat ) or n = 5 ( Pfkfb3 - mCat ) biologically independent cell culture preparations; one-way ANOVA followed by Bonferroni correction (Extended Data Fig. ).

Article Snippet: Next, a direct electrotransfer was performed, followed by immunoblotting for mitochondrial CI with anti-NDUFS1 antibody (1/500) (sc-50132, Santa Cruz Biotechnology), anti-NDUFA9 antibody (1/1,000) (ab14713, Abcam), anti-complex II antibody SDHA (1/1,000) (ab14715, Abcam), anti-complex III antibody UQCRC2 (1/1,000) (ab14745, Abcam), anti-complex IV antibody MTCO4 (1/1,000) (ab14705, Abcam) and anti-complex V antibody ATPβ (1/1,000) (MS503, MitoSciences).

Techniques: Cell Culture, In Vivo, Transduction, Isolation, Expressing, Activity Assay, Western Blot, Membrane

Journal: Nature Metabolism

Article Title: Weak neuronal glycolysis sustains cognition and organismal fitness

doi: 10.1038/s42255-024-01049-0

Figure Lengend Snippet: a , H 2 O 2 production. Data are mean ± s.e.m. P values are indicated; n = 5 biologically independent cell culture preparations; two-way ANOVA followed by Tukey’s test. NS, not significant. b , Mitochondrial ROS production. Data are mean ± s.e.m. P values are indicated; n = 10 (WT, Pfkfb3 ) or n = 9 ( mCat , Pfkfb3 - mCat ) biologically independent cell culture preparations; one-way ANOVA followed by Tukey’s test. c , In vivo AAV intravenous transduction strategy to express GFP in the neurons of Pfkfb3 lox/+ or CamkIIα-Pfkfb3 mice. HP, hippocampus. Created with https://www.biorender.com (Extended Data Fig. ). d , Mitochondrial ROS production in neurons isolated from mice transduced with AAV expressing GFP under the neuron-specific hSyn promoter. Data are mean ± s.e.m. P values are indicated; n = 5 (WT) or n = 4 ( CamkIIα-Pfkfb3 , CamkIIα-mCat , CamkIIα-Pfkfb3-mCat ) mice; one-way ANOVA followed by Bonferroni correction (Extended Data Fig. ). e , Mitochondrial CI activity. Data are mean ± s.e.m. P values are indicated; n = 6 (WT), n = 8 ( Pfkfb3 ), n = 3 ( mCat ) or n = 9 ( Pfkfb3 - mCat ) biologically independent cell culture preparations; one-way ANOVA followed by Bonferroni correction. f , Mitochondrial complex II–III activity. Data are mean ± s.e.m. n = 6 (WT, Pfkfb3 ), n = 3 ( mCat ) or n = 7 ( Pfkfb3 - mCat ) biologically independent cell culture preparations; one-way ANOVA followed by Bonferroni correction. g , In-gel activity of CI (IGA-CI) and BNGE followed by immunoblotting for CI subunits NDUFS1 and NDUFA9, complex (C)III subunit UQCRC2, complex IV subunit MTCO1, complex V subunit ATPβ or complex II subunit SDHA in primary neurons (Extended Data Fig. ). h , Deactive mitochondrial CI activity. Data are mean ± s.e.m. P values are indicated; n = 6 (WT, Pfkfb3 ) or n = 4 ( mCat , Pfkfb3 - mCat ) biologically independent cell culture preparations; one-way ANOVA followed by Bonferroni correction. i , OCR analysis and calculated parameters. Data are mean ± s.e.m. P values are indicated; n = 4 (WT, Pfkfb3 ) or n = 3 ( mCat , Pfkfb3 - mCat ) biologically independent cell culture preparations; one-way ANOVA followed by Bonferroni correction. Ant, antimycin; olig, oligomycin; rot, rotenone. j , Mitochondrial membrane potential (∆ ψ m ). Data are mean ± s.e.m. P values are indicated; n = 5 (WT), n = 8 ( Pfkfb3 ), n = 3 ( mCat ) or n = 5 ( Pfkfb3 - mCat ) biologically independent cell culture preparations; one-way ANOVA followed by Bonferroni correction (Extended Data Fig. ).

Article Snippet: Immunoblotting was performed with anti-LC3B (1/1,000) (2775, Cell Signaling), anti-p62 (1/1,000) (P0067, Sigma), anti-ATG7 (1/1,000) (2631, Cell Signaling), anti-acetylated lysine (1/1,000) (9441, Cell Signaling), anti-cyclin B1 (clone D-11, sc-7393, Santa Cruz Biotechnology, 1/500), anti-ROCK2 (clone D-11, sc-398519, Santa Cruz Biotechnology, 1/500), anti-PLIN2 (ab52356, Abcam, 1/500), anti-beclin (1/1,000) (3495, Cell Signaling), anti-TOMM20 (1/1,000) (ab56783, Abcam) anti-PFKFB3 (1/500) (H00005209-M08, Novus Biologicals), anti-VDAC (1/1,000) (PC548, Calbiochem), anti-heat-shock protein 60 (HSP60) (1/1,000) (ab46798, Abcam), anti-NDUFS1 (1/500) (sc-50132, Santa Cruz Biotechnology), anti-NDUFA9 (1/1,000) (ab14713, Abcam), anti-UQCRC2 (1/1,000) (ab14745, Abcam), anti-SDHA (1/1,000) (ab14715, Abcam), anti-ATPβ (1/1,000) (MS503, MitoSciences), anti-GFAP (1/500) (G6171, Sigma), anti-TUJ1 (1/300) (T2200, Sigma) and anti-β-actin (1/30,000) (A5441, Sigma) antibodies.

Techniques: Cell Culture, In Vivo, Transduction, Isolation, Expressing, Activity Assay, Western Blot, Membrane