Journal: Nature Communications

Article Title: Lipid droplet degradation by autophagy connects mitochondria metabolism to Prox1-driven expression of lymphatic genes and lymphangiogenesis

doi: 10.1038/s41467-022-30490-6

Figure Lengend Snippet: a – c RT-qPCR analysis of si CTRL and si ATG5 LEC treated with sodium acetate (AC, 20 mM, 48 h) or vehicle. mRNA expression of Prox1, VEGFR3, and LYVE1 (relative to HPRT). Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, ** p < 0.01 and *** p < 0.001 vs si CTRL, ## p < 0.01 and ### p < 0.001 vs si ATG5. d Representative blots for the indicated proteins in si CTRL or si ATG5 LEC treated with AC or vehicle. Densitometric quantification is indicated beneath the blots. Mean ± SD, N ≥ 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, * p < 0.05,*** p < 0.001. e Representative blots for indicated proteins in si CTRL and si ATG5 LEC treated with palmitate (PAL, 500 nM, 48 h) or BSA. Densitometric quantification is indicated beneath the blots. Mean ± SD, N ≥ 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. f Representative immunofluorescent images of si CTRL and si ATG5 treated with AC or vehicle and stained for mitochondrial protein TOMM20 and BODIPY 493/503. Nuclei are stained with DAPI. Scale bars represent 10 µm. g Quantification of mitochondrial fragmentation index (number of mitochondria/ total mitochondrial area). Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, * p < 0.05 vs si CTRL, # p < 0.05 vs si ATG5. Mean represents mean per independent experiment, with a minimum of 12 cells analyzed per condition per independent experiment. h Quantification of lipid droplet number per cell. Mean ± SD, N = 4 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, * p < 0.05 vs si CTRL. Mean represents mean per independent experiment, with a minimum of 29 cells analyzed per condition per independent experiment. i Representative super resolution AiryScan images of si CTRL and si ATG5, si CTRL + si DRP1, and si ATG5 + si DRP1 LEC stained for the mitochondrial protein TOMM20 and BODIPY 493/503. Nuclei are stained with DAPI. Scale bars represent 10 µm. j Quantification of mitochondrial index of fragmentation (number of mitochondria/ total mitochondrial area). Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, **** p < 0.0001 vs si CTRL, #### p < 0.0001 vs si ATG5. Mean represents mean per independent experiment, with a minimum of 35 cells analyzed per condition per independent experiment. k Quantification of lipid droplet number per cell. Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, ** p < 0.01 vs si CTRL. Mean represents mean per independent experiment, with a minimum of 47 cells analyzed per condition per independent experiment. l – o RT-qPCR analysis in si CTRL, si ATG5, si CTRL + si DRP1 and si ATG5 + si DRP1 LEC. mRNA expression of PROX1, VEGFR3, LYVE1, and CPT1A (relative to HPRT). Mean ± SD, N = 3 ( l , n , o ) and N = 4 ( m ) biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, ** p < 0.01 vs si CTRL and p = ns vs si ATG5.

Article Snippet: Primary antibodies used were rabbit anti-ATG5 (12994S, CST), rabbit anti-ATG7 (1:1000, 8558S, CST), rabbit anti-LC3 (1:1000, 3868S, CST), rabbit anti-GAPDH (1:5000, 2118S, CST), rabbit anti-p62 (1:1000, p0067, Millipore), rabbit anti-β-actin (1:5000, A5441, Sigma-Aldrich), goat anti-LYVE1 (1:1000, AF2089, R&D systems), rabbit anti-PROX1 (1:1000, 11067-2, Proteintech), rabbit anti-VEGFR3 (1:1000, ab154079, Abcam), mouse anti-NR2F2 (1:1000, ab41859, Abcam), rabbit anti-CPT1 (1:1000, D3B3, CST) antibody, rabbit anti-CPT2 (1:1000, ab18114, Abcam), rabbit anti-acetyl histone H3 (lysine 9) antibody (1:1000, 9671, CST), rabbit anti-pan-acetyl histone H3 antibody (1:1000, 39139, Active Motif)), mouse anti-CD36 antibody (1:1000; ab17044, Abcam), mouse anti-ULK1 antibody (1:1000; ab56344, Abcam), rabbit anti-TOMM20 (1:1000; BD612278, BD Biosciences), mouse anti-OPA1 (1:1000; 612607, BD Biosciences), mouse anti-ENOS (1:1000; 610297, BD biosciences), rabbit anti-phospho DRP1 (Ser616) (1: 1000; 3455S, CST), mouse anti-DRP1 (1:1000; 611113, BD Biosciences), rabbit anti-H3K4me antibody (1:1000; ab8580, Abcam), rabbit anti-H3K27 ac (1:1000; 8173T, CST) and rabbit anti-FABP4 antibody (1:1000; 2120S, CST).

Techniques: Quantitative RT-PCR, Expressing, Staining

Journal: bioRxiv

Article Title: Mitochondrial Raf1 Regulates Glutamine Catabolism

doi: 10.1101/2024.03.08.581297

Figure Lengend Snippet: (A) Schematic of Raf1 BioID and localization workflow. ( B ) SAINT plots demonstrating all hits with a positive SAINT score for proteins enriched in the proximal proteome of Raf1 in dependent MM485 or AsPC1 cells compared to Raf1-independent CHL1 and BxPC3 and eGFP controls. Red are select known Raf1 interactors and cyan indicates proteins of interest involved in metabolic processes. Dashed line drawn at SAINT score of 0.8 ( C ) Combined Cell Component, Biological Process, and Molecular Function Gene Ontology Enrichment in SAINT ≥ 0.8 proteins. Benjamini-Hochberg adjusted pValues are used. ( D ) Network of Raf1 proximal proteins with SAINT ≥ 0.9 in both pancreatic and melanoma cell lines of interest. Edges between non-Raf1 proteins represent known interactions. Proteins with known mitochondrial localization labeled with blue. ( E ) Mito-tag Mitochondrial isolation of MM485 melanoma cells. WCL denotes whole cell lysate, Mito indicates mitochondrial fraction, and PK denotes proteinase K treatment to digest outer mitochondrial membrane (OMM) proteins. Lamin A/C is a nuclear (nuc) protein, p70S6 kinase is a cytoplasmic (cyto) protein, and TOMM20 spans the mitochondrial outer mitochondrial membrane (OMM) ( F ) STED microscopy demonstrating inner-mitochondrial Raf1 (yellow arrows). Image stack side-view (XZ and YZ) are cropped laterally and then mean-projected along X or Y. Inset displays a Y-projection of a particular mitochondria of interest in XZ.

Article Snippet: Two-color confocal and 3D STED imaging of primary Mouse Raf1 (12552, Cell Signaling Technology) with Abberior STAR Red antiMouse and rabbit TOMM20 (ab78547) with antiAbberior STAR Orange was performed on an Abberior Facility Line STED microscope.

Techniques: Labeling, Isolation, Membrane, Microscopy

Journal: Acta physiologica (Oxford, England)

Article Title: Non-transgenic guinea pig strains exhibit divergent age-related changes in hippocampal mitochondrial respiration.

doi: 10.1111/apha.14185

Figure Lengend Snippet: FIGURE 5 Hartley but not PET guinea pigs have higher hippocampal mitochondrial protein content with age. An OXPHOS western blot quantified subunits for each of the 5 mitochondrial protein complexes. There was an overall effect of age or trend in complexes I (A), II (B), IV (D), and V (E) in that mitochondrial protein content was higher at 12 months compared to 5 months of age. The planned multiple comparison revealed trends suggesting that this higher mitochondrial content in 12 months compared to 5 months hippocampal samples was only in Hartleys and for complex I (p = 0.0718; A) and complex II (p = 0.0792; B). Representative OXPHOS blot and total protein stain (F). VDAC is the most abundant protein in the outer-mitochondrial membrane, and we observed that there was a significant age by strain interaction and a trend for an effect of age (p = 0.0472) (G). This effect of age was driven by PETs having lower VDAC at 12 months compared to 5 months (p = 0.0464). For TOM20, another mitochondrial protein, there was a significant age by strain interaction (p < 0.0001) (H). Specifically, at 5 months PETs had higher TOM20 than Hartleys (p = 0.0023) whereas at 12 months Hartleys had higher TOM20 than PETs (p = 0.0006). Further PETs had lower TOM20 at 12 months compared to 5 months (p = 0.0004), and Hartleys had higher TOM20 protein content at 12 months compared to 5 months (p = 0.0062), paralleling the OXPHOS results. Representative VDAC and TOM20 blot and total protein stains (I). Five months PET n = 6; 5 months Hartley n = 6; 12 months PET n = 6; 12 months Hartley n = 6 for (A, B, C, D, E, and H). (G) had 5 months PET n = 6; 5 months Hartley n = 5, 12 months PET n = 6; 12 months Hartley n = 6 as one 5 months Hartley sample was an outlier for the TOM20 blot. *p < 0.05, †p < 0.01, ‡p < 0.001.

Article Snippet: Membranes were rinsed and then incubated with appropriate secondary antibodies for (5% BSA in TBST; 1:10000) OXPHOS (goat anti- mouse Santa Cruz 2005, Lot # D1614), (5% nonfat milk in TBST; 1:10000) VDAC and TOM20 (goat anti- rabbit ProteinTech SA00001- 2) After the membranes were rinsed, Western Lightening ECL Enhanced (Perkin Elm NEL104001EA) was applied and the membranes were subsequently imaged using a FluorChem E Chemiluminescence Imager (Protein Simple, San Diego, CA, USA).

Techniques: Western Blot, Comparison, Staining, Membrane