Journal: Environmental Microbiology

Article Title: Hydrogen Oxidation Benefits Alphaproteobacterial Methanotrophs Under Severe Methane Limitation

doi: 10.1111/1462-2920.70163

Figure Lengend Snippet: Gene arrangement of all [NiFe] hydrogenases in the genomes of the three alphaproteobacterial methanotrophs M. aurea KYG T , “ M. acidophilus ” 29, and M. bryophila H2s T . (a) Group 1h uptake hydrogenases (Hyd‐1h) were present in all three genomes; (b) group 1d uptake hydrogenases (Hyd‐1d) and group 2b regulatory hydrogenases (Hyd‐2b) were found in one gene cluster in both “ M. acidophilus ” 29 and M. bryophila H2s T . “ M. acidophilus ” 29 and M. bryophila H2s T genomes encoded for (c) group 3b (Hyd‐3b) and (d) group 3d (Hyd‐3d) bidirectional hydrogenases. (e) The group 2a uptake hydrogenase (Hyd‐2a) encoded in the M. aurea KYG T genome. The arrows represent genes, indicate the direction of transcription, and are drawn to scale. Homologues are connected by lines. Double lines indicate breaks in the genomic assembly and dashed lines indicate a non‐scaled genomic region.

Article Snippet: The alphaproteobacterial methanotrophic species Methylocystis bryophila H2s T (DSM 21852), Methylocapsa aurea KYG T (DSM 22158), and “ Methylosinus acidophilus ” strain 29 (DSM 17628) were obtained from DSMZ (Braunschweig, Germany).

Techniques:

Journal: Environmental Microbiology

Article Title: Hydrogen Oxidation Benefits Alphaproteobacterial Methanotrophs Under Severe Methane Limitation

doi: 10.1111/1462-2920.70163

Figure Lengend Snippet: H 2 /D 2 oxidation by Methylocystis bryophila H2s T . (a) Oxidation rates accelerate over repeated H 2 pulses, and (b) further upon the addition of 1 μM CH 4 (orange arrows at 125 and 150 min). The culture in (b) was pre‐incubated with H 2 for 6 h before the addition of D 2 . The data points represent the sum of D 2 and HD, as HD is formed through H + /D + ion exchange between D 2 and H 2 O. Kinetic parameters were determined by fitting simulated Michaelis–Menten kinetics through the data points. Rates are displayed in μmol min −1 g −1 DW. Additional data on the oxidation kinetics of all three alphaproteobacterial methanotrophs are provided in Table .

Article Snippet: The alphaproteobacterial methanotrophic species Methylocystis bryophila H2s T (DSM 21852), Methylocapsa aurea KYG T (DSM 22158), and “ Methylosinus acidophilus ” strain 29 (DSM 17628) were obtained from DSMZ (Braunschweig, Germany).

Techniques: Incubation

Journal: Journal of Lipid Research

Article Title: Impaired ApoB secretion triggers enhanced secretion of ApoE to maintain triglyceride homeostasis in hepatoma cells

doi: 10.1016/j.jlr.2025.100795

Figure Lengend Snippet: TG biosynthesis regulates the secretion of both ApoE and ApoB. A: Immunoblots showing depletion of SOAT1 and SOAT2 in Huh-7.5 cells expressing indicated sgRNAs. B: RT-qPCR determination of DGAT1 and DGAT2 mRNA levels in control and SOAT1 / 2 sgRNA-expressing Huh-7.5 cells transfected with indicated siRNAs. ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). C: BODIPY 493/503 staining of LDs (green) in Huh-7.5 cells transfected with indicated siRNAs and expressing indicated sgRNAs. BODIPY values were normalized to the cell number as measured by DAPI staining (blue). ∗∗∗ P < 0.001 versus control (n = 4, one-way ANOVA with Dunnett’s multiple comparisons test). Scale bar, 100 μm. D: Relative abundances of ApoE and ApoB in supernatants (upper panels) and lysates (lower panels) from Huh-7.5 cells expressing indicated sgRNAs and transfected with indicated siRNAs. Immunoblots are shown below. ∗∗ P < 0.01, ∗∗∗ P < 0.001 (n = 3, two-way ANOVA with Sidak’s multiple comparisons test). E: Relative fluorescence intensity of Huh-7.5 cells expressing indicated sgRNAs stained with BODIPY 493/503. ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). F: Relative abundances of ApoE and ApoB in supernatants (upper panels) and lysates (lower panels) from Huh-7.5 cells expressing indicated sgRNAs. Immunoblots are shown below. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). G: (top) Incorporation of 13 C-OA into TG in Huh-7.5 cells expressing indicated sgRNAs. TG abundance was quantified by GC-MS. (bottom) TG abundance in the same set of cells was determined by a colorimetric assay. ∗ P < 0.05, ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). H: (top panel) Immunoblots showing expression of FLAG-tagged DGAT proteins in Huh-7.5 cells. (bottom panels) RT-qPCR determination of DGAT1 and DGAT2 mRNA levels. ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). I: TG abundance in Huh-7.5 cells expressing DGAT1-FLAG, DGAT2-FLAG or empty vector determined by a colorimetric assay. ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). J: Relative abundances of ApoE and ApoB in supernatants (upper panels) and lysates (lower panels) from Huh-7.5 cells expressing DGAT1-FLAG or DGAT2-FLAG. Immunoblots are shown below. ∗ P < 0.05, ∗∗ P < 0.01 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test).

Article Snippet: Primary antibodies to ApoB (1:200 dilution, sc-13538) and SOAT1 (1:200 dilution, sc-137013, non-reducing condition) were from Santa Cruz Biotechnology; ApoE (1:500 dilution, MCA5639GA) was from Bio-Rad; SOAT2 (1:1,000 dilution, 21852-1-AP, non-reducing condition), Albumin (1:50,000 dilution, 16475-1-AP), and LDLR (1:5,000 dilution, 10785-1-AP) were from Proteintech; Actin (1:10,000 dilution, A2066) and FLAG (1:1,000 dilution, F7425) were from Sigma; GAPDH (Clone 5A12; 1:5,000 dilution, 016–25523) was from Wako; IRDye 680 or 800 secondary antibodies including #926–68072, #926–32213, and #926–32212 (1:20,000 dilution) were from LI-COR.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Control, Transfection, Staining, Fluorescence, Gas Chromatography-Mass Spectrometry, Colorimetric Assay, Plasmid Preparation