Journal: bioRxiv

Article Title: The cyclic nitroxide antioxidant 4-methoxy-TEMPO decreases mycobacterial burden in vivo through host and bacterial targets

doi: 10.1101/464586

Figure Lengend Snippet: (A) CFU recovery from axenic early log phase cultures of M. marinum treated with MetT for 2 days. (B) Quantification of constitutive mCherry fluorescence in late log phase cultures of M. marinum Δ esx1 strain carrying pMV762-Peredox-mCherry treated with MetT. (C) Quantification of peredox fluorescence in late log phase cultures of M. marinum Δ esx1 strain carrying pMV762-Peredox-mCherry treated with MetT as an indicator of cellular NADH:NAD build up. (D) CFU recovery from early log phase cultures of M. marinum treated with MetT for 2 days (E) Representative images of 5 dpi zebrafish embryos infected with M. marinum -wasabi and exposed to 10% O 2 in a hypoxia chamber. (F) Quantification of bacterial burden by fluorescent pixel count in 4 dpi zebrafish embryos treated MetT and maintained in 10% O 2 as indicated.

Article Snippet: To visualise disruption of cellular respiration in M. marinum , we created a transgenic M. marinum Δesx1 strain carrying pMV762-Peredox-mCherry (Addgene 90217) to visualise build up of NADH:NAD ratio [ , ].

Techniques: Fluorescence, Infection

Journal: Scientific Reports

Article Title: Inhibition of mitochondrial function by metformin increases glucose uptake, glycolysis and GDF-15 release from intestinal cells

doi: 10.1038/s41598-021-81349-7

Figure Lengend Snippet: Metformin increases glycolysis in intestinal cells. ( a ) Changes in extracellular acidification rate (ECAR) during the glycolytic stress test wherein intestinal cells were pre-treated with control or 1 mM metformin for 24 h. Results were normalised to the protein concentration measured by BCA assay. Dotted lines indicate when compound were added: Gluc glucose (10 mM), OliA oligomycin-A (1 μM), 2-DG 2-deoxyglucose (50 mM). Inset: Effect of metformin on measured parameters of the glycolysis stress test. n = 10 wells from 3 independent experiments. NS not significant. ***P < 0.001. Two-way ANOVA and Bonferroni post-hoc test. ( b ) Example traces of Perceval fluorescence ratios from Control and Metformin treated cells in response to 2-DG (50 mM), with violin plots shows of the change in fluorescent intensity ratio (FI). ***P < 0.001, Student’s t test. Control; n = 14 from 4 dishes and metformin; n = 17 from 4 dishes. ( c ) Schematic of glycolysis and effects of inhibitors. ( d ) Effects of AR-C155858 (AR-C, 10 μM) and Syrosingopine (Syro, 50 μM) on Perceval fluorescence in Control (red) and Metformin (blue) treated cells. 10 mM glucose (Gluc) was present throughout, and 2-DG (50 mM) added as indicated. Error bars are mean ± SEM. **P < 0.01, ***P < 0.001, two-way ANOVA and Bonferroni post-hoc test. Control: n = 22 cells from 6 dishes; metformin: n = 16 cells from 5 dishes. Error bars are mean ± SEM. **P < 0.01, ***P < 0.001, two-way ANOVA and Bonferroni post-hoc test. ( e ) As ( d ), for oxamate (50 mM). Control: n = 23 cells from 6 dishes; metformin: n = 25 cells from 6 dishes. ( f ) Supernatant lactate levels in control (red) and metformin (blue) pre-treated cultures in glucose (10 mM, 10G), with oxamate (50 mM) or AR-C, (10 μM) and Syro (50 μM). Error bars are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, vs control. ††† P < 0.001 vs pre-treated conditions in 10G. Two-way ANOVA and Bonferroni post-hoc test. n = 2–3 wells from 4 independent experiments except AR–C/Syro (3 independent experiments). ( g ) Effects of oxamate on Peredox fluorescence in control (red) and metformin (blue) treated cells. Glucose (Gluc, 10 mM) and oxamate (50 mM) were applied as indicated. Error bars are median ± 95% confidence intervals. *P < 0.05, Mann–Whitney test. Control: n = 15 cells from 4 dishes; Metformin: n = 18 cells from 4 dishes.

Article Snippet: The T-Sapphire and mCitrine fluorescence of the Peredox sensor (32386, Addgene) was sequentially excited at 405 ± 20 nm and 480 ± 10 nm respectively, and images were acquired every 10 s. T-Sapphire fluorescence (emitted from excitation at 405 ± 20 nm) was increased correlating with cytosolic NADH concentrations, whilst the mCitrine fluorescence remained constant throughout the experiment.

Techniques: Control, Protein Concentration, BIA-KA, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Characterisation and use of a functional Gadd45g bacterial artificial chromosome

doi: 10.1038/s41598-018-35458-5

Figure Lengend Snippet: ( A ) Position of the mCherry reporter in the Gadd45g open reading frame of E15 BAC; ( B ) Detection of Cherry fluorescence in 11.5 dpc embryonic gonad. Green signal indicates location of PECAM. Blue is DAPI staining. ( C ) Cherry signal is detected in somatic cells of the gonad, which lack PECAM staining shown in ( D ). A few somatic cells (lacking PECAM) also lack Cherry signal (white arrows). ( E ) Lateral view of 10.5 dpc embryo showing Cherry fluorescence in developing neural tissue (forebrain (fb), midbrain (mb), hindbrain (hb)). ( F ) Dorsal view of same embryo reveals signal in neural tube (nt), trigeminal ganglion (tg) and facial ganglion (fg). ( G ) Section of embryo (in plane indicated by dotted line in ( E )) shows neural tube and dorsal root gangion (drg) fluorescence. Scale bar = 50 μm.

Article Snippet: A construct containing mCherry, nuclear localization signal and SV40pA terminator derived from Addgene plasmid GW1-Peredox-mCherry-NLS (#32381, a kind gift from Gary Yellen) was used to replace the rspL-neo cassette in a second round of transformation, restoring streptomycin resistance.

Techniques: Fluorescence, Staining