Journal: bioRxiv

Article Title: Fine-scale excitatory cortical circuits reflect embryonic progenitor pools

doi: 10.1101/363069

Figure Lengend Snippet: a , Tα1-Cre and CβA-FLEx plasmids were delivered by IUE at different embryonic ages (E12.5 to E15.5). Quantification of GFP + and TdTomato + cells in the VZ, 24 h after each time point for IUE, revealed that a significant proportion of labelled cells were GFP + . This is consistent with the idea that Tα1 + progenitors represent a significant progenitor population in the embryonic mouse VZ. Error bars indicate standard error of the mean. b , Quantifying GFP + cells at different time points (2 d, 3 d, >21 d) following IUE at E14.5, revealed that the proportion of SNP-derived cells remains relatively stable, consistent with the idea that the majority of Cre-mediated recombination occurs within 24 h of IUE. c , Quantification of GFP + and TdTomato + cells in the VZ, 24 h after IUE with different ratios of Tα1-Cre to CβA-FLEx plasmid. Consistent with the idea that labelling accurately reflects the promoter driving Cre expression, the proportion of GFP + and TdTomato + VZ cells was stable across a range of plasmid ratios. A plasmid ratio of 1:1 was used for electrophysiological studies.

Article Snippet: Plasmid DNA included: (i) ‘Tα1-Cre’, in which the gene for Cre recombinase is under the control of a portion of the Tα1 promoter ; (ii) ‘CβA-FLEx’ which uses the chicken β-actin promoter to control a flexible excision (FLEx) cassette in which Cre recombination switches expression from TdTomato fluorescent protein to enhanced green fluorescent protein ; (iii) ‘DIO-ChR2-mCherry’ (pAAV-EF1a-doublefloxed-hChR2(H134R)-mCherry-WPRE-HGHpA; Addgene #20297), in which Cre recombination turns on the expression of channelrhodopsin-2 (ChR2) under the control of the human elongation factor-1a promoter ; (iv) DO-ChR2-mCherry (‘Cre-Off’; pAAV-Ef1a-DO-hChR2(H134R)-mCherry-WPRE-pA; Addgene #37082 in which Cre recombination turns off the expression of ChR2 under the control of the human elongation factor-1a promoter ; and (v) DIO-ChR2-EYFP (pAAV-EF1a-double floxed-hChR2(H134R)-EYFP-WPRE-HGHpA; Addgene #20298), which is equivalent to DIO-ChR2-mCherry, except that EYFP replaces mCherry.

Techniques: Derivative Assay, Plasmid Preparation, Expressing

Journal: Diabetes & Metabolism Journal

Article Title: The Level of Autoantibodies Targeting Eukaryote Translation Elongation Factor 1 α1 and Ubiquitin-Conjugating Enzyme 2L3 in Nondiabetic Young Adults

doi: 10.4093/dmj.2016.40.2.154

Figure Lengend Snippet: Prevalence of EEF1A1-AAb and UBE2L3-AAb by age group

Article Snippet: Immunosorbent 96-well plates coated with EEF1A1 or UBE2L3 (Origene Rockville, MD, USA) were incubated with diluted serum samples (1:1,000 to 1:2,500) in phosphate-buffered saline buffer.

Techniques: Control

Journal: Diabetes & Metabolism Journal

Article Title: The Level of Autoantibodies Targeting Eukaryote Translation Elongation Factor 1 α1 and Ubiquitin-Conjugating Enzyme 2L3 in Nondiabetic Young Adults

doi: 10.4093/dmj.2016.40.2.154

Figure Lengend Snippet: The mean levels of (A) eukaryote translation elongation factor 1 alpha 1 autoantibody (EEF1A1-AAb) and (B) ubiquitin-conjugating enzyme 2L3 autoantibody (UBE2L3-AAb) by age group. The mean values with standard deviation in each age group are shown. There were no differences in the levels of EEF1A1-AAb and UBE2L3-AAb across the age groups in control subjects (black circles). However, the levels of both autoantibodies in subjects that were 18 to 29 years old with type 1 diabetes mellitus (T1DM; black squares) were significantly higher than all of the other age groups ( P <0.05) except for subjects 60 to 69 years old (black circles, nondiabetic control subjects; black squares, subjects with T1DM). a Significantly different compared to nondiabetic control subjects in each age group ( P <0.05), b Significantly different compared to 18- to 29-year-old in each disease status (non-diabetic control group and T1DM, respectively; P <0.05).

Article Snippet: Immunosorbent 96-well plates coated with EEF1A1 or UBE2L3 (Origene Rockville, MD, USA) were incubated with diluted serum samples (1:1,000 to 1:2,500) in phosphate-buffered saline buffer.

Techniques: Ubiquitin Proteomics, Standard Deviation, Control

Journal: Diabetes & Metabolism Journal

Article Title: The Level of Autoantibodies Targeting Eukaryote Translation Elongation Factor 1 α1 and Ubiquitin-Conjugating Enzyme 2L3 in Nondiabetic Young Adults

doi: 10.4093/dmj.2016.40.2.154

Figure Lengend Snippet: The level of (A) eukaryote translation elongation factor 1 alpha 1 autoantibody (EEF1A1-AAb) and (B) ubiquitin-conjugating enzyme 2L3 autoantibody (UBE2L3-AAb) in control and type 1 diabetes mellitus (T1DM) samples. Dotted lines represent the [mean absorbance+3×standard deviation] of the total nondiabetic control subject sample; dashed lines represent the [mean absorbance+3×standard deviation] of control subjects <40 years old; simple solid lines in each age group represent the mean level of EEF1A1-AAb and UBE2L3-AAb within the group (white circles, nondiabetic control subjects; black squares, subjects with T1DM).

Article Snippet: Immunosorbent 96-well plates coated with EEF1A1 or UBE2L3 (Origene Rockville, MD, USA) were incubated with diluted serum samples (1:1,000 to 1:2,500) in phosphate-buffered saline buffer.

Techniques: Ubiquitin Proteomics, Control

Journal: Nature Communications

Article Title: Mitochondria- and ER-associated actin are required for mitochondrial fusion

doi: 10.1038/s41467-024-55758-x

Figure Lengend Snippet: a , b Quantification of fission (Fis.) and fusion (Fus.) events associated with ER. Cells were transfected with mCherry-ER and mito-GFP. Each point represents an individual cell, with 15 cells quantified in 4 independent experiments. a ER-Positive events (%), ( b ) Total events per cell. Bars show the average ± SD. c Representative fusion event showing the enrichment of AC-ER at the fusion site (arrowhead). Cells were transfected with mCherry-mito (mitochondria, magenta) and AC-ER (actin, green). Scale bar 2 µm. d , e Quantification of fission (Fis.) and fusion (Fus.) events positive for AC-mito. Each point represents an individual cell, with 13 cells quantified in 3 independent experiments. d ER-Positive events (%), ( e ) Total events per cell. f AC-ER signal enrichment at fusion (Left) and fission (Right) sites in cells transfected as in ( c ). Signal intensity at the event site relative to an adjacent site on the ER network was quantified for AC-ER (AC) and mCherry-ER (mCh). Each point represents an individual cell, with 13 cells quantified in 3 independent experiments. Bars show the average ± SD. two-sided t-test. g Quantification of AC-ER signal associated with each of the fusing mitochondria (the stationary mitochondrion (Rec) and the mobile mitochondrion (Fus)) as well as with the fusion site one frame post-fusion (Post). Each point represents an individual cell, with 13 cells quantified in 3 independent experiments. Bars show the average ± SD. One-way ANOVA. h , i Kinetics of recruitment of AC-mito and AC-ER at the fusion site showing AC-ER recruitment relative to AC-mito ( g ) and recruitment of either marker relative to the time of fusion ( h ). Each point represents an individual event (20 events total in 4 cells within 3 independent experiments). two-sided t-test.

Article Snippet: These cells were stably transfected with mitochondria-targeted GFP (CCOeGFP, addgene, pLVX-EF1a-CCO-IRES, #134861) using Metafectene (Biontex).

Techniques: Transfection, Marker

Journal: Nature Communications

Article Title: Mitochondria- and ER-associated actin are required for mitochondrial fusion

doi: 10.1038/s41467-024-55758-x

Figure Lengend Snippet: a Representative images showing the loss of AC-mito signal in primary fibroblasts transfected with AC-mito (green) and mCherry-Fis1 (magenta), and treated with the Arp2 inhibitor CK-666. Scale bar 10 µm ( b ) Quantification of AC-mito signal accumulation in cells transfected as in ( a ). Each point represents an individual cell, with 15 cells quantified per condition in 3 independent experiments. Bars show the average ± SD. Two-sided t-test. ( c ) Quantification of the number of fusion (Left, blue) and fission (Right, orange) events, as well as the fusion/fission ratio ( d ) in cells transfected as in ( a ) and treated as indicated. Each point represents an individual cell, with 12 (control) and 9 (CK-666) cells quantified in 3 independent experiments. Bars show the average ± SD. Two-sided t-test. e Quantification of fusion events as tip-to-side (Side) or tip-to-tip (End). The total number of events for 9 cells in 3 experiments is shown for each condition. f Quantification of mitochondrial length (Left) and connectivity (Right) in cells from ( c ). Each point represents an individual cell. Bars show the average ± SD. One-way ANOVA. g – i Mitochondrial fusion assay. Human primary fibroblasts were transfected with photoactivatable-GFP (PA-GFP), treated as indicated and imaged before (pre) and after activation with the 405 nm laser. Fluorescence traces ( g , Left) and the quantification of the loss of fluorescence at 3 min relative to the initial time post-activation ( g , Right) are shown. Each point ( g , Right) represents an individual cell, with 14 ctrl and 16 CK-666 cells quantified in 4 independent experiments. Bars show the average ± SD. Two-sided t-test. Fusion was also directly quantified by measuring the events in which PA-GFP was transferred from one mitochondrion to another upon fusion (H, separation by fusion type; I, total fusion events). Each point represents an individual cell, with 12 ctrl and 11 CK-666 cells quantified in 3 independent experiments. Bars show the average ± SD. Two-sided t-test.

Article Snippet: These cells were stably transfected with mitochondria-targeted GFP (CCOeGFP, addgene, pLVX-EF1a-CCO-IRES, #134861) using Metafectene (Biontex).

Techniques: Transfection, Control, Single Vesicle Fusion Assay, Activation Assay, Fluorescence

Journal: Nature Communications

Article Title: Mitochondria- and ER-associated actin are required for mitochondrial fusion

doi: 10.1038/s41467-024-55758-x

Figure Lengend Snippet: a – d Temporal relationship between mitochondrial actin and the fusion dynamin MFN2. Cells were transfected with mCherry-MFN2 and AC-mito while mitochondria were visualised with Mitotracker Deep red. ( a ) Presence of AC-mito and mCherry-MFN2 at sites of mitochondrial fission, tip-to-side fusion and tip-to-tip fusion. b – d Kinetics of recruitment of AC-mito and mCherry-MFN2 at fission ( b ) and fusion ( c ) sites showing recruitment of either marker relative to the time of fusion, as well as mCherry-MFN2 recruitment relative to AC-mito ( d ). Each point represents an individual event in 4 cells within 3 independent experiments). Bars show the average ± SD. two-sided t-test. e – h Temporal relationship between the fusion dynamin MFN2 and the fission Dynamin DRP1. Cells were transfected with mCherry-MFN2 and GFP-DRP1 while mitochondria were visualised with Mitotracker Deep red. e Presence of GFP-DRP1 and mCherry-MFN2 at sites of mitochondrial fission, tip-to-side fusion and tip-to-tip fusion. ( f – h ) Kinetics of recruitment of mCherry-MFN2 andGFP-DRP1 at fission ( f ) and fusion ( g ) sites showing recruitment of either marker relative to the time of fusion, as well as GFP-DRP1 recruitment relative to mCherry-MFN2 ( d ). Each point represents an individual event in 3 cells within 3 independent experiments). Bars show the average ± SD. two-sided t-test. ( i ) Schematic representation of the timing of MFN2 and DRP1 recruitment to tip-to-side fusion (Top) and fission (Bottom) relative to actin recruitment (AC-mito-positive).

Article Snippet: These cells were stably transfected with mitochondria-targeted GFP (CCOeGFP, addgene, pLVX-EF1a-CCO-IRES, #134861) using Metafectene (Biontex).

Techniques: Transfection, Marker