mcherry tsg101 plasmid  (Addgene inc)

Journal: Nature Communications

Article Title: Multifaceted membrane interactions of human Atg3 promote LC3-phosphatidylethanolamine conjugation during autophagy

doi: 10.1038/s41467-023-41243-4

Figure Lengend Snippet: a Atg3 knockout (Atg3 −/− ) mouse embryonic fibroblasts (MEFs) stably expressing mCherry-EV (empty vector), mCherry-hAtg3, mCherry-hAtg3 F296L , or mCherry-hAtg3 F296S were cultured in complete media with 100 nM bafilomycin A1 for 3 h and subjected to immunoblotting for mCherry. b Representative immunoblot ( n = 4 blots). MEF cells were cultured in complete media (CM) or starvation media (SM) with/without 100 nM bafilomycin A1 (BafA1) to block LC3B-II (LC3B-PE) degradation for 3 h and subjected to immunoblotting with indicated antibodies. c Quantitative analysis of the relative LC3B-II level ( n = 4 blots) in in vivo LC3B lipidation experiments. d Quantitative analysis of induced autophagic flux ( n = 4 blots). Statistical analysis was performed using one-way ANOVA test followed by Turkey’s multiple comparisons test. In c , data are presented as mean ± SD, P values: **** P < 0.0001, *** P = 0.0001, 0.0002, 0.0001, 0.0001 (left to right, bottom to top); ** P = 0.0021, 0.0017, 0.002, 0.0052, and 0.0034 (left to right, bottom to top). In d , data are presented as mean ± SD, P values: * P = 0.0125, 0.0283, and 0.0118 (bottom to top), ** P = 0.0053. Source data are provided as a Source Data file.

Article Snippet: Cells were incubated in complete or starvation media in the absence and presence of 100 nM bafilomycin A1 (BafA1) (ThermoFisher scientific, J61835.MCR) for 3 h and subjected to immunoblotting using the mCherry antibody (Abcam, ab125096) with a ratio of 1:1000, the β-actin antibody (Sigma, A5441-100UL) with a ratio of 1:10,000, and the LC3 antibody (Novus Biologicals, NB100-2220) with a ratio of 1:5000.

Techniques: Knock-Out, Stable Transfection, Expressing, Plasmid Preparation, Cell Culture, Western Blot, Blocking Assay, In Vivo

Journal: Nature Communications

Article Title: Sulindac selectively induces autophagic apoptosis of GABAergic neurons and alters motor behaviour in zebrafish

doi: 10.1038/s41467-023-41114-y

Figure Lengend Snippet: a Gene dot plot showing that only Akt1 was abundant in GABAergic neurons, but not akt2, akt3a, akt3b, bdnf, erbb4 ( erbb4a, erbb4b, ERBB4 ) , fgf9, mtor , or pi3k ( pi3ca, pi3cb, pi3cd ). b Gene violin map distribution showed that akt1 was distributed mainly in the subpopulation of GABAergic neurons. c Double immunostaining revealed that Akt1 proteins were selectively expressed in GABAergic neurons in the midbrains of Tg(gad1b:mCherry) embryos, as indicated by yellow arrows. The experiment was repeated twice with similar results, and at least five zebrafish were observed each time. d Flow diagram showing a tissue-specific CRISPR vector microinjected to knoc kout akt1 in GABAergic neurons. e Specific knock out of akt1 in GABAergic neurons via injection with the gad1b:Cas9-T2A-mCherry,U6:gRNA akt1 vector led to apoptosis of GABAergic neurons, as indicated by yellow arrows. f Quantification of the yellow colour double-positive cells in the midbrains of embryos. The values represent the means ± SDs ( n = 5 larvae for per group, each dot denotes one larva). Statistics calculated by unpaired two-tailed Student’s t test. Source data are provided as a Source Data file. g The number of apoptotic GABAergic neurons was restored by overexpression of akt1 mRNA after sulindac treatment, as indicated by yellow arrows. h Quantification of the yellow double-positive cells in the midbrains of embryos. The values represent the means ± SDs ( n = 5 larvae for per group, each dot denotes one larva). Statistics calculated by unpaired two-tailed Student’s t test. Source data are provided as a Source Data file. Apoptosis of GABAergic neurons in the midbrain is indicated by yellow arrows. ( i ) Western blot analysis showed that overexpression of akt1 inhibited the declines in p-Akt1 and Gad67 protein levels. j The data are presented as the means ± SDs ( n = 3 independent biological replicates). Statistics calculated by unpaired two-tailed Student’s t test. Source data are provided as a Source Data file. β-Actin was used as an internal control.

Article Snippet: Rabbit anti-Akt1 (Invitrogen, #PA5-29169, 1:100) and mouse anti-mCherry (Abcam, #ab125096, 1:500) were used as primary antibodies, and Alexa Fluor 488-conjugated anti-rabbit (Invitrogen, #A32731, 1:400) and Alexa Fluor 555-conjugated anti-mouse (Invitrogen, #A32727, 1:400) were used as secondary antibodies.

Techniques: Double Immunostaining, CRISPR, Plasmid Preparation, Knock-Out, Injection, Two Tailed Test, Over Expression, Western Blot

Journal: eLife

Article Title: Functional imaging of conduction dynamics in cortical and spinal axons

doi: 10.7554/eLife.86512

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-mCherry (Mouse Monoclonal) , Abcam , ab125096 , IHC (1:500).

Techniques: Recombinant, Plasmid Preparation, Software

Journal: Nature Cell Biology

Article Title: Lipid saturation controls nuclear envelope function

doi: 10.1038/s41556-023-01207-8

Figure Lengend Snippet: a , Sequence alignment of a conserved phosphorylation motif located at the C-termini of Sct1 and Gpt2. Red asterisks: serine residues that were mutated to alanine in Sct1 3A or Gpt2 3A. Amino acid positions in Sct1 indicated. b , Live imaging of sct1 Δ cells expressing genomically tagged H2B -mScarlet and overexpressing plasmid-based mGFP -SCT1 constructs. Numbers: circularity index of H2B signal, taken as a proxy for nuclear shape. Circularity of Sct1 3A-expressing cells is significantly reduced, consistent with NE rigidification. Sct1* = Sct1 G253L. N, nucleus. Scale bar, 2 µm. c , Quantification of circularity index of nuclear H2B signal in (b). Value of 1 indicates a perfect circle. As the value approaches 0, the structure becomes increasingly irregular. p-value (****<0.0001) determined by two-sided Mann-Whitney test. Box-plot indicates median and interquartile range, whiskers extend to the smallest and largest value. n = number of biological replicates; >450 cells analyzed for each condition. d , Imaging of cells expressing genomically tagged SEC63 -GFP and overexpressing plasmid-based Homo sapiens ( H.s .) GPAT constructs tagged with BFP. N, nucleus. Scale bar, 2 µm. e , Prevalence of polygonal NE/ER phenotype in (d and Fig. 1e). Mean value and standard deviation indicated. n = number of biological replicates; >300 cells analyzed for each condition. f , Immunoblotting for Gpt2 phosphorylation. Samples analyzed on a 3–8% Tris-acetate gel in Tris-acetate SDS buffer. Upper band: phosphorylated Gpt2; lower band: unphosphorylated Gpt2. Gpt2 3A = Gpt2 S664A S668A S671A. Pgk1 (3-phosphoglycerate kinase) as loading control. g , Imaging of gpt2 Δ cells overexpressing plasmid-based mGFP- GPT2 constructs. N, nucleus. Scale bar, 2 µm. h , Prevalence of polygonal NE/ER phenotype in (g). Mean value and standard deviation indicated. n = number of biological replicates; >500 cells analyzed for each condition. i , TEM analysis of sct1 Δ cells overexpressing plasmid-based mGFP- SCT1 from the GAL1 promoter. Insets show a magnified view of marked areas. Schemes illustrate the most frequently observed NE/ER morphologies in cells with increased lipid saturation. Hypothetically, the order of phenotypes (from top to bottom) reflects the evolution of membrane rigidification, starting with the ER (spherical nucleus/planar ER) and progressing through one stiff NE membrane (half-spherical nucleus/planar ER; see Fig. 1f) to two or more stiff NE membranes (polyhedral nucleus/planar ER). ER, endoplasmic reticulum. Scale bar, 1 µm; 200 nm for insets. j , Imaging of sct1 Δ cells expressing plasmid-based mGFP- SCT1 constructs and the reticulon RTN1 -mCherry. Constructs were overexpressed from the GAL1 promoter. Note that Rtn1 does not overlap with rigidified ER sheets. Sct1* = Sct1 G253L. N, nucleus. Scale bar, 2 µm. k , Growth of wild-type cells overexpressing plasmid-based constructs from the GAL1 promoter. Empty vector used as control. Growth on plates containing glucose (repressed) and galactose (induced). Source numerical data and unprocessed blots are available in source data.

Article Snippet: Antibodies were used according to the manufacturer’s instructions: mouse monoclonal anti-mCherry (clone 1C51) (1:2,000, Abcam cat. no. ab125096), mouse monoclonal anti-GFP (clones 7.1 and 13.1) (1:2,000, Roche cat. no. ab11814460001), mouse monoclonal anti-Pgk1 (clone 22C5D8) (1:10,000, Abcam cat. no. ab113687), peroxidase AffiniPure Goat anti-Mouse IgG (polyclonal) (1:5,000, Jackson ImmunoResearch cat. no. 115035008) and mouse monoclonal anti-HA (clone 12CA5) (1:1,000, Max Perutz Labs Monoclonal Antibody Facility).

Techniques: Sequencing, Imaging, Expressing, Plasmid Preparation, Construct, MANN-WHITNEY, Standard Deviation, Western Blot, Membrane

Journal: Nature Cell Biology

Article Title: Lipid saturation controls nuclear envelope function

doi: 10.1038/s41556-023-01207-8

Figure Lengend Snippet: a , Live imaging of GAL1 OLE1 cells expressing genomically tagged SEC63 –GFP. Growth on medium containing galactose (highly expressed OLE1 ) or raffinose (lowly expressed OLE1 ). N, nucleus. Scale bar, 2 µm. b , Imaging of sct1 Δ cells overexpressing plasmid-based mGFP– SCT1 constructs. Cells were grown for 2 h in galactose-containing medium, then treated with 0.5% LA (+1% Tween 80) for 2 h. Sct1*, Sct1 G253L. Scale bar, 2 µm. c , Growth analysis of wild-type or sct1 Δ cells transformed with plasmid-based constructs overexpressed from the GAL1 promoter. Plates contained glucose (repressed) or galactose (induced), or galactose supplemented with 0.5% LA (+1% Tween 80). d , Imaging of sct1 Δ cells expressing a plasmid-based INM LipSat sensor and SCT1 or OLE1 overexpressed from a GAL1 promoter. Sensor fluorescence intensity was measured across a line spanning the nucleus. FU, arbitrary fluorescence units; N, nucleus; NP loc., nucleoplasmic localization; NE loc., nuclear envelope localization. The FU value of 1 is marked with a dashed line. Scale bar, 2 µm. e , Quantification of INM LipSat sensor distribution in d . Ratios of NE and nucleoplasmic fluorescence intensities were calculated. Mean value and standard deviation indicated. P values (** P = 0.0011, **** P < 0.0001) determined by two-sided Mann–Whitney test. n , number of biological replicates; >170 cells analysed for each condition. f , Imaging of sct1 Δ cells containing an empty vector or expressing plasmid-based mGFP– SCT1 and OLE1 –mCherry from a GAL1 promoter. Scale bar, 2 µm. g , GUV reconstitution from whole cell lipid extracts from the indicated Sct1-overexpressing strains. Rhod–PE prefers the liquid-disordered phase. Scale bar, 4 µm. h , Quantification of phase-separated GUVs in g . Mean value and standard deviation indicated. n , number of biological replicates; >190 GUVs analysed for each condition. i , Quantification of angular GUVs among phase-separated GUVs. Mean value and standard deviation indicated. n , number of biological replicates; >190 GUVs analysed for each condition. Source numerical data are available in source data.

Article Snippet: Antibodies were used according to the manufacturer’s instructions: mouse monoclonal anti-mCherry (clone 1C51) (1:2,000, Abcam cat. no. ab125096), mouse monoclonal anti-GFP (clones 7.1 and 13.1) (1:2,000, Roche cat. no. ab11814460001), mouse monoclonal anti-Pgk1 (clone 22C5D8) (1:10,000, Abcam cat. no. ab113687), peroxidase AffiniPure Goat anti-Mouse IgG (polyclonal) (1:5,000, Jackson ImmunoResearch cat. no. 115035008) and mouse monoclonal anti-HA (clone 12CA5) (1:1,000, Max Perutz Labs Monoclonal Antibody Facility).

Techniques: Imaging, Expressing, Plasmid Preparation, Construct, Transformation Assay, Fluorescence, Standard Deviation, MANN-WHITNEY

Journal: Nature Cell Biology

Article Title: Lipid saturation controls nuclear envelope function

doi: 10.1038/s41556-023-01207-8

Figure Lengend Snippet: a , Generalized polarization (GP) values of NE ‘arc’, NE ‘base’, and peripheral ER regions in a cell overexpressing Sct1 3A in (Fig. 4a). Note that the GP curves for the ER and the NE ‘base’ are shifted to the right, indicating a higher membrane order than the NE ‘arc’. b , GP values of NE ‘arc’, ‘base’, and ER regions in cells overexpressing Sct1 3A. Mean value and standard deviation indicated. 25 ‘arc’ cells analyzed. c , Single-plane, pseudocolored generalized polarization (GP) images of Laurdan-stained cells. Color bar designates the range of GP values. Red indicates the highest, blue the lowest membrane order. sct1 Δ cells expressed genomically tagged SEC63 -mScarlet, the nucleoplasmic marker PUS1 -GFP, and overexpressed plasmid-based SCT1 constructs. White arrowhead highlights the lack of co-localization between the highly ordered ER membrane and the Sec63 translocon. White asterisks: lipid droplets, which accumulate Laurdan due to their apolar interior . Sct1* = Sct1 G253L. L d , liquid-disordered phase; L o , liquid-ordered phase; N, nucleus. Scale bar, 2 µm. d , Live imaging of sct1 Δ cells expressing genomically tagged PMA1 -mScarlet and overexpressing plasmid-based mGFP -SCT1 constructs. Numbers: circularity index of the Pma1 fluorescence signal, taken as a proxy for the plasma membrane contour. Sct1* = Sct1 G253L. N, nucleus. Scale bar, 2 µm. e , Quantification of circularity index of Pma1 contour in (d). Circularity index of 1 indicates a perfect circle. ns (not significant, p-value 0.205) as determined by two-sided Mann-Whitney test. Box-plot indicates median and interquartile range, whiskers extend to the smallest and largest value. n = number of biological replicates; >250 cells analyzed. f , TEM images of mitochondria in cells overexpressing mGFP- SCT1 from the GAL1 promoter. Wild-type cells used as a control. Arrows label rigidified ER membranes. Mito, mitochondrion; N, nucleus; ER, endoplasmic reticulum. Scale bar, 200 nm. g , Imaging of sct1 Δ cells expressing genomically tagged mitochondrial marker COX4 -mGFP and overexpressing plasmid-based mCherry -SCT1 constructs. Sct1* = Sct1 G253L. N, nucleus. Scale bar, 2 µm. Source numerical data are available in source data.

Article Snippet: Antibodies were used according to the manufacturer’s instructions: mouse monoclonal anti-mCherry (clone 1C51) (1:2,000, Abcam cat. no. ab125096), mouse monoclonal anti-GFP (clones 7.1 and 13.1) (1:2,000, Roche cat. no. ab11814460001), mouse monoclonal anti-Pgk1 (clone 22C5D8) (1:10,000, Abcam cat. no. ab113687), peroxidase AffiniPure Goat anti-Mouse IgG (polyclonal) (1:5,000, Jackson ImmunoResearch cat. no. 115035008) and mouse monoclonal anti-HA (clone 12CA5) (1:1,000, Max Perutz Labs Monoclonal Antibody Facility).

Techniques: Membrane, Standard Deviation, Staining, Marker, Plasmid Preparation, Construct, Imaging, Expressing, Fluorescence, MANN-WHITNEY

Journal: Nature Cell Biology

Article Title: Lipid saturation controls nuclear envelope function

doi: 10.1038/s41556-023-01207-8

Figure Lengend Snippet: a , TEM examples of NE rupture upon overexpression of mGFP- SCT1 from the GAL1 promoter. White brackets mark NE gaps (240 nm in left image; 440 nm in right image). White asterisk: open flap of the torn NE. NE, nuclear envelope; N, nucleus; ER, endoplasmic reticulum; V, vacuole. Scale bar, 1 µm. b , Nuclear leakage assay. Live imaging of sct1 Δ cells expressing genomically tagged PUS1 -GFP (a tRNA:pseudouridine synthase), NUP188 -mScarlet and overexpressing plasmid-based BFP- SCT1 constructs. Examples of ‘arc’ and ‘irregular’ NPC patterns are shown. Empty arrowhead: straight NE ‘base’. N, nucleus. Scale bar, 2 µm. c , Quantification of Pus1 leakage into cytoplasm in (b). Nuclear integrity was considered normal when Pus1-GFP was enriched in the nucleus, and defective (that is leakage) when nucleoplasmic and cytoplasmic fluorescence intensities were equal. Mean value and standard deviation indicated. n = number of biological replicates; >460 cells analyzed. d , Quantification of nuclear leakage in (Fig. 5c). Nuclear integrity was considered normal when the MGM4 reporter was excluded from the nucleus, and as defective (that is leakage) when nucleoplasmic and cytoplasmic fluorescence intensities were equal. Mean value and standard deviation indicated. n = number of biological replicates; >450 cells analyzed. e , Immunoblotting for Sct1 phosphorylation. sct1 Δ cells overexpressing plasmid-based mCherry- SCT1 and/or mGFP -DGA1 . Samples taken from cell cultures used in (Fig. 6b) and analyzed on a 3–8% Tris-acetate gel in Tris-acetate SDS buffer. Both constructs were overexpressed from the GAL1 promoter. Note that Dga1 co-expression did not noticeably affect Sct1 protein levels. Upper band: phosphorylated form; lower band: unphosphorylated form. Pgk1 (3-phosphoglycerate kinase) as loading control. f , Lipidomic analysis. Relative abundance of four major phospholipid species (PC, PE, PI and PS). Cells supplemented with palmitic acid (C16:0) were grown under normoxia (air) or hypoxia (N 2 ). Sct1 3A and control (wild-type) cells were grown for 4 h in galactose-containing media. Mean value and standard deviation indicated. n = number of biological replicates. Source numerical data and unprocessed blots are available in source data.

Article Snippet: Antibodies were used according to the manufacturer’s instructions: mouse monoclonal anti-mCherry (clone 1C51) (1:2,000, Abcam cat. no. ab125096), mouse monoclonal anti-GFP (clones 7.1 and 13.1) (1:2,000, Roche cat. no. ab11814460001), mouse monoclonal anti-Pgk1 (clone 22C5D8) (1:10,000, Abcam cat. no. ab113687), peroxidase AffiniPure Goat anti-Mouse IgG (polyclonal) (1:5,000, Jackson ImmunoResearch cat. no. 115035008) and mouse monoclonal anti-HA (clone 12CA5) (1:1,000, Max Perutz Labs Monoclonal Antibody Facility).

Techniques: Over Expression, Imaging, Expressing, Plasmid Preparation, Construct, Fluorescence, Standard Deviation, Western Blot