Journal: bioRxiv

Article Title: Photoactivated SOPP3 enables APEX2-mediated proximity labeling with high spatio-temporal resolution in live cells

doi: 10.1101/2024.06.23.595995

Figure Lengend Snippet: a Schematic of APEX2+SOPP3 mediated photoactivated-proximal labeling (photo-PL). B, Biotin. b Photosensitizer screening for APEX2 activation. Streptavidin blot showed photo-PL efficiency. anti-V5 and anti-HA indicated expression level of APEX2 and photosensitizers, respectively. Anti-α-tubulin, internal loading control for western blot. c , d Schematic ( c ) and construct designs ( d ) of APEX2+SOPP3 mediated PL to map proteome on ER-Mito contact sites. BP, Biotin Phenol. e Evaluation on the efficiency of photo-PL at ER-Mito contact sites. Anti-HA and anti-V5 indicated expression level of SOPP3-ERM and OMM-APEX2 respectively. f Left: confocal fluorescence imaging of APEX2+SOPP3 mediated photo-PL on ER-Mito contact sites. Outer membrane of mitochondria (OMM)-localized APEX2 and ER membrane (ERM)-localized SOPP3 were visualized by anti-V5 and anti-HA antibody. Biotinylation signals on the contact sites were visualized by Alexa Fluor 555-conjugated streptavidin. Illumination time, 5 seconds. Scale bar, 9 μm. Right: the ‘Surface’ tool in Imaris software was used to create a 3D rendering from each channel of confocal images of boxed region. Contact area algorithm was further performed to determine the interface (yellow) between ER (green) and Mito (cyan). Scale bar, 0.8 μm. g Validation on MAMs proteins from streptavidin-enriched PL samples. h , i Schematic and construct designs ( h ) and evaluation on photo-PL efficiency on cell-cell contact sites via western blotting analysis ( i ). Anti-V5 and anti-HA indicated expression level of APEX2-TM and SOPP3-TM respectively. j Confocal fluorescence imaging of APEX2+SOPP3 mediated photo-PL on cell-cell contact sites, stained with Alexa Fluore 555-conjugated streptavidin. Scale bar, 2 μm. Zoomed image from boxed region showed biotinylation labeling precisely on cell-cell contact sites. Scale bar, 1 μm. Illumination time, 10 seconds. k Schematic and construct design of chimeric APEX2-SOPP3-mediated photo-PL in ERM. l Evaluation on the efficiency of photo-PL mediated by chimeric APEX2-SOPP3 targeted to ERM via western blotting analysis. Anti-HA/Flag/V5 indicated expression level of chimeric APEX2-SOPP3-ERM, SOPP3-ERM and OMM-APEX2 respectively. m Confocal fluorescence imaging of photo-PL via APEX2-SOPP3 targeted to ERM. Chimeric APEX2-SOPP3-ERM and biotinylation signals was visualized by anti-HA antibody and Alexa Fluor 555-conjugated streptavidin respectively. Anti-Flag blot indicated expression level of chimeric APEX-SOPP3 targeted to various subcellular compartments. Illumination time, 5 seconds. Scale bar, 10 μm. n , o Schematic and construct designs ( n ) and evaluation on chimeric APEX2-SOPP3 on cell-cell contact sites via western blotting analysis ( o ). Anti-HA indicated expression level of chimeric APEX2-SOPP3-TM. p Confocal fluorescence imaging of chimeric APEX2-SOPP3 mediated photo-PL on cell surface. Chimeric APEX2-SOPP3 was visualized by anti-HA antibody. Biotinylation signals were visualized by Alexa Fluor 555-conjugated streptavidin. Scale bar, 8 μm. Illumination time, 5 seconds. q Flow cytometry analysis on p (30,000 cells per condition). After photo-PL, cells were stained with Alexa Fluor 555-conjugated streptavidin, followed with flow cytometry gating for quantification of biotinylation labeling on the surface of APEX2-SOPP3-TM cells (anti-HA positive, q up) and neighboring WT cells (anti-HA negative, q down). Blue light-activated SOPP3 showed more efficient to facilitate APEX2-mediated proximity labeling compared to that of 1 mM H 2 O 2 treatment. Illumination time, 10 seconds.

Article Snippet: Freshly isolated crude mitochondria from HeLa cells were resuspended in mitochondria storage buffer (Beyotime Biotechnology, C3601).

Techniques: Labeling, Activation Assay, Expressing, Control, Western Blot, Construct, Fluorescence, Imaging, Membrane, Software, Biomarker Discovery, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Mitoxantrone modulates a heparan sulfate-spike complex to inhibit SARS-CoV-2 infection

doi: 10.1038/s41598-022-10293-x

Figure Lengend Snippet: Mitoxantrone inhibits SARS-CoV-2 infection in an EpiAirway 3D tissue model. (A) A schematic diagram of the experimental design. (B) Remdesivir (2 μM) but not Bleomycin (100 μM) inhibits SARS-CoV-2 infection. 24 or 96 h after drug treatment and viral infection (MOI 0.1), the cell surface was washed. The viral titer (TCID50) in the wash was determined. (C,D) Mitoxantrone inhibits SARS-CoV-2 infection in the EpiAirway 3D model. TCID50 was determined either 24 h (C) or 96 h (D) after the organoids were treated with the drug at the indicated concentrations and then air-infected with SARS-CoV-2 at MOI 0.1 for 1 h. The cells were washed from the apical side to remove the virus in the cell exterior and then incubated for 24 (C) or 96 h (D) . Cells were rinsed from the apical side again and viral titers in the wash were determined. The dashed lines indicate the viral titer from cells infected without Mitoxantrone or in the presence of 2 mM Remdesivir (Rem.), as indicated. (E) Bleomycin (100 μM) but not Remdesivir (2 μM) induces cell death, releasing LDH as determined by a luciferase assay. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001 by unpaired student’s t-test. n = 2 tissues per test, each with 3 technical repeats. (F,G) Mitoxantrone inhibits SARS-CoV-2-induced cytotoxicity in the EpiAirway 3D model. As in (D,E) except that LDH in washes collected from Mitoxantrone-treated tissues was measured.

Article Snippet: Medium from the basolateral layer of the tissue culture inserts was removed 24- and 96-h post-infection and diluted in LDH Storage Buffer as per the manufacturer’s instructions (LDH-Glo Cytotoxicity Assay, Promega).

Techniques: Infection, Virus, Incubation, Luciferase

Journal: eLife

Article Title: Cytoplasmic mRNA decay represses RNA polymerase II transcription during early apoptosis

doi: 10.7554/eLife.58342

Figure Lengend Snippet:

Article Snippet: Other , KillerTRAIL Storage and Dilution Buffer , Enzo Life Sciences , Cat# ALX-505–005 R500 , .

Techniques: Binding Assay, Recombinant, Modification, Western Blot, TUNEL Assay, Cell Fractionation, Clone Assay, Expressing, Transfection, Sequencing, Software