h2b mruby  (Addgene inc)

Journal: bioRxiv

Article Title: Drug repurposing screen identifies masitinib as a 3CLpro inhibitor that blocks replication of SARS-CoV-2 in vitro

doi: 10.1101/2020.08.31.274639

Figure Lengend Snippet: A. Schematic of the screen. A549 cells expressing H2B-mRuby were infected with OC43 (MOI 0.3), treated with drugs, incubated for 4 days at 33°C, and stained for the viral nucleoprotein. B. Screen results showing the %OC43 staining of mock-infected cells (green), no-drug controls (black), drugs with no effect on OC43 infection (blue), and screen hits (red). Overall agreement between the two repeats is high (R =0.81) C. Dose response curves of remdesivir and the top hits from the screen, n = 3. Individual measurements are shown as semi-transparent circles (note that some circles overlap).

Article Snippet: A549 expressing H2B-mRuby were generated by first infecting A549 cells (ATCC CCL-185) with a lentivirus (carrying H2B-mRuby), and FACS-sorting mRuby+ cells.

Techniques: Expressing, Infection, Incubation, Staining

Journal: bioRxiv

Article Title: Drug repurposing screen identifies masitinib as a 3CLpro inhibitor that blocks replication of SARS-CoV-2 in vitro

doi: 10.1101/2020.08.31.274639

Figure Lengend Snippet: Of the 26 drugs that inhibited OC43 and tested against SARS-CoV-2, 20 inhibited SARS-CoV-2 replication in a dose-dependent manner, showing good concordance between OC43 and SARS-CoV-2 inhibition. A. A549 cells over-expressing ACE2 were pre-treated with indicated drugs for 2 hours, infected with SARS-CoV-2 (MOI 0.5) and incubated for 2 days. Cells were stained for the presence of the spike protein and the % of infected cells was analyzed. Most of the drugs effective against OC43 showed similar effectivity against SARS-CoV-2, n=3. Individual measurements are shown as semi-transparent circles (note that some circles overlap). B. Effect of selected drugs on SARS-CoV-2 progeny production. Cells were treated with 10 μM of the indicated drugs for 2 hours, infected with SARS-CoV-2 (MOI 0.5) and cell supernatant were collected for titration 2 days later, n = 3. Individual measurements are shown as semi-transparent circles. All drugs showed a statistically significant (p-values<0.001, one-tailed t-test, FDR-corrected) reduction in viral titers.

Article Snippet: A549 expressing H2B-mRuby were generated by first infecting A549 cells (ATCC CCL-185) with a lentivirus (carrying H2B-mRuby), and FACS-sorting mRuby+ cells.

Techniques: Inhibition, Expressing, Infection, Incubation, Staining, Titration, One-tailed Test

Journal: medRxiv

Article Title: Pathogenic variants in TMEM184B cause a neurodevelopmental syndrome via alteration of metabolic signaling

doi: 10.1101/2024.06.27.24309417

Figure Lengend Snippet: A-D and G-J Multichannel images of non-starved HEK293T cells overexpressing WT or K184E, respectively. TFEB-GFP (A and G), H2B-mRuby (B and H), TMEM184B-miRFP6670 (C and I), and merge (D and J). E-F and K-L are WT and K184E cells starved for one hour, respectively. TFEB-GFP (E and K) and merge (F and L). Arrows in all images point to TMEM184B-miRFP670 transfected cells. All H2B-mRuby images had their Lookup Tables adjusted to 4000 for visual clarity. M Ratio of mean TFEB-GFP fluorescence in the nucleus vs. cytoplasm in TMEM184B-miRFP670 expressing cells, normalized to non-starved WT cells. N Timelapse of TFEB localization (nucleus vs. cytoplasm) of K184E and IVS8+1G>A (exon 7 deletion) expressing cells at baseline, one hour, and six hour starvation timepoints (N=3 trials). M and N error bars represent SEM. ****p<0.0001, *p<0.01. 50 cells were quantified in every trial for every patient variant and time point except for K184E at six hour starvation (42-50 cells) and Ex7del at all time points. At no starvation, one hour, and six hours, we quantified between 43-50, 42-50, and 14-42 cells respectively.

Article Snippet: TFEB-sfGFP expressing cells were then infected with H2B-mRuby lentivirus (Addgene Plasmid #90236) prepared as above except that VSV-G was replaced with PSPAX2.

Techniques: Transfection, Fluorescence, Expressing, Variant Assay

Journal: Cell reports

Article Title: Regulation of transcription patterns, poly(ADP-ribose), and RNA-DNA hybrids by the ATM protein kinase

doi: 10.1016/j.celrep.2024.113896

Figure Lengend Snippet:

Article Snippet: For R-ChIP studies, human RNaseH1 was expressed using a lentivirus derived from pTP5135, containing a tet-on allele of RNaseH D210N in a pLenti Hygro H2B mRuby backbone. pLentiPGK Hygro DEST H2B-mRuby2 was a gift from Markus Covert (Addgene plasmid # 90236; http://n2t.net/addgene:90236 ; RRID:Addgene_90236).

Techniques: Recombinant, Protease Inhibitor, Binding Assay, Magnetic Beads, Single Cell Gel Electrophoresis, Multiplex Assay, Software, RNA Extraction