Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) Violin plots depict limb muscle single cell RNAseq data for Dlk1 - Dio3 megacluster-encoded lncRNAs Meg3 , Rian , and Mirg . All lncRNAs showed enrichment in satellite (sat.) and mesenchymal (mes.) cell types, with Meg3 as the most abundant. CPM = counts per million reads mapped. B) qPCR temporal Meg3 expression profiling was performed on regenerating mouse Tibialis anterior (TA) muscle tissue harvested before (uninjured), and on the indicated days after cardiotoxin injection (+CTX). Meg3 lncRNA transcripts were upregulated following CTX-induced injury, which corresponds with satellite and mesenchymal cell expansion (n=3 mice per time point). C) qPCR temporal expression profiling of Meg3 in C2C2 myoblast differentiation. Meg3 transcripts were most enriched during proliferation (prolif.), and progressively downregulated during course of differentiation (n=4). D) RNA immunoprecipitation (RNA-IP) was performed on subconfluent C2C12 myoblast lysates to examine for Meg3 -PRC2 interaction. Immunoprecipitated RNA was quantified by qPCR, using supernatant as an internal normalization control. Compared to normal IgG controls, Meg3 was enriched in anti-Ezh2 immunoprecipitates (n=3 sets of 60 plates).

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Expressing, Injection, RNA Immunoprecipitation, Immunoprecipitation, Control

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) qPCR quantification of Meg3 transcript levels in heterogeneous cell populations derived from G418 selection (C2C12 het.), and subsequently derived clonal populations (C2C12 clones) indicate that stable shRNA integration resulted in Meg3 knockdown (n=3). B) Immunofluorescence quantification of MYH4 indicates markedly reduced expression in sh Meg3 C2C12 clones. Quantification of nuclei within α-actinin cell-boundaries show reduced fusion index in sh Meg3 clones (n=3). qPCR expression profiling indicated unchanged Myf5 transcript levels, but significant reduction in other myogenic differentiation markers (n=3). C) Western blot quantification of MF20 signal (normalized to β-tubulin) showed marked reduction specific to sh Meg3 clones (n=4). D) qPCR quantification confirmed overexpression of human MEG3 in sh LacZ and sh Meg3 myoblasts, and restoration of endogenous Meg3 transcript levels relative to β-galactosidease controls (n=3). E) Human MEG3 restored both MYH4 expression and fusion index in sh Meg3 but not sh LacZ myoblasts (n=6 MYH4, n=3 fusion index). F) qPCR expression profiling of heterogeneous rescue clones revealed an increase in Mef2C, Ckm , MyoD and Myog levels in sh Meg3 + MEG3 myotubes. MYH4 = myosin heavy chain 4 (Proteintech antibody), MF20 = myosin heavy chain 4 (DHSB antibody), Myf5 = myogenic factor 5 , MyoD = myogenic differentiation 1, Mef2C = myogenic enhancing factor- 2 C , Myog = Myogenin , Ckm = Muscle Creatine Kinase , Acta1 = skeletal muscle actin.

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Derivative Assay, Selection, Clone Assay, shRNA, Knockdown, Immunofluorescence, Expressing, Western Blot, Over Expression

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) Quantification of BrdU+ nuclei (green arrowheads) indicated that sh Meg3 myoblasts divided at a reduced frequency (n=3). B) Cleaved caspase 3 assay revealed higher apoptosis in sh Meg3 myoblasts and myotubes relative to control (n=3). Cell Titer Blue viability assay indicated reduced viability in sh Meg3 myoblasts and myotubes relative to control (n=3). C) sh Meg3 myoblasts seeded at increasing densities was not sufficient to restore MYH4 expression or fusion index (n=3).

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Caspase-3 Assay, Control, Viability Assay, Expressing

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: C2C12 myoblasts and myotubes were pulsed with MitoTracker CMXRos for 40 minutes, and co-stained with α-actinin. Quantification of Mitotracker (restricted to α-actinin+ cells) indicated reduced mitochondrial signal in sh Meg3 myoblasts, but not myotubes (n=3). Both treatment groups displayed increased MitoTracker signal with differentiation.

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Staining

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: RNAseq data indicated that transcripts of master regulators of myogenic fusion, notably Myomaker and Myomixer , were not significantly downregulated in sh Meg3 myotubes.

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques:

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) Cadherin switching was assessed by qPCR (left) and Western blot (right) quantification of E-cadherin (Cdh1) and N-cadherin (Cdh2) in day 3 myotubes. Cdh1 transcripts were significantly downregulated in sh Meg3 myotubes relative to sh LacZ controls (n=3), but Cdh1 protein signal from myotubes lysates was extremely faint (n=4). Cdh2 transcripts were upregulated (n=3), as was Cdh2 protein (n=4). B) qPCR expression profiling indicated significant downregulation of epithelial Plakophilin and PatJ transcripts in sh Meg3 myotubes, with simultaneous upregulation of mesenchymal Fibronectin and Snai2 (n=3). C) Western blot of Vimentin normalized to β-tubulin indicated no change in mesenchymal Vimentin (n=4). D) Scratch-wound assays revealed no detectable change in wound-healing efficiency, as measured by % scratch area (n=3). Brightfield (BF) microscopy of wound-healing morphology differed between sh LacZ control and sh Meg3 clones, with a higher proportion of myoblasts invading the scratch territory with fewer than 2 cell contacts (black arrowheads, n=3).

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Western Blot, Expressing, Microscopy, Control, Clone Assay

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) Following incubation with 10μM LY2157299 (LY), myoblasts were induced to differentiate and examined for MYH4 expression and fusion index. LY-treated sh Meg3 myotubes adopted an elongated morphology, and displayed increased MYH4, decreased myotubes with 1-nuclei, and increased myotubes with 2- and ≥3 nuclei relative to untreated sh Meg3 controls (n=3). B) Western blot for MF20 indicated that LY-treatment restored myosin heavy chain 4 expression to sh Meg3 myotubes (n=3). C) qPCR expression profiling of LY-treated myotubes indicate significant upregulation of all myogenic markers surveyed ( Myf5 , MyoD , Mef2C , Myog , Ckm , Acta1 ) relative to untreated sh LacZ myotubes.

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Incubation, Expressing, Western Blot

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) qPCR indicated that LY2157299 (LY) treatment resulted in reduced E-cadherin ( Cdh1 ) transcripts regardless of sh RNA treatment, with simultaneous upregulation of N-cadherin ( Cdh2 ) transcripts (n=3). Western blot revealed modest Cdh1 band detection, and quantification of β-tubulin-normalized signal revealed that LY-treatment restored Cdh1 levels to sh Meg3 myotubes, while Cdh1 in sh LacZ myotubes remained unchanged. While LY treatment did not change Cdh2 expression in sh LacZ myotubes, LY-treatment enhanced Cdh2 signal in sh Meg3 myotubes. B) qPCR profiling indicated upregulation of epithelial transcripts Plakophilin and PatJ regardless of sh RNA background (n=3). Fibronectin transcript levels returned to normal levels in LY-treated sh Meg3 myotubes. LY treatment intensified upregulation of Snai2 transcripts in sh Meg3 cells, but did not affect Twist2 or Mmp9 levels relative to sh Meg3 myotubes. sh LacZ + LY myotubes displayed reduced Mmp9 , with simultaneous upregulation of Twist2 when compared to untreated sh LacZ cells (n=3). C) Western blot quantification of Vimentin suggests that LY treatment reduced Vimentin expression in sh LacZ controls, but did not change Vimentin expression in sh Meg3 myotubes (n=3). D) Myoblasts pre-treated with 5ng/mL BMP4 (BMP) were subjected to differentiation, and examined for changes in MYH4 expression and fusion index. BMP4 treated sh Meg3 myotubes had improved MYH4 expression (n=3), reduced mononucleated myotubes, and improved 2-cell fusion, but not ≥3 nuclei fusion.

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) Following incubation with 40μM ROCK1/2 inhibitor (Y-27632), myoblasts were induced to differentiate and examined for MYH4 expression and fusion index. Y-27632-treated sh Meg3 myotubes adopted an elongated morphology, and fusion quantification indicated decreased myotubes with 1-nuclei, and increased myotubes with 2- and ≥3 nuclei relative to sh Meg3 control (n=3). While MYH4 expression was enhanced in sh LacZ + Y-27631 myotubes, MYH4 levels remained unchanged with Y-27632 treatment in sh Meg3 cells (n=3). B) Following incubation with 5μM p38 inhibitor (SB203580), myoblasts were induced to differentiate and examined for MYH4 expression and fusion index. SB203580-treated sh Meg3 myotubes adopted an elongated spindle-like morphology, and fusion quantification indicated decreased myotubes with 1-nuclei, and increased myotubes with 2- and ≥3 nuclei relative to sh Meg3 control (n=3). MYH4 expression was unaffected by SB203580 treatment (n=3).

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Incubation, Expressing, Control

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) qPCR expression profiling indicated reduced Meg3 expression in TA muscles co-injected with sh Meg3 adenovirus (n=3). B) Whole mount morphology of regenerating muscles co-injected with adeno-sh LacZ (top) or adeno-sh Meg3 . C) Hematoxylin and eosin (H&E) staining of muscle sections. D) Cross-sectional area (CSA) of laminin-ensheathed regenerating myofibers was measured for days 3, 7, and 14 post-CTX injury. sh Meg3 muscle displayed reduced CSA for all time points surveyed. E) Immunofluorescence quantification indicated sh Meg3 TA sections harbor increased Ki67 signal (left bar graph). Co-staining for Pax7 indicated no change in satellite-cell specific Ki67 signal (left graph) and no change in satellite cell abundance (white, red arrowheads). Marker quantification revealed an increase in proliferating cells lacking Pax7 co-stain (green arrowheads). F ) Immunofluorescence quantification indicated an increase in PDGFR signal, as well as increased abundance of PDGFRα+ cells (red arrowheads).

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Expressing, Muscles, Injection, Staining, Immunofluorescence, Marker

Journal: bioRxiv

Article Title: The long noncoding RNA Meg3 regulates myoblast plasticity and muscle regeneration through epithelial-mesenchymal transition

doi: 10.1101/2020.06.15.152884

Figure Lengend Snippet: A) Expression profiling by qPCR indicated no change in E-cadherin , while day 7 sh Meg3 muscle was enriched for N-cadherin transcripts (n=3). Immunofluorescence revealed that, while numerous satellite cells were not N-cad+ (red arrowheads), N-cad signal was largely restricted to Pax7+ satellite cells in regenerating muscle (white arrowheads). Cell quantification (% mononuc. cells) revealed that sh Meg3 muscle harbored increased abundance of N-cadherin+ satellite cells, whereas levels of N-cadherin per cell was unchanged (n=3). B) qPCR expression profiling indicated no change in epithelial markers Plakophilin and PatJ , but mesenchymal markers Fibronectin and Snai2 were significantly upregulated in sh Meg3 muscle (n=3). C) Immunofluorescence revealed the presence of satellite cells lacking Snai2 (red arrowheads), Snai2+ satellite cells (white arrowheads), Snai2+ non-satellite cells (green arrowheads), and Snai2+ nuclei in regenerating myofibers (green asterisks). Quantification of Snai2+ cells indicated no change in the occurance of Snai2+ nuclei (bar graph, % Snai2+ nuclei). Generalized analysis (DAPI) indicated significant upregulation of cytoplasmic Snai2 signal per cell, but no change nuclear intensity; Snai2 signal in satellite cells (Pax7+) was increased for both cytoplasmic and nuclear compartments (n=3). D) Immunofluorescence revealed the presence of satellite cells lacking Vimentin (red arrowheads), Vimentin+ satellite cells (white arrowheads), and Vimentin+ non-satellite cells (green arrowheads). Cell quantification (% mononuc. cells) revealed increased abundance of Vimentin+ mononucleated cells that were not Pax7+, and that Pax7+/Vimentin+ cells were reduced in sh Meg3 muscle. Vimentin signal per cell was downregulated in mononucleated cells (bottom right bar graph), which may reflect sh Meg3 -specifici differences in Vimentin+ cell morphology (green channel) (n=3).

Article Snippet: For overexpression, human MEG3 cDNA was PCR amplified from the pCI-Meg3 (Addgene Plasmid #44727) using NEB Q5 high-fidelity polymerase, and cloned into pShuttleCMV vector (Agilent AdEasy Adenoviral Vector System).

Techniques: Expressing, Immunofluorescence

Journal: Nature Communications

Article Title: Molecular architecture of a cylindrical self-assembly at human centrosomes

doi: 10.1038/s41467-019-08838-2

Figure Lengend Snippet: Cooperative formation of a dynamic cylindrical self-assembly by Cep63 and Cep152. a , b Sedimentation velocity analyses with Cep63 P1•Cep152 M4d or its respective P1(4A)•M4d(5A) mutant complex, using the complexes shown in Supplementary Fig. , left. A high-molecular weight (MW) species (arrow) with rapid sedimentation velocity (~4S) detected at higher concentrations may likely represent a dimer of the ~2.74S heterotetramer (i.e., heterooctamer). Weighted-average sedimentation coefficients obtained from the c ( s ) profiles are presented in b for the Cep63 P1•Cep152 M4d (red) and P1(4A)•M4d(5A) mutant (blue) complexes as a function of concentration. c Three-dimensional structured-illumination microscopy (3D-SIM (top) and surface rendering (bottom) of the in vitro self-assembly generated by the mCherry-Cep63 P1•mGFP-Cep152 M4d complex. Bar, 2 μm; bar for rendered image, 0.5 μm. d – f The dimension of the self-assemblies ( d , e ) and the diameter difference for mCherry-Cep63 P1 and mGFP-Cep152 M4d fluorescence ( f ) were determined from a total of 429 cylindrical assemblies obtained from three independent experiments. The Zeiss Zen software allowed to determine the inter-signal distance of up to 33nm/pixel (see Methods for details). A sharp drop in the number of small assemblies (<100 nm in diameter) in d is due to the resolution limit of SIM. The interpolated line of best fit is shown in e . Bars, s.d. g Summary of diameter differences between mGFP and mCherry fluorescence for the indicated cylindrical assemblies. Center line, median; cross, mean; box limits, upper and lower quartiles; whiskers, maximum or minimum of the data. ** P < 0.01; **** P < 0.0001 (unpaired two-tailed t test). h , i SIM-total internal reflection fluorescence (TIRF) time lapse of the self-assembling process of the mCherry-Cep63 P1•mGFP-Cep152 M4d complex in vitro ( h ) and the diameters of the assemblies as a function of time ( i ). Data are representative of a total of eight independent time-lapse movies analyzed. Bar, 1 μm. j Fluorescent recovery after photobleaching (FRAP) analysis for the mCherry-Cep63 P1•mGFP-Cep152 M4d self-assemblies in vitro. Representative data from a total of 14 independent FRAP experiments are shown. Bar, 1 μm. mGFP monomeric green fluorescent protein

Article Snippet: To generate mCherry-fused Cep63 expression constructs, a pCI-neo-mCherry vector was first created by inserting an Asc I– Pme I fragment containing an mCherry open reading frame (ORF) into the pCI-neo vector (Addgene).

Techniques: Sedimentation, Mutagenesis, Molecular Weight, Concentration Assay, Microscopy, In Vitro, Generated, Fluorescence, Software, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: African Swine Fever Virus Induces STAT1 and STAT2 Degradation to Counteract IFN-I Signaling

doi: 10.3389/fmicb.2021.722952

Figure Lengend Snippet: Arm/07/CBM/c2 and NH/P68 infection triggers proteasomal-dependent STAT2 degradation. STAT1 and STAT2 mRNA detection by qRT-PCR (A) . PAMs were mock infected or infected with attenuated NH/P68 or with virulent Arm/07/CBM/c2 ASFV strains (1 PFU/cell). Cells were collected at 3 hpi for a qRT-PCR analysis of STAT1 and STAT2 mRNA levels. As a control of infection, viral p32 mRNA (CP204L) was measured. STAT2 (B) and STAT1 levels (C) were analyzed in presence of the proteasome inhibitor MG132 or the lysosome/autophagosome inhibitor chloroquine (CQ) by Western blot analysis. PAMs were mock infected (M) or infected with Arm/07/CBM/c2 (A) or NH/P68 (N) ASFV strains (3 PFU/cell) and treated with MG132 (20 μM) or chloroquine (50 μM) at 12 hpi. At 16 hpi, cells were collected and lysed for the Western blot analysis. The Western blot bands corresponding to STAT1 (91k Da) and STAT2 (113 kDa) were quantified according with their actin levels and relativized with the corresponding mock control using ImageJ. Immunoprecipitation of STAT2 in mock-infected or infected COS-1 cells (D) . COS-1 cells were co-transfected either with pCI-His-hUbiquitin vector and pCAGGS empty vector or hSTAT2-FLAG vector. At 6 h post-transfection cells were infected with Arm/07/CBM/c2 strain (2 PFU/cell). Cells were collected at 16 hpi and lysed for STAT2 immunoprecipitation assay with A/G magnetic beads. Western blot labeling with anti-ubiquitin, anti-STAT2 and anti-actin antibodies is shown. Ubiquitinated STAT2 is indicated with an arrowhead in the figure.

Article Snippet: 8 × 10 6 COS-1 cells were co-transfected with 3 μg of pCAGGS empty vector (chicken β-actin promoter) or with human-STAT2-FLAG ( ) and pCI-His-hUbiquitin vector (Addgene, #31815).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Magnetic Beads, Labeling

Journal: bioRxiv

Article Title: CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

doi: 10.1101/2020.09.25.308734

Figure Lengend Snippet: ( A ) Domain architecture of the CARD8 protein. ( B ) HIV-1 protease cleaves the N-terminus of CARD8. HEK293T cells were transfected with plasmids encoding HA-CARD8 (100ng), together with either the pNL4-3 (1μg) or Pro-D25H (1μg). Cells were collected 24 hours after transfection. Anti-HA, anti-CARD8-N and anti-p24 antibodies were used sequentially on the same blot. ( C ) HIV-1 protease is necessary and sufficient to cleave CARD8. HEK293T cells were transfected with constructs encoding CARD8 (100ng) together with indicated viral plasmids (1μg). Cells were collected 24 hours after transfection. ( D ) RPV enhances HIV-1 protease-mediated cleavage of CARD8. HEK293T cells were transiently transfected with HA-CARD8 (100ng) and indicated viral plasmids (1μg). DMSO or RPV was added 24 hours post transfection. Cell lysates were collected 6 hours after RPV treatment. ( E and F ) HIV-1 protease triggers CASP1-dependent pro-IL-1β processing. HEK293T cells were co-transfected with plasmids encoding CASP1 (2ng), pro-IL-1β (200ng) and an HIV-1 plasmid (1μg). After 24 hours, cells were treated with indicated drugs for another 6 hours. ( G ) RPV induces HIV-1 protease-dependent cleavage of CARD8 in infected cells. HEK293T cells were infected with VSV-G-pseudotyped HIV-1 reporter virus. Infected cells were then transfected with HA-CARD8 (100ng). DMSO or RPV was added 24 hours post transfection. Cell lysates were collected 6 hours after RPV treatment. In B - F , cell lysates were evaluated by immunoblotting. CARD8-FL, full-length CARD8; CARD8-N, N terminal CARD8; free Nt, freed N terminus. Data are representative of three or more independent experiments.

Article Snippet: The CASP1 expression vector was from Addgene (#41552).

Techniques: Transfection, Construct, Plasmid Preparation, Infection, Virus, Western Blot

Journal: bioRxiv

Article Title: CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

doi: 10.1101/2020.09.25.308734

Figure Lengend Snippet: Expression of CARD8 and killing of unstimulated CD4 + T cells. (A) CARD8 expression in cell lines, as well as primary CD4 + T cells, monocytes derived macrophages and total PBMCs. Cell lysates were evaluated by immunoblotting using antibodies against CARD8 C-terminus or CASP1. CARD8-FL, full-length CARD8; CARD8-C, C terminal CARD8. (B) CARD8 transcription in primary CD4 + T cells. Central memory (T CM , CD45RO + CCR7 + ), effector memory (T EM , CD45RO + CCR7 - ) and naïve (T N , CD45RO - CCR7 + ) CD4 + T cells were purified from the CD3 + CD4 + CD8 - fraction of tonsil mononuclear cells by sorting. Blood CD4 + T cells were isolated from healthy donor PBMCs. U: unstimulated; A: activated. CARD8 transcription was measured by qPCR. (C) , Unstimulated CD4 + T cells were infected with HIV-1 reporter virus NL4-3-Pol. 4 days post infection, cells were treated with indicated ARVs for 24 hours before FACS analysis. P values were calculated using one-way ANOVA and Turkey multiple comparison tests. ****p < 0.0001. N=3. Error bars show mean values with SEM.

Article Snippet: The CASP1 expression vector was from Addgene (#41552).

Techniques: Expressing, Derivative Assay, Western Blot, Purification, Isolation, Infection, Virus, Comparison

Journal: bioRxiv

Article Title: CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

doi: 10.1101/2020.09.25.308734

Figure Lengend Snippet: ( A to E ) HIV-1 protease activation by NNRTIs induced rapid pyroptosis of infected monocytes derived macrophages (MDMs). MDMs were infected with HIV NL4-3/BaL . At day 4, Raltegravir (RAL) and T-20 were added to block new infection. Cells were then treated with RPV, EFV, LPV, or combinations for up to 24 hours. In A to C , GFP + cells were detected by flow cytometry. In D , images of infected MDMs were taken using a Cytation 5 Imaging Multi-Mode Reader (Biotek). In E , culture supernatant was collected for IL-1β ELISA. ( F and G ) Pyroptosis of HIV-1-infected MDMs is CASP1-dependent. MDMs were infected and treated as described above. In F , immunoblot analysis of pro-(p45) and cleaved CASP1 (p10 and p20) in infected MDMs after RPV treatment for 1 hour. In G , infected MDMs were pre-treated with VX-765 (100μM) or Z-VAD-FMK (100μM) for 3 hours and then treated with RPV for 4 hours before flow cytometry analysis. ( H and I ) HIV-1 protease mediated inflammasome activation is proteasome-dependent. MDMs were infected and treated as described above. Infected MDMs were pretreated with proteasome inhibitors MG132, Bort, or Me-Bs for 30 minutes and then treated with RPV for 4 hours. In H , GFP expression was analyzed by flow cytometry. In I , culture supernatant was collected for the detection of IL-1β by ELISA. ( J to I ) The CARD8 inflammasome is required for pyroptosis of HIV-1-infected macrophages. ( J ) knockout of CARD8, ASC, CASP1 or NLRP3 in THP-1 cells was confirmed by immunoblotting. Knockout or control THP-1 cells were infected with VSV-G pseudotyped HIV-1 reporter virus NL4-3-Pol. 3 days after infection, cells were pre-treated with LPS (100ng/ml) for 3 hours before RPV treatment. In K , GFP expression was analyzed by flow cytometry 24 hours post RPV treatment; Data were normalized to the control group. In L , culture supernatant was collected 48 hours post RPV treatment for IL-1β detection. In B, G, H and K , P values were calculated using one-way ANOVA and Turkey multiple comparison tests. In C, E, I and L P values were calculated using two-way ANOVA and Turkey multiple comparison tests. *p < 0.05, ****p < 0.0001. In each bar graph, n≥3. Error bars show mean values with SEM. Data are representative of three or more independent experiments.

Article Snippet: The CASP1 expression vector was from Addgene (#41552).

Techniques: Activation Assay, Infection, Derivative Assay, Blocking Assay, Flow Cytometry, Imaging, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Knock-Out, Control, Virus, Comparison

Journal: bioRxiv

Article Title: CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

doi: 10.1101/2020.09.25.308734

Figure Lengend Snippet: ( A ) Analysis of CARD8, Caspase 1 and Caspase 3 expression level in unstimulated (U) and activated (A) primary CD4 + T cells by immunoblotting. ( B and C ) HIV-1 protease activation by NNRTIs leads to killing of infected primary CD4 + T cells. Activated CD4 + T cells were used for viral infection. In B , cells were infected with HIV-1 reporter virus NL4-3-Pol. Various NNRTIs at indicated concentration were added 3 days post infection. In C , cells were infected with different HIV-1 reporter viruses for 3 days and treated with RPV. Cells were analyzed for GFP expression by flow cytometry 48 hours post NNRTI treatment. ( D to F ) HIV-1 protease activation by NNRTIs induced CASP1 activation in infected primary CD4 + T cells. Activated primary CD4 + T cells infected with the NL4-3-Pol. In D and E , infected cells were treated with EFV, RPV, LPV and combinations for 3 hours before FLICA660 Caspase 1 staining for active CASP1 detection. In F , infected cells were pre-treated with CASP1 inhibitor VX765 (50μM) and Pan-Caspase inhibitor Z-VAD-FMK (50μM) for 3 hours before adding RPV. GFP expression was measured by flow cytometry at indicated time. ( G to J ) The CARD8 inflammasome is required for pyroptosis of HIV-1-infected primary CD4 + T cells. In G , knockout of CARD8 in primary CD4 + T cells was confirmed by immunoblotting. H to J , CARD8, ASC or CASP1 knockout primary CD4 + T cells were co-stimulated and then infected with NL4-3-Pol. At day 3 post infection, cells were treated with RPV. In H , FLICA660 Caspase 1 was used for staining of active CASP1 at 3 hours post RPV treatment. In I and J , GFP expression was measure by flow cytometry 24 hours after RPV treatment. In C, E, I and J , P values were calculated using one-way ANOVA and Turkey multiple comparison tests. In F and H , P values were calculated using two-way ANOVA and Turkey multiple comparison tests. **p < 0.01, ***p < 0.001, and ****p < 0.0001. In each bar graph, n≥3. Error bars show mean values with SEM. Data are representative of five or more independent experiments.

Article Snippet: The CASP1 expression vector was from Addgene (#41552).

Techniques: Expressing, Western Blot, Activation Assay, Infection, Virus, Concentration Assay, Flow Cytometry, Staining, Knock-Out, Comparison

Journal: bioRxiv

Article Title: CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

doi: 10.1101/2020.09.25.308734

Figure Lengend Snippet: Proteasome inhibitors block HIV-1 protease-mediated inflammasome activation in HIV-1-infected primary CD4 + T cells. Activated primary CD4 + T cells were infected with VSV-G pseudotyped HIV-1 reporter virus NL4-3-Pol. At day 3 post infection, cells were pre-treated with MG132 (10μM), Bort (5μM), or Me-Bs (20μM) for 30 minutes and then treated with RPV for 6 hours. In A , FLICA660 Caspase 1 was used to measure active CASP1. In B , cell killing was determined by FACS. In A , P values were calculated using two-way ANOVA and Turkey multiple comparison tests. In B , P values were calculated using one-way ANOVA and Turkey multiple comparison tests. ****p < 0.0001. In each bar graph, n=3. Error bars show mean values with SEM.

Article Snippet: The CASP1 expression vector was from Addgene (#41552).

Techniques: Blocking Assay, Activation Assay, Infection, Virus, Comparison

Journal: bioRxiv

Article Title: CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

doi: 10.1101/2020.09.25.308734

Figure Lengend Snippet: Killing of HIV-1-infected primary CD4 + T cells is blocked by knockdown of CASP1. (A) Validation of CASP1 knockdown in primary CD4 + T cells by immunoblotting. (B) CASP1-KD primary CD4 + T cells were infected with VSV-G pseudotyped HIV-1 reporter virus NL4-3-Pol. 3 days after infection, cells were treated with RPV. GFP expression was analyzed by FACS 24 hours post RPV treatment. P values were calculated using two-way ANOVA and Turkey multiple comparison tests. ****p < 0.0001. N=3. Error bars show mean values with SEM.

Article Snippet: The CASP1 expression vector was from Addgene (#41552).

Techniques: Infection, Knockdown, Biomarker Discovery, Western Blot, Virus, Expressing, Comparison

Journal: Cell Reports

Article Title: Cancer-Specific Loss of p53 Leads to a Modulation of Myeloid and T Cell Responses

doi: 10.1016/j.celrep.2019.12.028

Figure Lengend Snippet:

Article Snippet: Retroviral introduction of ovalbumin sequence for membrane bound ovalbumin (mOVA) was cloned out of the pCl-neo-mOVA plasmid (Addgene No 25099) ( ) and into the MSCV-based MIGR1 (Addgene 27490) vector ( ).

Techniques: Control, Functional Assay, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software