Journal: Autophagy

Article Title: HIV-1 Nef counteracts autophagy restriction by enhancing the association between BECN1 and its inhibitor BCL2 in a PRKN-dependent manner

doi: 10.1080/15548627.2020.1725401

Figure Lengend Snippet: HIV uses Nef to block autophagy. (A) Left panel: HEK293T cells were transfected with the full-length proviral DNA of HIV-1 NL4-3, different mutants of this molecular clone (Δ vpu , Δ env , Δ nef and Δ vpr ), or an empty retroviral vector. As controls for autophagy, cells were treated with 4 μM of rapamycin or mock (DMSO) for 4 h. 48 h post-transfection, cells were lysed and analyzed by western blot for SQSTM1, Gag (p55 and p24), LC3 and ACTB/β-actin. Right Panel: Densitometric analyses were performed to determine the ratio of LC3-II over LC3-I relative to the empty vector control. Data represent the mean and standard error of the mean (SEM) from 4 independent biological replicates. (B) Jurkat CD4 + T cells, (C) THP-1-derived macrophages and (D) primary CD4 + T cells were infected with 100 ng of p24 equivalents of HIV-1 NL4-3 or HIV-1 NL4-3 Δ nef . In the case of macrophages, infections were performed with VSV-G pseudotyped HIV-1 NL4-3 Δ env or HIV-1 NL4-3 Δ env Δ nef . Cell lysates were collected at the indicated time intervals and analyzed by western blot for SQSTM1, Gag (p55 and p24), Nef, LC3, and ACTB (left panels). The right panels correspond to the mean and SEM of 3 independent experiments showing the ratios of LC3-II:I over time relative to the first data point, which were calculated by densitometric analyses. *: p ≤ 0.05; **: p ≤ 0.01. Red arrow in panel A indicates decreased Gag and SQSTM1 levels as well as rapid LC3-I-to-LC3-II transition. Red arrows in panels C and D indicate the accumulation of LC3-I

Article Snippet: In addition, an HA-tagged version of NL4-3 Nef was obtained from Addgene: pCI-NL4-3 Nef-HA-WT (24162, Dr. Warner Greene’s lab).

Techniques: Blocking Assay, Transfection, Plasmid Preparation, Western Blot, Derivative Assay, Infection

Journal: Autophagy

Article Title: HIV-1 Nef counteracts autophagy restriction by enhancing the association between BECN1 and its inhibitor BCL2 in a PRKN-dependent manner

doi: 10.1080/15548627.2020.1725401

Figure Lengend Snippet: Autophagy limits virion production in nef -defective HIV. (A) Top panel: HEK293T cells were transfected with the full-length proviral DNA of HIV-1 NL4-3 or NL4-3 Δ nef and treated with the indicated concentrations of rapamycin for 12 h. The percentage of maximal virus production was then measured by the accumulation of HIV p24 in the culture supernatant relative to the no-rapamycin treatment for each virus. Bottom panel: Cells were also analyzed by western blot for Gag p55, Nef, ACTB, and LC3. (B) Top panel: HEK293T cells were transfected with the proviral DNA of HIV-1 NL4-3 Δ nef and trans-complemented with either NL4-3 nef or an empty vector. Similar to panel A, cells were treated with increasing concentrations of rapamycin for 12 h, and the percentage of maximal virus production relative to the no-rapamycin treatment was measured as explained above. Bottom panel: Cell lysates were analyzed as in panel A. (C) Top panel: HEK293T cells were transfected with the proviral DNA of HIV-1 NL4-3 Δ nef and stimulated with increasing concentrations of rapamycin for 12 h. Next, cells were either treated with 3-MA (3 mM) or water 4 h before measuring the percentage of particle release. Bottom panel: Cells were also analyzed by western blot as in panel A. (D) Left panel: HEK293T cells were transfected with either siRNA for BECN1 or a control siRNA. 24 h later, the cells were transfected again with the proviral DNA of HIV-1 NL4-3 Δ nef and treated with rapamycin for 12 h. Next, the percentage of maximal virus production was measured as indicated above. Right panel: Cell lysates were also analyzed for BECN1, Gag p55, Nef, ACTB, and LC3. (E) Left panel: Jurkat CD4 + T cells were infected with 100 ng of p24 equivalents of HIV-1 NL4-3 or HIV-1 NL4-3 Δ nef . 24 h later, the cell medium was replaced and supplemented with different concentrations of rapamycin. 12 h later, the percentage of maximal virus production was measured as detailed above. Right panel: cell lysates were analyzed as in panel A. In each case, the percentage of maximal virus production is indicated as the mean and SEM from 4 independent biological replicates. *: p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001. Numbers underneath the blots indicate the ratio of LC3-II:I relative to the no-rapamycin treatment

Article Snippet: In addition, an HA-tagged version of NL4-3 Nef was obtained from Addgene: pCI-NL4-3 Nef-HA-WT (24162, Dr. Warner Greene’s lab).

Techniques: Transfection, Western Blot, Plasmid Preparation, Infection

Journal: Autophagy

Article Title: HIV-1 Nef counteracts autophagy restriction by enhancing the association between BECN1 and its inhibitor BCL2 in a PRKN-dependent manner

doi: 10.1080/15548627.2020.1725401

Figure Lengend Snippet: Nef impairs the lipidation of LC3, affecting autophagosome formation. (A, B) HEK293T cells were transfected with NL4-3 nef or an empty vector. 48 h later, cells were (A) exposed to the indicated concentrations of rapamycin, or (B) starved for the selected times. Left panels: Cells were analyzed by western blot for SQSTM1, Nef, LC3, and ACTB. Right panels: Data represent the mean and SEM of the ratios of LC3-II over LC3-I relative to the empty vector control (without rapamycin or at 1 d post-starvation, respectively) from 3 independent biological replicates. (C) HEK293T cells were transfected with NL4-3 nef or an empty vector. Next, the mRNA levels of LC3B and ATG16L1 were assessed at the selected time points by RT-qPCR and expressed as fold change after normalization to GAPDH and GFP . Data represent the mean and SEM from 3 independent biological replicates. The dashed line represents cutoff for biologically relevant differences. (D) HEK293T cells were transfected with HIV-1 NL4-3 or NL4-3 Δ nef proviral constructs. Cells treated with rapamycin or transfected with an empty retroviral vector were included as controls. 24 h later, cells were treated with DMSO or chloroquine (60 μM) for 12 h. Next, cells were analyzed by western blot for Gag (p55 and p24), SQSTM1, LC3, and ACTB. The SQSTM1:ACTB and LC3-II:I ratios relative to the vector control are provided underneath the blots. (E) HEK293T cells were co-transfected with EGFP -LC3B and an empty vector or NL4-3 nef -HA. As controls, cells were also treated with rapamycin (4 μM) and 3-MA (3 mM). 48 h post-transfection, cells were analyzed by flow cytometry for autophagosome-associated EGFP-LC3B. Data correspond to the mean and SEM of the percentage of EGFP + cells from 3 independent experiments. (F, G) HEK293T cells were co-transfected with EGFP -LC3B and either an empty vector, GST -HA (F) or NL4-3 nef -HA (G). Cells were exposed for 4 h to rapamycin (4 μM) in the presence and absence of 3-MA (3 mM) prior to microscopy visualization. Next, cells were stained for GFP (green), HA (DyLight-550; red) and the nuclei (DAPI; blue). (H) Data correspond to the mean and SEM of EGFP-LC3B puncta present in 20 randomly selected cells for each experimental condition. *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001; n.s. not significant. Red arrows: accumulation of LC3-I. White scale bar: 10 μm. Cells surrounded by white borders are HA +

Article Snippet: In addition, an HA-tagged version of NL4-3 Nef was obtained from Addgene: pCI-NL4-3 Nef-HA-WT (24162, Dr. Warner Greene’s lab).

Techniques: Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Construct, Flow Cytometry, Microscopy, Staining

Journal: Autophagy

Article Title: HIV-1 Nef counteracts autophagy restriction by enhancing the association between BECN1 and its inhibitor BCL2 in a PRKN-dependent manner

doi: 10.1080/15548627.2020.1725401

Figure Lengend Snippet: Nef enhances the association between BECN1 and BCL2 to arrest autophagy initiation. (A) HEK293T cells were transfected with NL4-3 nef or an empty vector. 48 h later, cells were exposed to rapamycin (4 μM) for 4 h. The cell lysates were analyzed by western blot to assess the expression levels of the proteins ULK1, ATG12–ATG5, ATG7, ATG3, BECN1, ATG16L1, BCL2, Nef, and ACTB. (B) Schematic diagram of the regulatory effect of BCL2 on the BECN1-dependent initiation of autophagy. (C) HEK293T cells were transfected with NL4-3 nef or an empty vector. 48 h later, BCL2 was immunoprecipitated from the cell lysates to assess its levels of ubiquitination and SUMOylation. The cell lysates were also analyzed by western blot to evaluate the levels of Nef, p-BCL2, BCL2, and ACTB. (D) HEK293T cells were co-transfected with BECN1 -Flag and NL4-3 nef or an empty vector. 48 h later, the cell lysates were subjected to immunoprecipitation for BECN1. The pulldown fraction was then examined for the presence of BECN1 and BCL2. The cell lysates were also analyzed by western blot to assess the cellular levels of BECN1, Nef, BCL2, and ACTB. Graph: Data represent the mean and SEM of 6 independent biological replicates to calculate the relative binding between BECN1 and BCL2 obtained by densitometry analyses. (E) Left panel: HEK293T cells were transfected with the full-length proviral DNA of HIV-1 NL4-3 or NL4-3 Δ nef . 24 h later, the cell medium was replaced, and cells were treated for 12 h with either the indicated concentrations of rapamycin and DMSO or a combination of rapamycin and GX15-070 at its IC 50 (3 μM), an inhibitor of BCL2. The percentage of maximal virus production relative to the no-rapamycin treatment was then measured for each virus and condition by the accumulation of HIV p24 in the culture supernatant. Data represent the mean and SEM of 3 independent biological replicates. Right panel: Cell lysates for samples transfected with the HIV-1 NL4-3 proviral construct were also analyzed for Gag p55, Nef, ACTB, and LC3. V: vector. Rel: relative. Ab ctr: cell lysates were incubated with the antibody used for the pulldown assays to exclude bands corresponding to the heavy and light chain (HC and LC) of the antibodies from the target proteins. Asterisk: differential migration pattern of BCL2. Pound sign: ubiquitinated BCL2. *: p ≤ 0.05; *** p ≤ 0.001; n.s. not significant

Article Snippet: In addition, an HA-tagged version of NL4-3 Nef was obtained from Addgene: pCI-NL4-3 Nef-HA-WT (24162, Dr. Warner Greene’s lab).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Immunoprecipitation, Binding Assay, Construct, Incubation, Migration

Journal: Autophagy

Article Title: HIV-1 Nef counteracts autophagy restriction by enhancing the association between BECN1 and its inhibitor BCL2 in a PRKN-dependent manner

doi: 10.1080/15548627.2020.1725401

Figure Lengend Snippet: The Nef-enhanced association between BECN1 and BCL2 is PRKN-dependent. (A) HEK293T and HeLa cells were co-transfected with BECN1 -Flag and either NL4-3 nef or an empty vector. In addition, HeLa cells were trans-complemented with PRKN -MYC. 48 h post-transfection, the cell lysates were immunoprecipitated for endogenous BCL2, and the pulldown fraction was examined for the presence of BECN1 and BCL2. The cell lysates were also subjected to western blot to assess the cellular levels of the proteins PRKN, BECN1, ACTB, Nef, BCL2, and LC3. The ratios of LC3-II over LC3-I relative to the empty vector are provided underneath the blots. Graph: Densitometric analyses were used to determine the relative levels of interaction between BECN1 and BCL2. Data represent the mean and SEM of 4 independent biological replicates. (B) HEK293T cells were depleted of PRKN by shRNA and subsequently co-transfected with BECN1 -Flag and NL4-3 nef or an empty vector. Next, the cell lysates were analyzed for the BECN1-BCL2 interaction by immunoprecipitation and western blot. Similar to panel A, the relative PRKN expression levels normalized to ACTB as well as the ratios of LC3-II over LC3-I are provided underneath the blots. Graph: Densitometric analyses were used to determine the relative levels of interaction between BECN1 and BCL2. Data represent the mean and SEM of 4 independent biological replicates. (C) Left panel: HEK293T cells were depleted of PRKN by shRNA followed by transfection with the full-length proviral DNA of HIV-1 NL4-3. Cells were treated with the indicated concentrations (0–4 μM) of rapamycin for 12 h. The percentage of maximal virus production relative to the no-rapamycin treatment was then measured by the accumulation of HIV p24 in the culture supernatant. Data represent the mean and SEM of 3 independent biological replicates. Right panel: Cell lysates were also analyzed for Gag p55, PRKN, ACTB, and LC3. The ratios of LC3-II over LC3-I relative to the respective no-rapamycin treatments are provided underneath. (D) HEK293T cells were co-transfected with NL4-3 nef -HA and PRKN -MYC or an empty vector. Human CD4 was included as a positive control for Nef binding. 48 h later, cells were lysed and immunoprecipitated for HA. The affinity-isolated fraction was examined for the presence of PRKN-MYC, CD4 and Nef-HA. The cell lysates were also analyzed for the expression of PRKN-MYC, CD4, ACTB, and Nef-HA. V: vector. Rel: relative. Ab ctr: antibody control. LC: light chain. *: p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; n.s. not significant

Article Snippet: In addition, an HA-tagged version of NL4-3 Nef was obtained from Addgene: pCI-NL4-3 Nef-HA-WT (24162, Dr. Warner Greene’s lab).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, shRNA, Expressing, Positive Control, Binding Assay, Isolation

Journal: Autophagy

Article Title: HIV-1 Nef counteracts autophagy restriction by enhancing the association between BECN1 and its inhibitor BCL2 in a PRKN-dependent manner

doi: 10.1080/15548627.2020.1725401

Figure Lengend Snippet: Nef promotes BECN1-BCL2 binding in the ER. (A) HEK293T cells were co-transfected with BECN1 -Flag and Tot-GFP- BCL2 (total BCL2), ER-GFP- BCL2 (Endoplasmic Reticulum-associated BCL2) or Mit-GFP- BCL2 (Mitochondrial BCL2). In addition, cells were transfected with NL4-3 nef or an empty vector. 48 h later, the cell lysates were immunoprecipitated for BCL2. The pulldown fraction was examined for the presence of BECN1, GFP-BCL2 and Ub-BCL2. The cell lysates were also analyzed by western blot to assess the cellular levels of BECN1, p-BCL2, ACTB, GFP-BCL2, Nef, and LC3. Graphs: Densitometric analyses were used to determine the relative interaction between BECN1 and BCL2 and the relative levels of ubiquitinated BCL2. Data represent the mean and SEM of 5 independent biological replicates. (B) HEK293T cells were co-transfected with BECN1 -Flag, ER-GFP- BCL2 and either GST -HA or NL4-3 nef -HA. 48 h later, cells were stained for HA (DyLight-550; red), ER-GFP-BCL2 (green), BECN1-Flag (Alexa Fluor 633; magenta) and nuclei (DAPI; blue). Merge of HA, ER-GFP-BCL2 and BECN1-Flag is provided. The Pearson’s correlation coefficient for the colocalization of BECN1 and BCL2 is provided in a graph from 15 independent events for each experimental condition. (C) Proposed mechanism by which Nef arrests autophagy initiation. Rel: relative. Ab ctr: antibody control. *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001. White scale bar: 10 μm

Article Snippet: In addition, an HA-tagged version of NL4-3 Nef was obtained from Addgene: pCI-NL4-3 Nef-HA-WT (24162, Dr. Warner Greene’s lab).

Techniques: Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Staining

Journal: Autophagy

Article Title: HIV-1 Nef counteracts autophagy restriction by enhancing the association between BECN1 and its inhibitor BCL2 in a PRKN-dependent manner

doi: 10.1080/15548627.2020.1725401

Figure Lengend Snippet: The Nef-enhanced sequestration of BECN1 by BCL2 also affects autophagy maturation. (A) HEK293T cells were co-transfected with BECN1 -Flag and an empty vector, NL4-3 nef and/or ER-GFP- BCL2 . 48 h later, cells were immunoprecipitated for UVRAG and the pulldown fraction was analyzed for the presence of UVRAG and BECN1-Flag. The cell lysates were also examined for UVRAG, BECN1, ACTB, ER-GFP-BCL2, Nef, and LC3. Graph: Densitometric analyses were used to determine the relative interaction between BECN1 and UVRAG. Data represent the mean and SEM of 3 independent biological replicates. (B-D) Nef A36-A39 was examined for its ability to block autophagosome formation by flow cytometry through the accumulation of autophagosome-associated EGFP-LC3B (B), its ability to prevent the lipidation of LC3 by western blot (C), and its ability to enhance the BECN1-BCL2 interaction by co-immunoprecipitation (D). Assays were carried out in HEK293T cells, as detailed previously. pCGCG-EGFP or pcDNA5 were used as vector controls, NL4-3 nef or NL4-3 nef -HA were used to express HIV-1 Nef. Data correspond to the mean and SEM of 4 independent experiments. Rel: relative. Ab ctr: antibody control. LC: light chain. *: p ≤ 0.05; **: p ≤ 0.01

Article Snippet: In addition, an HA-tagged version of NL4-3 Nef was obtained from Addgene: pCI-NL4-3 Nef-HA-WT (24162, Dr. Warner Greene’s lab).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Blocking Assay, Flow Cytometry, Western Blot

Journal: Autophagy

Article Title: HIV-1 Nef counteracts autophagy restriction by enhancing the association between BECN1 and its inhibitor BCL2 in a PRKN-dependent manner

doi: 10.1080/15548627.2020.1725401

Figure Lengend Snippet: Nef’s ability to inhibit autophagy is mainly observed in HIV-1/SIV cpz . (A) Representative data of the analyses of the anti-autophagic properties of lentiviral Nef proteins. HEK293T cells were transfected with NL4-3 nef , an empty vector and SIV mac 239 nef . 48 h later, cells were exposed for 4 h to increasing concentrations of rapamycin (0–4 μM). Next, cells were analyzed by western blot for the levels of SQSTM1, Nef, LC3, and ACTB. Densitometric analyses were performed to determine the levels of SQSTM1:ACTB and the ratio of LC3-II over LC3-I relative to the empty vector control under no rapamycin treatment. (B, C) Data correspond to the mean and SEM of the relative LC3-II:I ratios from 3 independent biological replicates of different lentiviral nef alleles of the HIV-1/SIV cpz lineage (B) and HIV-2/SIV smm lineage (C). (D) The amino acid sequence of Nef from the different lentiviral species was compared and clustered in a cladogram where the relative evolutionary distance between each variant is shown. Color-coded squares are used to identify species in which Nef is able to inhibit autophagy (solid line) or exhibit partial activity (dashed line). *: p ≤ 0.05; **: p ≤ 0.01; n.s. not significant. Red arrows indicate Nef alleles active against autophagy

Article Snippet: In addition, an HA-tagged version of NL4-3 Nef was obtained from Addgene: pCI-NL4-3 Nef-HA-WT (24162, Dr. Warner Greene’s lab).

Techniques: Transfection, Plasmid Preparation, Western Blot, Sequencing, Variant Assay, Activity Assay

Journal: European journal of immunology

Article Title: p300, but not PCAF, collaborates with IRF-1 in stimulating TRIM22 expression independently of its histone acetyltransferase activity.

doi: 10.1002/eji.201343308

Figure Lengend Snippet: Figure 1. IFN-γ enhances p300 and PCAF association with 5′eISRE-harboring TRIM22 promoter sequence. (A) Nuclear extracts from IFN-γ-treated or -untreated HepG2 cells were incubated with biotin-labeled oligonucleotide probe harboring 5′eISRE or 5′eISRE-mut and streptavidin-agarose beads for 1 h. The complex was obtained after centrifugation, and proteins in the complex as well as in the nuclear extracts were examined by Western blotting. (B) Formaldehyde crosslinked chromatin was prepared from HepG2 cells untreated or treated with IFN-γ for 1, 3, or 6 h. ChIP assays were performed with the indicated antibodies. Immunoprecipitated chromatin was subjected to real-time PCR analysis using primers spanning the TRIM22 promoter region containing 5′eISRE. Data are shown as fold change to control (IFN-γ untreated) after normalized to input and rabbit normal IgG. Data are shown as mean +SD of triplicates, and are representative of three independent experiments performed. *p < 0.05 versus control (Student’s t-test).

Article Snippet: Plasmid constucts and transfection Plasmid constructs p300 (Addgene; plasmid 23252 deposited by Warner Greene), PCAF (Addgene; plasmid 8941 deposited by Yoshihiro Nakatani), p300 HAT-(Addgene; plasmid 23252 deposited by Warner Greene), and TRIM22 reporter luciferase plasmid pLUC160 [24] were used.

Techniques: Sequencing, Incubation, Labeling, Centrifugation, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Control

Journal: European journal of immunology

Article Title: p300, but not PCAF, collaborates with IRF-1 in stimulating TRIM22 expression independently of its histone acetyltransferase activity.

doi: 10.1002/eji.201343308

Figure Lengend Snippet: Figure 2. p300, not PCAF, augments IFN-γ- induced TRIM22 expression. (A) The expres- sion of p300 or PCAF in HepG2 cells after transfected with different dose of plas- mids expressing p300 or PCAF was deter- mined by Western blotting. (B) HepG2 cells were transfected with different dose of plas- mids expressing p300 or PCAF. Twenty-four hours posttransfection, cells were treated or untreated with 1000 U IFN-γ for another 24 h, TRIM22 protein expression level was deter- mined by Western blotting. (A, B) Actin was used as a loading control. (C) Cells were treated as in (B), and TRIM22 mRNA level was determined by quantitative RT-PCR. Data are shown as mean +SD of triplicates, and are representative of three independent exper- iments performed. (D) HepG2 cells were cotransfected with TRIM22 reporter plas- mids pLUC-160 and increasing dose of p300 or PCAF expression plasmids. Twenty-four hours posttransfection, cells were treated with or without IFN-γ for another 24 h, fol- lowed by the detection of luciferase activity in the cell lysate. The activity of unstimu- lated pLuc 160 was set to 1. Data are shown as mean +SD of triplicates, and are represen- tative of three independent experiments per- formed. (E) HepG2 cells were transfected with pLuc160 together with plasmids expressing p300, PCAF or p300 plus PCAF. Twenty-four hours posttransfection, HepG2 cells were treated with or without IFN-γ for another 24 h followed by the detection of luciferase activ- ity in the cell lysate. Data are shown as mean +SD of triplicates, and are representative of three independent experiments performed. NS = not statistically significant. *p < 0.05 (Student’s t-test).

Article Snippet: Plasmid constucts and transfection Plasmid constructs p300 (Addgene; plasmid 23252 deposited by Warner Greene), PCAF (Addgene; plasmid 8941 deposited by Yoshihiro Nakatani), p300 HAT-(Addgene; plasmid 23252 deposited by Warner Greene), and TRIM22 reporter luciferase plasmid pLUC160 [24] were used.

Techniques: Expressing, Transfection, Western Blot, Control, Quantitative RT-PCR, Luciferase, Activity Assay

Journal: European journal of immunology

Article Title: p300, but not PCAF, collaborates with IRF-1 in stimulating TRIM22 expression independently of its histone acetyltransferase activity.

doi: 10.1002/eji.201343308

Figure Lengend Snippet: Figure 3. The effect of p300 or PCAF on IRF-1-mediated TRIM22 expression. (A) Nuclear extracts from IFN-γ-treated or -untreated HepG2 cells were immunoprecipitated with anti-IRF-1 and probed with anti-p300 or anti-PCAF. (B) HepG2 cells were transfected with IRF-1 alone or together with increasing doses of p300 or PCAF expression plasmids for 24 h, and the protein expression levels of TRIM22, IRF-1, p300, PCAF, and actin were determined by Western blotting. (A, B) Actin was used as a loading control. (C) HepG2 cells were treated as in (B). TRIM22 mRNA expression level was determined by quantitative RT-PCR. Data are shown as mean +SD of triplicates, and are representative of three independent experiments performed. (D) pLuc160 and IRF-1 expression plasmids were transfected into HepG2 cells together with increasing doses of p300 or PCAF expression plasmids. Twenty-four hours posttransfection, cells were harvested and luciferase activity in the cell lysate was measured, and the activity of unstimulated pLuc 160 was set to 1. Data are shown as mean +SD of triplicates, and are representative of three independent experiments performed. *p < 0.05 (Student’s t-test).

Article Snippet: Plasmid constucts and transfection Plasmid constructs p300 (Addgene; plasmid 23252 deposited by Warner Greene), PCAF (Addgene; plasmid 8941 deposited by Yoshihiro Nakatani), p300 HAT-(Addgene; plasmid 23252 deposited by Warner Greene), and TRIM22 reporter luciferase plasmid pLUC160 [24] were used.

Techniques: Expressing, Immunoprecipitation, Transfection, Western Blot, Control, Quantitative RT-PCR, Luciferase, Activity Assay

Journal: European journal of immunology

Article Title: p300, but not PCAF, collaborates with IRF-1 in stimulating TRIM22 expression independently of its histone acetyltransferase activity.

doi: 10.1002/eji.201343308

Figure Lengend Snippet: Figure 4. The effect of siRNA knockdown of p300 or PCAF on IFN-γ or IRF-1-mediated TRIM22 expression. (A) HepG2 cells were transfected with control, p300, and PCAF siRNA, respectively. After 72 h, the protein expression level of p300, PCAF, and actin was detected by Western blotting. (B) At 72 h after control, p300 or PCAF siRNA transfection, HepG2 cells were treated with IFN-γ for 24 h. TRIM22 protein expression was determined by Western blotting. (C) Cells were treated as in B. TRIM22 mRNA expression level was determined by quantitative RT-PCR. Data are shown as mean +SD of triplicates, and are representative of three independent experiments performed. *p < 0.05 (Student’s t-test). (D) At 48 h after control, p300 or PCAF siRNA transfection, cells were further transfected with pLUC160. At 72 h after siRNA transfection, cells were treated with IFN-γ for 24 h. The luciferase activity in the cell lysate was then measured, and the luciferase activity of control siRNA-transfected and IFN-γ-untreated cells was set to 1. Data are shown as mean +SD of triplicates, and are representative of three independent experiments performed. *p < 0.05 (Student’s t-test). (E) At 48 h after control, p300 or PCAF siRNA transfection, HepG2 cells were transfected with empty vector or IRF-1 expression plasmids. Twenty-four hours posttransfection, TRIM22 protein expression was determined by Western blotting. (A, B, E) Actin was used as a loading control. (F) Cells were treated as in E. TRIM22 mRNA expression level was determined by quantitative RT-PCR. Data are shown as mean +SD of triplicates, and are representative of three independent experiments performed. *p < 0.05 (Student’s t-test). (G) At 48 h after control, p300 or PCAF siRNA transfection, cells were further cotransfected with pLUC160 and either pIRF-1 or pCDNA. At 72 h after siRNA transfection, the luciferase activity in the cell lysate was then measured, and the luciferase activity of control siRNA-transfected and IRF-1-untreated cells was set to 1. Data are shown as mean +SD of triplicates, and are representative of three independent experiments performed. *p < 0.05 (Student’s t-test).

Article Snippet: Plasmid constucts and transfection Plasmid constructs p300 (Addgene; plasmid 23252 deposited by Warner Greene), PCAF (Addgene; plasmid 8941 deposited by Yoshihiro Nakatani), p300 HAT-(Addgene; plasmid 23252 deposited by Warner Greene), and TRIM22 reporter luciferase plasmid pLUC160 [24] were used.

Techniques: Knockdown, Expressing, Transfection, Control, Western Blot, Quantitative RT-PCR, Luciferase, Activity Assay, Plasmid Preparation

Journal: European journal of immunology

Article Title: p300, but not PCAF, collaborates with IRF-1 in stimulating TRIM22 expression independently of its histone acetyltransferase activity.

doi: 10.1002/eji.201343308

Figure Lengend Snippet: Figure 5. The HAT activity of p300 is not required for its effect on IFN-γ- and IRF-1-mediated TRIM22 expression. (A) HepG2 cells were transfected with empty vector or p300 expression plasmid. Twenty-four hours posttransfection, cells were treated with IFN-γ for 6 h. The cell lysate was immunoprecipitated using anti-acetyllysine (AcK) or anti-IRF-1 antibody, and probed by anti-IRF-1, anti-Ack, and anti-histone 3 (H3), respectively. (B) HepG2 cells were transfected with empty or p300 expressions plasmids. Twenty-four hours posttransfection, cells were treated with IFN-γ for the indicated time points. The recruitment of IRF-1 to TRIM22 promoter was determined by ChIP analysis as in Figure 1B. The control value was obtained from the empty vector-transfected, IFN-γ-untreated group, and is set to 1. Data are shown as mean +SD of triplicates, and are representative of three independent experiments performed. (C) HepG2 cells were transfected with plasmids expressing p300-WT or p300-HAT-. Twenty-four hours posttransfection, the protein expression levels of p300, TRIM22, and actin were determined by Western blotting; histone protein was isolated and subjected to Western blotting using anti-H3 and anti-AC-H3. (A, C) Actin was used as a loading control. (D) pLuc 160 was transfected into HepG2 cells together with empty vector, p300-WT or p300-HAT-. Twenty-four hours posttransfection, cells were treated with or without IFN-γ for another 24 h. Luciferase activity in the cell lysate was measured. Data are shown as mean +SD of triplicates, and are representative of three independent experiments performed. NS = not statistically significant. *p < 0.05 (Student’s t-test). (E) pLuc 160 and pIRF-1 were transfected into HepG2 cells together with empty vector, p300-WT or p300-HAT–. Twenty-four hours posttransfection, luciferase activity in the cell lysate was measured, and the luciferase activity of empty vector-transfected cells was set to 1. Data are shown as mean +SD of triplicates, and are representative of three independent experiments performed. NS = not statistically significant. *p < 0.05 (Student’s t-test).

Article Snippet: Plasmid constucts and transfection Plasmid constructs p300 (Addgene; plasmid 23252 deposited by Warner Greene), PCAF (Addgene; plasmid 8941 deposited by Yoshihiro Nakatani), p300 HAT-(Addgene; plasmid 23252 deposited by Warner Greene), and TRIM22 reporter luciferase plasmid pLUC160 [24] were used.

Techniques: Activity Assay, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Control, Western Blot, Isolation, Luciferase

Journal: European journal of immunology

Article Title: p300, but not PCAF, collaborates with IRF-1 in stimulating TRIM22 expression independently of its histone acetyltransferase activity.

doi: 10.1002/eji.201343308

Figure Lengend Snippet: Figure 6. p300 is required for the recruitment of pol II to TRIM22 promoter region. (A) HepG2 cells were transfected with empty vec- tor, p300-WT, or p300-HAT plasmid. Twenty-four hours posttrans- fection, cells were treated with IFN-γ for 6 h. The recruitment of pol II to TRIM22 promoter was determined by ChIP analysis using the rabbit anti-pol II. Control value is obtained from empty vector- transfected, IFN-γ-untreated group, and was set to 1. Data are shown as mean +SD of triplicates, and are representative of three inde- pendent experiments performed. NS = not statistically significant. *p < 0.05 (Student’s t-test). (B) At 72 h after control or p300 siRNA transfection, cells were treated with or without IFN-γ for 6 h. The recruitment of pol II to TRIM22 promoter was determined by ChIP analysis. The value obtained from control siRNA-transfected and IFN-γ-untreated cells was set to 1. Data are shown as mean +SD of trip- licates, and are representative of three independent experiments per- formed. *p < 0.05 (Student’s t-test). (C) At 48 h after control or p300 siRNA transfection, cells were transfected with empty vector or IRF-1 expres- sion plasmids. Twenty-four hours posttransfection, the recruitment of pol II to TRIM22 promoter was determined by ChIP analysis. The value obtained from control siRNA-transfected and IRF-1-untreated cells was set to 1. Data are shown as mean +SD of triplicates, and are representa- tive of three independent experiments performed. *p < 0.05 (Student’s t-test).

Article Snippet: Plasmid constucts and transfection Plasmid constructs p300 (Addgene; plasmid 23252 deposited by Warner Greene), PCAF (Addgene; plasmid 8941 deposited by Yoshihiro Nakatani), p300 HAT-(Addgene; plasmid 23252 deposited by Warner Greene), and TRIM22 reporter luciferase plasmid pLUC160 [24] were used.

Techniques: Transfection, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: Novel mechanism of inflammatory activation by Ebola virus matrix protein linked to the ebolavirus virulence

doi: 10.1101/2024.10.06.616882

Figure Lengend Snippet: a Diagram of the canonical and non-canonical NF-κB pathways. The canonical pathway involves the IκB kinase (IKK) complex, which comprises IKKα, IKKβ, and IKKγ. Activation of the IKK complex leads to the phosphorylation/ubiquitination and subsequent proteasomal degradation of Iκβα, allowing either p65:p50 or cRel:p50 NF-κB heterodimers to enter the nucleus. The non-canonical pathway relies on the cooperation of NF-κB-inducing kinase (NIK) and its downstream kinase IKKα. Activation of NIK and IKKα triggers the proteasomal processing of p100 into p52, leading to the nuclear translocation of RelB:p52 NF-κB heterodimers. b Western blotting for NF-κB subunits in cytoplasmic and nuclear fractions isolated from 293 cells expressing EBOV VP40 or TRAF6 at 24 hpt. c NF-κB-responsive luciferase reporter activity in 293 cells expressing EBOV VP40 at 48 hpt, following siRNA-mediated knockdown of p65 or GAPDH. d Western blotting for IKK complex and Iκβα in 293 cells expressing EBOV VP40, EBOV GP, or TRAF6 at 24 hpt. e NF-κB-responsive luciferase reporter activity in 293 cells expressing 0.25 μg of IKK wild-type or IKK mutant (K44M) together with either 0.25 μg of EBOV VP40 or TRAF6 at 48 hpt. f NF-κB-responsive luciferase reporter activity in Iκβα-knockout 293 cells expressing 0.25 μg of Iκβα wild-type or Iκβα mutant (SS32/36AA) together with 0.25 μg of EBOV VP40 at 48 hpt. g NF-κB-responsive luciferase reporter activity in 293 cells expressing 0.01, 0.1, or 0.5 μg of Iκβα together with 0.5 μg of EBOV VP40 at 48 hpt. h Western blotting for p65 in cytoplasmic and nuclear fractions isolated from 293 cells expressing EBOV VP40 with or without Iκβα, TRAF6, or Iκβα at 24 hpt. Control: Empty vector transfected. For c and e - g , data are shown with mean ± SD (n = 3 independent biological replicates). ns > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001; ordinary one-way ANOVA.

Article Snippet: The following open reading frames were cloned into an expression vector under the control of the phosphoglycerate kinase promoter: IKK-2 and its mutant IKK-2 K44M (Addgene plasmids #11103 and #11104, gifts from Anjana Rao ); 3xHA-Iκβα and its mutants 3xHA-Iκβα-SS32/36AA (Addgene plasmids #21985 and #24143, gifts from Warner Greene ); TNFR1-YFP (Addgene plasmid #111209, gift from Johannes A. Schmid).

Techniques: Activation Assay, Translocation Assay, Western Blot, Isolation, Expressing, Luciferase, Activity Assay, Knockdown, Mutagenesis, Knock-Out, Control, Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: Novel mechanism of inflammatory activation by Ebola virus matrix protein linked to the ebolavirus virulence

doi: 10.1101/2024.10.06.616882

Figure Lengend Snippet: a Western blotting for molecules involved in the non-canonical NF-κB pathway. 293 cells were transfected with plasmid EBOV VP40, EBOV GP, TRAF6, or NIK and harvested at 24 hpt or treated with 15 ng/ml of TNFα for 1 hour and then harvested. b Western blotting for phosphorylated-Iκβα or Iκβα in 293 cells expressing EBOV VP40 or TRAF6. c Quantification of Iκβα mRNA in EBOV VP40-expressing 293 cells. Extracted RNA was reverse transcribed using OligodT primer, and cDNA was used for qRT-PCR with iTaq Universal Probes Supermix (BioRad), Hs00355671_g1 NFKBIA (Thermo Scientific) and Hs00355671_g1 NFKBIA (Thermo Scientific). GAPDH was used as a reference gene (the primer/TaqMan probe sequences are shown in Supplementary Table 3) . Delta-delta C t values were used to determine their relative expression as fold changes. Details on RNA preparation are provided in the Materials and Methods section. d Western blotting for phosphorylated-Iκβα or Iκβα in 293 cells expressing EBOV VP40, EBOV GP, TRAF6, NIK or RESTV VP40 at 24 hpt. e Western blotting for phosphorylated-Iκβα or Iκβα in 293 cells expressing EBOV VP40, EBOV VP40 Δ1-20aa , or chimeric VP40 proteins where the HVR of EBOV VP40 was replaced with that of RESTV or BDBV (EVP40 HVR-RESTV and EVP40 HVR-BDBV ) at 24 hpt. Control: Empty vector transfected. For c , data are shown with mean ± SD (n = 3 independent biological replicates).

Article Snippet: The following open reading frames were cloned into an expression vector under the control of the phosphoglycerate kinase promoter: IKK-2 and its mutant IKK-2 K44M (Addgene plasmids #11103 and #11104, gifts from Anjana Rao ); 3xHA-Iκβα and its mutants 3xHA-Iκβα-SS32/36AA (Addgene plasmids #21985 and #24143, gifts from Warner Greene ); TNFR1-YFP (Addgene plasmid #111209, gift from Johannes A. Schmid).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Reverse Transcription, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: Novel mechanism of inflammatory activation by Ebola virus matrix protein linked to the ebolavirus virulence

doi: 10.1101/2024.10.06.616882

Figure Lengend Snippet: a Synthego ICE analysis for CRISPR editing targeting a AAVS1, b TNFR1, c LTβR, d TNFα. e Western blotting for Iκβα in Iκβα-knockout 293 and wild-type 293 cells.

Article Snippet: The following open reading frames were cloned into an expression vector under the control of the phosphoglycerate kinase promoter: IKK-2 and its mutant IKK-2 K44M (Addgene plasmids #11103 and #11104, gifts from Anjana Rao ); 3xHA-Iκβα and its mutants 3xHA-Iκβα-SS32/36AA (Addgene plasmids #21985 and #24143, gifts from Warner Greene ); TNFR1-YFP (Addgene plasmid #111209, gift from Johannes A. Schmid).

Techniques: CRISPR, Western Blot, Knock-Out