Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: Western blot showing the relative expression levels of the Venus tagged SQSTM1 and LC3 constructs compared to endogenous SQSTM1 and LC3. HeLa cells transfected with the indicated Venus-tagged SQSTM1 or LC3 constructs in duplicate were lysed and resolved by SDS-PAGE followed by western blotting with (A) anti-SQSTM1 or (B) anti-LC3 antibodies. Mm, molecular mass markers in kilodaltons.

Article Snippet: Venus- and Cerulean-SQSTM1 vectors were constructed by first amplifying the SQSTM1 cDNA using PCR with the following forward and reverse primers: Forward – 5′-GAGAGAAGATCTATGGCGTCGTTCACGG-TGAAG-3′ Reverse – 5′-CTGAATGTCGACTGTGGAGGGTGCTT-3′ We then inserted the SQSTM1 cDNA into Venus-C1 or Cerulean-C1 vectors (Addgene, 27794 and 27796; deposited by Steven Vogel) by double-restriction digestion with enzymes, BglII (NEB R0144S) and SalI (NEB R0138S).

Techniques: Western Blot, Expressing, Construct, Transfection, SDS Page

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: The nucleocytoplasmic ratio of soluble Venus-LC3 is decreased in the presence of overexpressed Cerulean-SQSTM1. Representative confocal images of the localization of Venus, Venus-LC3, Venus-LC3G120A, Venus-LC3F52A,L53A, and Venus-LC3R70A when transiently co-expressed with (A) Cerulean or (B) Cerulean-SQSTM1 in HeLa cells. Scale bar: 10 μm. (C) An automated image analysis routine was used to quantify the N/C ratio of the soluble (puncta independent) Venus fluorescence (YFP channel) in the cells co-expressing the indicated Venus-tagged constructs in combination with either Cerulean (light gray bars) or Cerulean-SQSTM1 (dark gray bars). Bars are the mean ± 95% confidence interval for N = 20 cells from 2 independent experiments. Statistical comparisons are between the grouped bars. ns, p > 0.05; ***, p ≤ 0.001.

Article Snippet: Venus- and Cerulean-SQSTM1 vectors were constructed by first amplifying the SQSTM1 cDNA using PCR with the following forward and reverse primers: Forward – 5′-GAGAGAAGATCTATGGCGTCGTTCACGG-TGAAG-3′ Reverse – 5′-CTGAATGTCGACTGTGGAGGGTGCTT-3′ We then inserted the SQSTM1 cDNA into Venus-C1 or Cerulean-C1 vectors (Addgene, 27794 and 27796; deposited by Steven Vogel) by double-restriction digestion with enzymes, BglII (NEB R0144S) and SalI (NEB R0138S).

Techniques: Fluorescence, Expressing, Construct

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: FRET reports on the close physical proximity of both soluble and puncta-associated Cerulean-SQSTM1 and Venus-LC3. FRET analysis was performed on cells co-expressing Cerulean-SQSTM1 with the indicated Venus-LC3 constructs and semi-automatically analyzed across multiple cells. The data were separately analyzed for (A) soluble SQSTM1 and (B) puncta-associated pools of SQSTM1. FRET was also measured between Cerulean-SQSTM1 and empty Venus as a negative control. Bars are the mean ± 95% confidence interval for N = 9–30 cells from 3 independent experiments. Statistical comparisons are with the negative control. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0 .001.

Article Snippet: Venus- and Cerulean-SQSTM1 vectors were constructed by first amplifying the SQSTM1 cDNA using PCR with the following forward and reverse primers: Forward – 5′-GAGAGAAGATCTATGGCGTCGTTCACGG-TGAAG-3′ Reverse – 5′-CTGAATGTCGACTGTGGAGGGTGCTT-3′ We then inserted the SQSTM1 cDNA into Venus-C1 or Cerulean-C1 vectors (Addgene, 27794 and 27796; deposited by Steven Vogel) by double-restriction digestion with enzymes, BglII (NEB R0144S) and SalI (NEB R0138S).

Techniques: Expressing, Construct, Negative Control

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: Venus-LC3's rate of diffusion decreases when co-expressed with Cerulean-SQSTM1. A quantitative FRAP assay was carried out on cells co-expressing the indicated Venus- and Cerulean-tagged constructs using a rectangular imaging ROI centered on the cytoplasm, and small circular bleach region (rn = 1 μm) placed in a region devoid of puncta. (A) Examples of the early time-points from normalized FRAP recoveries are shown for Venus-LC3 co-expressed with Cerulean (light gray circles) or with Cerulean-SQSTM1 (dark gray circles). The solid lines are fits to a single component diffusion model. Residuals for the fits are indicated in the lower panel. (B) D values for the indicated Venus-LC3 constructs in cells co-expressing either Cerulean (light gray bars) or Cerulean-SQSTM1 (dark gray bars). Empty Venus was included as a control. Bars show the mean ± 95% confidence intervals for N = 19–64 cells from 2–4 independent experiments. Statistical comparisons are between the grouped bars. ns, p > 0.05; ***, p ≤ 0.001.

Article Snippet: Venus- and Cerulean-SQSTM1 vectors were constructed by first amplifying the SQSTM1 cDNA using PCR with the following forward and reverse primers: Forward – 5′-GAGAGAAGATCTATGGCGTCGTTCACGG-TGAAG-3′ Reverse – 5′-CTGAATGTCGACTGTGGAGGGTGCTT-3′ We then inserted the SQSTM1 cDNA into Venus-C1 or Cerulean-C1 vectors (Addgene, 27794 and 27796; deposited by Steven Vogel) by double-restriction digestion with enzymes, BglII (NEB R0144S) and SalI (NEB R0138S).

Techniques: Diffusion-based Assay, FRAP Assay, Expressing, Construct, Imaging

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: Apparent molecular mass and mobile fractions for Venus, Venus-LC3, and Venus-LC3 mutants co-expressing either Cerulean or Cerulean-SQSTM1 based on the FRAP diffusion measurements in live HeLa cells under basal conditions.

Article Snippet: Venus- and Cerulean-SQSTM1 vectors were constructed by first amplifying the SQSTM1 cDNA using PCR with the following forward and reverse primers: Forward – 5′-GAGAGAAGATCTATGGCGTCGTTCACGG-TGAAG-3′ Reverse – 5′-CTGAATGTCGACTGTGGAGGGTGCTT-3′ We then inserted the SQSTM1 cDNA into Venus-C1 or Cerulean-C1 vectors (Addgene, 27794 and 27796; deposited by Steven Vogel) by double-restriction digestion with enzymes, BglII (NEB R0144S) and SalI (NEB R0138S).

Techniques: Diffusion-based Assay

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: Soluble Venus-LC3 interacts with overexpressed Cerulean-SQSTM1 even when SQSTM1's PB1 domain is disrupted. (A) Representative confocal images of the localization of Venus-LC3 in HeLa cells co-expressing either Cerulean, as a negative control, or the indicated Cerulean-SQSTM1 constructs. Scale bar: 10 μm. (B) Quantification of the N/C ratios for soluble Venus-LC3 (YFP channel) in the presence of the indicated constructs. Bars show the mean ± 95% confidence intervals for N = 20–31 cells from 2–3 independent experiments. (C) Quantification of FRET between soluble Venus-LC3 and the indicated Cerulean-SQSTM1 constructs. FRET between Venus-LC3 and Cerulean was assessed as a negative control. Bars show the mean ± 95% confidence intervals for N = 30 cells from 3 independent experiments. (D) D for Venus-LC3 in cells co-expressing the indicated Cerulean-SQSTM1 constructs or empty Cerulean. Bars show the mean ± 95% confidence interval for N=30–35 cells from 3 independent experiments. Statistics are for comparisons with Venus-LC3 co-expressing wild-type SQSTM1. ns, p > 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

Article Snippet: Venus- and Cerulean-SQSTM1 vectors were constructed by first amplifying the SQSTM1 cDNA using PCR with the following forward and reverse primers: Forward – 5′-GAGAGAAGATCTATGGCGTCGTTCACGG-TGAAG-3′ Reverse – 5′-CTGAATGTCGACTGTGGAGGGTGCTT-3′ We then inserted the SQSTM1 cDNA into Venus-C1 or Cerulean-C1 vectors (Addgene, 27794 and 27796; deposited by Steven Vogel) by double-restriction digestion with enzymes, BglII (NEB R0144S) and SalI (NEB R0138S).

Techniques: Expressing, Negative Control, Construct

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: Nucleocytoplasmic distribution of Venus-SQSTM1 constructs. Representative images of the subcellular distribution of the indicated SQSTM1 and SQSTM1 isoform 2 constructs (A) following 2 h in the presence of vehicle or (B) following 2 h of LMB treatment. Scale bar: 10 μm. Data are representative of 3 independent experiments. (C) A semi-automated image analysis routine was used to quantify the N/C ratio of the soluble (puncta independent) Venus fluorescence (YFP channel) in the cells expressing the indicated Venus-tagged SQSTM1 constructs and nuclei labeled with DRAQ5 in the absence of LMB (light gray bars) or presence of LMB (dark gray bars). Bars are the mean ±95% confidence interval for N = 20 cells from 2 independent experiments. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01.

Article Snippet: Venus- and Cerulean-SQSTM1 vectors were constructed by first amplifying the SQSTM1 cDNA using PCR with the following forward and reverse primers: Forward – 5′-GAGAGAAGATCTATGGCGTCGTTCACGG-TGAAG-3′ Reverse – 5′-CTGAATGTCGACTGTGGAGGGTGCTT-3′ We then inserted the SQSTM1 cDNA into Venus-C1 or Cerulean-C1 vectors (Addgene, 27794 and 27796; deposited by Steven Vogel) by double-restriction digestion with enzymes, BglII (NEB R0144S) and SalI (NEB R0138S).

Techniques: Construct, Fluorescence, Expressing, Labeling

Journal: Autophagy

Article Title: Size, organization, and dynamics of soluble SQSTM1 and LC3-SQSTM1 complexes in living cells

doi: 10.1080/15548627.2016.1199299

Figure Lengend Snippet: FRET and FRAP analysis of soluble SQSTM1 homo-oligomers. Analysis of FRET between Cerulean- and Venus-tagged versions of the indicated (A) SQSTM1 constructs, or (B) SQSTM1iso. FRET was separately calculated for the soluble pools of protein (light gray bars) and puncta-associated pools (dark gray bars). FRET was also measured in cells co-expressing Cerulean-SQSTM1 and empty Venus as negative controls. Bars show the mean ± 95% confidence intervals for N = 27–30 cells from 3 independent experiments in (A), or N = 20 cells from 2 independent experiments in (B). Statistics are for the comparisons with the negative controls. FRAP analysis was used to quantify the diffusion coefficients of the indicated (C) Venus-tagged SQSTM1 constructs or (D) Venus-SQSTM1iso2. Diffusion coefficients for empty Venus were measured as internal controls. Bars show the mean ± 95% confidence intervals for N = 10–20 cells from 2 independent experiments in (A), and N = 31–35 cells from 3 independent experiments in (B). Statistics are for the comparisons with wild-type SQSTM1. ns, p > 0.05; ***, p ≤ 0.001.

Article Snippet: Venus- and Cerulean-SQSTM1 vectors were constructed by first amplifying the SQSTM1 cDNA using PCR with the following forward and reverse primers: Forward – 5′-GAGAGAAGATCTATGGCGTCGTTCACGG-TGAAG-3′ Reverse – 5′-CTGAATGTCGACTGTGGAGGGTGCTT-3′ We then inserted the SQSTM1 cDNA into Venus-C1 or Cerulean-C1 vectors (Addgene, 27794 and 27796; deposited by Steven Vogel) by double-restriction digestion with enzymes, BglII (NEB R0144S) and SalI (NEB R0138S).

Techniques: Construct, Expressing, Diffusion-based Assay

Journal: Journal of Virology

Article Title: Disruption of Nuclear Organization during the Initial Phase of African Swine Fever Virus Infection

doi: 10.1128/jvi.00704-11

Figure Lengend Snippet: FIG. 1. Peripheral lamin A/C phosphorylation and disruption at early times in ASFV infection. (A) 3D immuno-FISH of Vero cells uninfected (NI) and infected at 6, 8, and 12 h p.i. with 5 PFU of the Ba71V strain of ASFV. Nuclei were counterstained with DAPI (blue signal in a, e, i, and m), labeled with the FITC-conjugated ASFV genome probe (green signal in b, f, j, and n), and stained with a monoclonal antibody against lamin A/C (red signal in c, g, k, and o). Scale bars, 5 m. (B) ASFV infection induces increased phosphorylation of lamin A and C in Vero cells. (Top) Autoradiography showing the immunoprecipitated 32P-labeled lamin A/C proteins obtained from mock-infected () and ASFV-infected () Vero cells at 4 h p.i. (Bottom) Western blot analysis showing the total amounts of lamin A/C and -actin contained in each cell extract. (C) Degradation of nuclear lamin A/C at late times after ASFV infection. Western blot analysis of cell extracts from mock-infected (M) and ASFV-infected (V) Vero cells at different times postinfection (0, 2, 4, 6, 8, 12, and 18 h p.i.) using a polyclonal antibody against nuclear lamin A/C and an anti--actin antibody as an endogenous control.

Article Snippet: Cell extracts were quantified, and equal quantities of protein from each sample were resolved in 6% or 12% SDSpolyacrylamide gels, transferred to nitrocellulose, and probed with the following primary antibodies: a rabbit polyclonal antibody against lamin A/C (1:1,000) from Cell Signaling; the monoclonal antibodies H5 and 8WG16 (1:500 in both cases) from Covance, recognizing the hyperphosphorylated form at serine 2 and the hypophosphorylated form of RNA Pol II, respectively; the polyclonal antibody N-20 (1:500) from Santa Cruz, recognizing the N-terminal part of the largest RNA Pol II subunit; a mouse monoclonal antibody against -actin (1: 5,000) from Sigma; and a rabbit polyclonal antibody against -tubulin (1:1,000) from Sigma.

Techniques: Phospho-proteomics, Disruption, Infection, Labeling, Staining, Autoradiography, Immunoprecipitation, Western Blot, Control

Journal: Journal of Virology

Article Title: Disruption of Nuclear Organization during the Initial Phase of African Swine Fever Virus Infection

doi: 10.1128/jvi.00704-11

Figure Lengend Snippet: FIG. 2. Localization of nuclear envelope markers in viral factories at 12 h p.i. with ASFV. Shown is 3D double immuno-FISH of Vero cells infected at 12 h p.i. with 5 PFU of the BA71V strain of ASFV. FISH was performed using the ASFV probe, followed by double IF using antibodies against nuclear envelope proteins and the ASFV protein p54 to label viral factories. (A) Lamin A/C localization in viral factories. (i) DAPI- counterstained DNA is shown in blue. (ii) ASFV DNA was visualized with streptavidin-TRITC, shown in gray. (iii and iv) 3D double immuno- fluorescence was developed using a polyclonal antibody against the ASFV marker p54, revealed with anti-rabbit-FITC (green signal in iii) and a monoclonal antibody against lamin A/C revealed with anti-mouse-Cy5 (red signal in iv). (v) Colocalization of both markers (yellow signal). (B) Nucleoporin p62 localization in viral factories. (i) DAPI-counterstained DNA is shown in blue. (ii) ASFV DNA was visualized with streptavidin-TRITC, shown in red. (iii and iv) 3D double immunofluorescence was developed using a polyclonal antibody against the ASFV marker p54, revealed with anti-rabbit-FITC (green signal in iii) and a monoclonal antibody against nucleoporin p62 revealed with anti-mouse-Cy5 (gray signal in iv). (v) Merged image. Scale bars, 5 m.

Article Snippet: Cell extracts were quantified, and equal quantities of protein from each sample were resolved in 6% or 12% SDSpolyacrylamide gels, transferred to nitrocellulose, and probed with the following primary antibodies: a rabbit polyclonal antibody against lamin A/C (1:1,000) from Cell Signaling; the monoclonal antibodies H5 and 8WG16 (1:500 in both cases) from Covance, recognizing the hyperphosphorylated form at serine 2 and the hypophosphorylated form of RNA Pol II, respectively; the polyclonal antibody N-20 (1:500) from Santa Cruz, recognizing the N-terminal part of the largest RNA Pol II subunit; a mouse monoclonal antibody against -actin (1: 5,000) from Sigma; and a rabbit polyclonal antibody against -tubulin (1:1,000) from Sigma.

Techniques: Infection, Marker