Journal:

Article Title: RNAi-mediated depletion of the 15 KH domain protein, vigilin, induces death of dividing and non-dividing human cells but does not initially inhibit protein synthesis

doi: 10.1093/nar/gkg768

Figure Lengend Snippet: Rapid disappearance of vigilin in cells transfected with vigilin-specific siRNA. (A) HeLa cells were either not transfected (Untrans), mock transfected (Mock), transfected with either of two vigilin-specific siRNAs (Vig 1 and Vig 2), or with a non-specific pGL3 luciferase-specific siRNA (pGL3). Forty-eight hours after transfection, the cells were harvested, extracts were prepared, and 10 µg of each sample was fractionated on an 8% polyacrylamide gel and analyzed for vigilin content by western blotting with polyclonal rabbit anti-vigilin antibody. Purified recombinant FLAG epitope-tagged vigilin (fVig) was run as a control. (B) HeLa cells were left untransfected (Untrans), mock transfected (Mock), or were transfected with Vig 2 siRNA (0.84 µg) or with increasing concentrations of control pGL3 siRNA (0.14–0.84 µg). Forty-eight hours post-transfection, cell extracts were prepared, and 10 µg of each sample was fractionated by SDS–PAGE and analyzed for vigilin by western blotting. (C) HeLa cells were transfected with pGL3 or Vig 2 siRNAs, cells were harvested at 12, 24, 36 and 48 h post-transfection, extracts were prepared, and 30 µg of each sample was fractionated on an 8% polyacrylamide gel and analyzed for vigilin by western blotting. As a control for the specificity of the siRNAs, 20 µg of each extract was run on a 12% SDS–PAGE gel and analyzed for actin content by western blotting.

Article Snippet: The membranes were blocked overnight at 4°C in 1% non-fat dry milk (1 h at room temperature in 5% non-fat dry milk for actin and PARP western blots), then probed with either rabbit polyclonal anti-vigilin, or anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-PARP (Cell Signaling, Beverly, MA, USA) antibody for 1 h at room temperature (overnight at 4°C for actin and PARP), washed and probed with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature.

Techniques: Transfection, Luciferase, Western Blot, Purification, Recombinant, FLAG-tag, Control, SDS Page

Journal:

Article Title: RNAi-mediated depletion of the 15 KH domain protein, vigilin, induces death of dividing and non-dividing human cells but does not initially inhibit protein synthesis

doi: 10.1093/nar/gkg768

Figure Lengend Snippet: Vigilin knockdown in HeLa cells results in programmed cell death. HeLa cells were treated with 6.25 nM etoposide (a standard inducer of programmed cell death), mock transfected, or transfected with pGL3 or Vig 2 siRNAs. A western blot was performed 48 h post-transfection using a polyclonal antibody to poly(ADP-ribose) polymerase (PARP). A western blot using a polyclonal antibody to actin was also performed as a loading standard. Because the etoposide-treated cells were in the late stages of apoptosis, they exhibited reduced levels of actin. Levels of actin in the pGL3 and Vig 2-treated cells were similar and the ratio of the cleaved PARP to uncleaved PARP is dramatically increased in the Vig 2-treated cells relative to the mock or pGL3-treated cells.

Article Snippet: The membranes were blocked overnight at 4°C in 1% non-fat dry milk (1 h at room temperature in 5% non-fat dry milk for actin and PARP western blots), then probed with either rabbit polyclonal anti-vigilin, or anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-PARP (Cell Signaling, Beverly, MA, USA) antibody for 1 h at room temperature (overnight at 4°C for actin and PARP), washed and probed with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature.

Techniques: Knockdown, Transfection, Western Blot

Journal: Nature microbiology

Article Title: An RNA-centric dissection of host complexes controlling flavivirus infection.

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: Fig. 2 | Intersection of ChIRP-MS with genome-wide CRISPR screens nominates functionally relevant pro-viral host proteins. a, Genome-scale CRISPR KO screens of all four DENV serotypes (DENV-1276RKI, DENV-2429557, DENV-3Philippines/H871856 and DENV-4BC287/97) in Huh7.5.1 cells. The genetic screens were independently performed for each serotype, analysed with MAGeCK and combined to obtain the robust rank aggregation (RRA) significance scores (y axis). The 50 most-enriched genes were coloured and grouped by function. b, Scatter plot depicting the enrichment scores of high-confidence ChIRP-MS DENV hits (x axis) and the 200 top-scoring hits from DENV CRISPR genetic screens (y axis). Common hits shared by both the DENV genetic screens and DENV ChIRP-MS are indicated in red (vigilin), blue (RRBP1) and purple (others). c, Western blot analysis of WT and clonal RRBP1-KO (top) or vigilin-KO (bottom) Huh7.5.1 cells. Representative western blot of n = 2 biologically independent replicates showing similar results. d, Analysis by qRT–PCR of WT and RRBP1-KO cells infected with DENV (48 h.p.i.; m.o.i. of 0.1), ZIKVPRVABC59 (48 h.p.i.; m.o.i. of 0.1), POWVLB (48 h.p.i.; m.o.i. of 0.1) or CHIKV181/25 (24 h.p.i.; m.o.i. of 0.01). e, Analysis by qRT–PCR of WT and vigilin-KO cells infected as in d. The WT datasets for POWV in d,e were derived from the same experiments. f, Western blot analysis of the cell lysates of DENV-2429557-infected (72 h.p.i.; m.o.i. of 0.1) WT, RRBP1-KO and vigilin-KO Huh7.5.1 cells probed with antibodies to DENV prM and NS3. Representative western blot of n = 4 biologically independent replicates showing similar results. g, Titres of the infectious particles produced from WT, RRBP1-KO and vigilin-KO Huh7.5.1 cells infected with DENV-2429557 for 72 h at an m.o.i. of 0.1. d,e,g, The data represent the mean ± s.e.m. of n = 3 independent biological replicates, except POWV, where n = 4 independent biological replicates. All P values were determined by two-tailed, unpaired t-tests using GraphPad Prism; n.s., not significant.

Article Snippet: Western blot analysis of the two different fractions was then performed with antibodies against RRBP1, vigilin, the cytoplasmic markers GAPDH (Genetex, GTX 627408) and tubulin (Abcam ab97872), and the ER marker RPN1 (Bethyl Laboratories, A305-026A).

Techniques: Genome Wide, CRISPR, Western Blot, Quantitative RT-PCR, Infection, Derivative Assay, Produced, Two Tailed Test

Journal: Nature microbiology

Article Title: An RNA-centric dissection of host complexes controlling flavivirus infection.

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: Fig. 3 | RRBP1 and vigilin interact at the ER. a, Single-cell quantification and correlation between RRBP1 or vigilin and the ER–GFP or DAPI immunofluorescence signals. The total numbers of cells that were randomly chosen for each analysis and the mean ± s.e.m. are indicated. b, Western blot analysis of the ER and cytosolic cell fractions probed with GAPDH or tubulin (cytosolic markers), RPN1 (ER marker), RRBP1 and vigilin antibodies. Representative western blot of n = 3 biologically independent replicates showing similar results. c, Western blot analysis of three independent co-IP experiments from uninfected Huh7.5.1 cells with RRBP1 as the bait showing similar results. The samples were treated with or without RNase A. d,e, Representative immunofluorescence of RRBP1 (d) and vigilin (e) co-stained with RNA fluorescent-in-situ-hybridization targeting of DENV or ZIKV positive-stranded RNA genomes. Representative images of n = 2 biologically independent replicates showing similar results. Scale bars, 10 μm. IP, immunoprecipitation.

Article Snippet: Western blot analysis of the two different fractions was then performed with antibodies against RRBP1, vigilin, the cytoplasmic markers GAPDH (Genetex, GTX 627408) and tubulin (Abcam ab97872), and the ER marker RPN1 (Bethyl Laboratories, A305-026A).

Techniques: Immunofluorescence, Western Blot, Marker, Co-Immunoprecipitation Assay, Staining, In Situ Hybridization, Immunoprecipitation

Journal: Nature microbiology

Article Title: An RNA-centric dissection of host complexes controlling flavivirus infection.

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: Fig. 4 | DENV and ZIKV co-opt the RNA-binding properties of RRBP1 and vigilin in human cells. a, RRBP1 (left) and vigilin (right) irCLIP RT-stop mapping statistics annotated to the human, and DENV and ZIKV genomes (gRNA) and the ribosomal RNAs (rRNA) from Huh7.5.1 cells infected with an m.o.i. of 0.1 for 48 h. b, Histogram of RT stops mapping to the rRNAs from the RRBP1 (top) and vigilin (bottom) irCLIP in uninfected Huh7.5.1 cells. The three cytosolic rRNAs are highlighted. The red dashed line denotes the strongest vigilin binding site, which is adjacent to that of RRBP1. c, Annotation of peaks called from the RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapping to functional elements of human mRNAs including the 5′ UTR, exons, 3′ UTR and introns. The enrichment values were calculated based on the size of each function domain relative to the human genome. d, RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapped at base resolution to the DENV genome. The RT stop intensity was normalized to the total number of unique reads mapping to the viral genome. The 5′ and 3′ UTR regions are highlighted in red and blue, respectively.

Article Snippet: Western blot analysis of the two different fractions was then performed with antibodies against RRBP1, vigilin, the cytoplasmic markers GAPDH (Genetex, GTX 627408) and tubulin (Abcam ab97872), and the ER marker RPN1 (Bethyl Laboratories, A305-026A).

Techniques: RNA Binding Assay, Infection, Binding Assay, Functional Assay

Journal: PLoS ONE

Article Title: BioID identifies proteins involved in the cell biology of caveolae

doi: 10.1371/journal.pone.0209856

Figure Lengend Snippet: A . HeLa cells labelled with anti-caveolin1 and anti-CD2AP antibodies. The region in the yellow box is shown magnified in the lower panels. Bar 20 microns. Maximum intensity projections of multiple confocal sections acquired at 1 micron intervals, with 63x objective. Yellow arrowheads indicate co-localisation. B . HeLa cells labelled with anti-caveolin1 and anti-CD2AP antibodies. The region in the yellow box is shown magnified in the lower panels. Bar 20 microns. Total Internal Reflection imaging, with 63x objective. Yellow arrowheads indicate co-localisation. C . Quantification of Pearson’s correlation coefficient in multiple cell areas from TIR images as shown in B, in either images where the two fluorescence channels are correctly aligned or where they were manually offset by approximately 0.5 microns. Statistical comparison is by t-test (* denote P<0.05). Each dot represents one cell. D . Cell projection induced by overexpression of GFP-CD2AP Bar 5 microns. E . Co-localisation between GFP-CD2AP, caveolin1 antibody labelling, and cavin1-mCherry, in the cell projection shown in D. Single confocal sections acquired with 63x objective, bar is 5 microns. White arrows indicate co-localisation.

Article Snippet: The following antibodies were used: rabbit anti-caveolin1 (BD Biosciences Cat# 610060), mouse anti-βeta-catenin (BD Biosciences Cat# 610153), rabbit anti-cavin1 (Abcam Cat# ab48824), rabbit anti CD2AP (A599 Cell Signalling, Cat# 5478) for WB and mouse anti-CD2AP (Santa Cruz Biotechnology Cat# sc-25272) for immunostaining, YL1-2 anti-alpha tubulin (in-house cell culture supernatant), mouse anti-GFP (Roche Cat# 11814460001), mouse anti-flotillin-2 (BD, 610384), goat anti-EHD2 (Abcam Cat# ab23935), rabbit anti-CSDE1 (N2C1 GeneTex, Cat# GTX116218), mouse anti-TXNRD1 (Novus Biologicals Cat# NBP2-59489), mouse anti-vigilin (LSBio Cat# LS-C342610-100), rabbit anti-PRRC2C (Abcam Cat#ab117790), rat anti-Nav1 (Abcam Cat#ab201920) and mouse anti-myc clone 9E10 (in-house cell culture supernatant).

Techniques: Imaging, Fluorescence, Comparison, Over Expression

Journal: PLoS ONE

Article Title: BioID identifies proteins involved in the cell biology of caveolae

doi: 10.1371/journal.pone.0209856

Figure Lengend Snippet: A . HeLa cells overexpressing Rac1Q61L-myc and GFP-CD2AP, labelled with anti-caveolin1 and anti-beta-catenin antibodies. The region in the yellow box is shown magnified in the lower panels. Bar 20 microns. Maximum intensity projections of multiple confocal sections acquired at 1 micron intervals, with 63x objective. B . HeLa cells overexpressing Rac1Q61L-myc, labelled with anti-caveolin1 and anti-EHD2 antibodies. The region in the yellow box is shown magnified in the lower panels. Bar 20 microns. Maximum intensity projections of multiple confocal sections acquired at 1 micron intervals, with 63x objective. C . MDCK cells labelled with anti-caveolin1 and anti-beta-catenin antibodies. The region in the yellow box is shown magnified in the lower panels. Bar 10 microns. Single confocal section, with 63x objective.

Article Snippet: The following antibodies were used: rabbit anti-caveolin1 (BD Biosciences Cat# 610060), mouse anti-βeta-catenin (BD Biosciences Cat# 610153), rabbit anti-cavin1 (Abcam Cat# ab48824), rabbit anti CD2AP (A599 Cell Signalling, Cat# 5478) for WB and mouse anti-CD2AP (Santa Cruz Biotechnology Cat# sc-25272) for immunostaining, YL1-2 anti-alpha tubulin (in-house cell culture supernatant), mouse anti-GFP (Roche Cat# 11814460001), mouse anti-flotillin-2 (BD, 610384), goat anti-EHD2 (Abcam Cat# ab23935), rabbit anti-CSDE1 (N2C1 GeneTex, Cat# GTX116218), mouse anti-TXNRD1 (Novus Biologicals Cat# NBP2-59489), mouse anti-vigilin (LSBio Cat# LS-C342610-100), rabbit anti-PRRC2C (Abcam Cat#ab117790), rat anti-Nav1 (Abcam Cat#ab201920) and mouse anti-myc clone 9E10 (in-house cell culture supernatant).

Techniques:

Journal: PLoS ONE

Article Title: BioID identifies proteins involved in the cell biology of caveolae

doi: 10.1371/journal.pone.0209856

Figure Lengend Snippet: A . Western blot of cells transfected with the siRNAs shown, using antibodies as indicated. CD2AP siRNAs were either three separate single species or a pooled population containing all three. B . Hela cells overexpressing Rac1Q61L-myc, showing different degrees of recruitment of caveolin1 to cell-cell junctions. These categories were used in the analysis shown in C below. Bars 5 microns, single confocal sections acquired with 63x objective C . Analysis of the recruitment of caveolin1 to cell-cell junctions, as in B, in cells treated with the siRNAs shown stained with anti beta-catenin and anti caveolin 1 antibodies. N = total number of beta-catenin-positive cell-cell junctions analysed.

Article Snippet: The following antibodies were used: rabbit anti-caveolin1 (BD Biosciences Cat# 610060), mouse anti-βeta-catenin (BD Biosciences Cat# 610153), rabbit anti-cavin1 (Abcam Cat# ab48824), rabbit anti CD2AP (A599 Cell Signalling, Cat# 5478) for WB and mouse anti-CD2AP (Santa Cruz Biotechnology Cat# sc-25272) for immunostaining, YL1-2 anti-alpha tubulin (in-house cell culture supernatant), mouse anti-GFP (Roche Cat# 11814460001), mouse anti-flotillin-2 (BD, 610384), goat anti-EHD2 (Abcam Cat# ab23935), rabbit anti-CSDE1 (N2C1 GeneTex, Cat# GTX116218), mouse anti-TXNRD1 (Novus Biologicals Cat# NBP2-59489), mouse anti-vigilin (LSBio Cat# LS-C342610-100), rabbit anti-PRRC2C (Abcam Cat#ab117790), rat anti-Nav1 (Abcam Cat#ab201920) and mouse anti-myc clone 9E10 (in-house cell culture supernatant).

Techniques: Western Blot, Transfection, Staining

Journal: Molecular Cancer Research

Article Title: TSC2 Interacts with HDLBP/Vigilin and Regulates Stress Granule Formation

doi: 10.1158/1541-7786.mcr-20-1046

Figure Lengend Snippet: Figure 1. Co-Immunoprecipitation (IP) of vigilin with TSC2. A, HEK-293T cells were transiently transfected with c-Myc-vigilin or c-Myc. c-Myc was pulled down. Endogenous TSC2 was detected by immunoblot. All lanes shown are from same gel. B, Vigilin was immunoprecipitated from HEK293T and HeLa cells. Endogenous TSC2 was detected by immunoblot. An empty lane was used between inputs and 293T IP and between 293T IP and HeLa IP.

Article Snippet: The following antibodies were used: TSC2 (rabbit), FXR2 (rabbit), phospho-S6(S235/236) (rabbit), phospho-eIF2a(S51), Caspase 3 (rabbit), PARP (rabbit; Cell Signaling Technology), vigilin/HDLBP (rabbit), G3BP1 (rabbit), Caprin1 (rabbit; Proteintech), G3BP1 (mouse; Abcam), TSC2 (mouse; Lifespan Biosciences), b-Actin (mouse; Milipore/Sigma-Aldrich).

Techniques: Immunoprecipitation, Transfection, Western Blot

Journal: Molecular Cancer Research

Article Title: TSC2 Interacts with HDLBP/Vigilin and Regulates Stress Granule Formation

doi: 10.1158/1541-7786.mcr-20-1046

Figure Lengend Snippet: Figure 3. TSC2 localizes to mammalian SGs after oxidative stress. HeLa cells were treated with 1 mmol/L NaAsO2 for 50 minutes (A) and 30 mmol/L NaAsO2 for 2 hours (B). Cells were stained for TSC2. FXR2 was used for SG detection. DAPI was used to visualize the nuclei. Arrows indicate SGs containing TSC2. Scale bars, 20 mm, 5 mm.

Article Snippet: The following antibodies were used: TSC2 (rabbit), FXR2 (rabbit), phospho-S6(S235/236) (rabbit), phospho-eIF2a(S51), Caspase 3 (rabbit), PARP (rabbit; Cell Signaling Technology), vigilin/HDLBP (rabbit), G3BP1 (rabbit), Caprin1 (rabbit; Proteintech), G3BP1 (mouse; Abcam), TSC2 (mouse; Lifespan Biosciences), b-Actin (mouse; Milipore/Sigma-Aldrich).

Techniques: Staining

Journal: Molecular Cancer Research

Article Title: TSC2 Interacts with HDLBP/Vigilin and Regulates Stress Granule Formation

doi: 10.1158/1541-7786.mcr-20-1046

Figure Lengend Snippet: Figure 4. Knocking down vigilin impacts SG translocation of TSC2. A, Immunoblot showing levels of vigilin after siVigilin in MEFs. B, siVigilin and control MEFs were treated with 0.5 mmol/L NaAsO2 for 50 minutes. Arrows indi- cate SGs containing TSC2. Arrow- heads indicate SGs without TSC2. C, SGs positive for G3BP1 or TSC2 were quantified with CellProfiler. Scale bar, 20 mm. , P < 0.001 (n ¼ 64 cells per genotype).

Article Snippet: The following antibodies were used: TSC2 (rabbit), FXR2 (rabbit), phospho-S6(S235/236) (rabbit), phospho-eIF2a(S51), Caspase 3 (rabbit), PARP (rabbit; Cell Signaling Technology), vigilin/HDLBP (rabbit), G3BP1 (rabbit), Caprin1 (rabbit; Proteintech), G3BP1 (mouse; Abcam), TSC2 (mouse; Lifespan Biosciences), b-Actin (mouse; Milipore/Sigma-Aldrich).

Techniques: Translocation Assay, Western Blot, Control

Journal: Molecular Cancer Research

Article Title: TSC2 Interacts with HDLBP/Vigilin and Regulates Stress Granule Formation

doi: 10.1158/1541-7786.mcr-20-1046

Figure Lengend Snippet: Figure 5. TSC2 loss leads to an increased number of SGs upon cellular stress. MEFs (n ¼ 125 cells/genotype; A) and 105K cells (n ¼ 75 cells/genotype; B) were treated with 0.5 mmol/L NaAsO2 (arsenite) for 50 minutes. C, MEFs (n ¼ 330 cells/genotype) were heat shocked for 45 minutes at 42C. Cells were stained for G3BP1 to detect SGs, quantified with CellProfiler. DAPI was used to visualize the nuclei. Scale bar, 20 mm. , P < 0.0001.

Article Snippet: The following antibodies were used: TSC2 (rabbit), FXR2 (rabbit), phospho-S6(S235/236) (rabbit), phospho-eIF2a(S51), Caspase 3 (rabbit), PARP (rabbit; Cell Signaling Technology), vigilin/HDLBP (rabbit), G3BP1 (rabbit), Caprin1 (rabbit; Proteintech), G3BP1 (mouse; Abcam), TSC2 (mouse; Lifespan Biosciences), b-Actin (mouse; Milipore/Sigma-Aldrich).

Techniques: Staining

Journal: Molecular Cancer Research

Article Title: TSC2 Interacts with HDLBP/Vigilin and Regulates Stress Granule Formation

doi: 10.1158/1541-7786.mcr-20-1046

Figure Lengend Snippet: Figure 7. G3BP1 downregulation decreases the proliferation of Tsc2-deficient cells in vitro and in vivo. A, Immunoblot confirming downregulation of G3BP1 in TSC2 reexpressing 105K cells and in 105 cells. Knockdown of G3BP1 induces apoptosis (as assessed by cleaved caspase 3 and cleaved PARP indicated by stars, respectively) in 105K cells, but not in 105K cells with reexpression of TSC2. B, Knockdown of G3BP1 increased the number of TSC2 reexpressing 105K cells by 17% and decreased the number of 105K cells by 25% at 72 hours, , P < 0.0001 (crystal violet staining). C, Mice were injected subcutaneously with 2.5 106 Tsc2-deficient 105K cells shCtrl or shG3BP1. Tumor latency was increased in Tsc2-deficient 105K shG3BP1 compared with shCtrl tumors. Statistical significance was assessed by Mantel–Cox Text. D, Tumor volume was decreased in the shG3BP1 tumors compared with shCtrl tumors. Statistical significance was assessed by Student unpaired t test with , P < 0.05; , P < 0.01.

Article Snippet: The following antibodies were used: TSC2 (rabbit), FXR2 (rabbit), phospho-S6(S235/236) (rabbit), phospho-eIF2a(S51), Caspase 3 (rabbit), PARP (rabbit; Cell Signaling Technology), vigilin/HDLBP (rabbit), G3BP1 (rabbit), Caprin1 (rabbit; Proteintech), G3BP1 (mouse; Abcam), TSC2 (mouse; Lifespan Biosciences), b-Actin (mouse; Milipore/Sigma-Aldrich).

Techniques: In Vitro, In Vivo, Western Blot, Knockdown, Staining, Injection

Journal: Molecular Cancer Research

Article Title: TSC2 Interacts with HDLBP/Vigilin and Regulates Stress Granule Formation

doi: 10.1158/1541-7786.mcr-20-1046

Figure Lengend Snippet: Figure 3. TSC2 localizes to mammalian SGs after oxidative stress. HeLa cells were treated with 1 mmol/L NaAsO2 for 50 minutes (A) and 30 mmol/L NaAsO2 for 2 hours (B). Cells were stained for TSC2. FXR2 was used for SG detection. DAPI was used to visualize the nuclei. Arrows indicate SGs containing TSC2. Scale bars, 20 mm, 5 mm.

Article Snippet: The following antibodies were used: TSC2 (rabbit), FXR2 (rabbit), phospho-S6(S235/236) (rabbit), phospho-eIF2a(S51), Caspase 3 (rabbit), PARP (rabbit; Cell Signaling Technology), vigilin/HDLBP (rabbit), G3BP1 (rabbit), Caprin1 (rabbit; Proteintech), G3BP1 (mouse; Abcam), TSC2 (mouse; Lifespan Biosciences), b-Actin (mouse; Milipore/Sigma-Aldrich).

Techniques: Staining