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  • 99
    Millipore hela cells hela cells
    A) <t>HeLa</t> cells were <t>transfected</t> with pcDNA3 (-) or CD74 (+), together with Notch . The total cell lysates (TL) and FLAG IPs were analyzed as in Figure 1B. Notch was detected with αmyc antibody. B) HeLa cells were transfected with pcDNA3 (-) or CD74 (+), together with APLP2, and were analyzed as in A. C) Left panels: HEK293APP cells were transfected with the indicated plasmids. The transfected cells were further incubated for 24 hours with fresh media, and secreted sAPPα/β were measured by Westerm blot. The cell lysates were analyzed as in Figure 1B. Right panels: the quantification of sAPPα/β. The levels in pcDNA3 transfected cells were set to 100. D) HEK293APP cells were transfected with pcDNA3 or CD74 and treated as in C. Aβ40 and 42 secreted in the media, and Aβ40 in the lysates were measured with specific ELISA. The amount of Aβ was normalized to the amount of the protein in the total lysates. The asterisks (**) indicate that p values by Student's t test are less than 0.01. E) HeLa cells were transfected with pcDNA3 or CD74, and the media was replaced with fresh media containing synthetic Aβ40 and Aβ42. The media of pcDNA3 (open circles) and CD74 (closed circles) transfected cells were incubated for indicated hours, and the remaining Aβ40 and Aβ42 were measured as in D. F) HeLa cells were transfected with pcDNA3 or FLAG-CD74 together with APP. The cells were pulsed for 30 min with 35 S methionine+cysteine, and chased in fresh media for indicated hours. APP was immunoprecipitated with αAPPct antibody and analyzed by SDS-PAGE followed by autoradiography. G) HeLa cells were transfected with pcDNA3 (-) or FLAG-CD74 (+) together with wild type APP (wt APP) or the indicated mutants. The total lysates and FLAG IPs were processed as in Figure 1B. Note that APP Ncas lacks C-terminal 31 amino acids of APP and has therefore a smaller in size.
    Hela Cells Hela Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boster Bio hela cells
    A) <t>HeLa</t> cells were <t>transfected</t> with pcDNA3 (-) or CD74 (+), together with Notch . The total cell lysates (TL) and FLAG IPs were analyzed as in Figure 1B. Notch was detected with αmyc antibody. B) HeLa cells were transfected with pcDNA3 (-) or CD74 (+), together with APLP2, and were analyzed as in A. C) Left panels: HEK293APP cells were transfected with the indicated plasmids. The transfected cells were further incubated for 24 hours with fresh media, and secreted sAPPα/β were measured by Westerm blot. The cell lysates were analyzed as in Figure 1B. Right panels: the quantification of sAPPα/β. The levels in pcDNA3 transfected cells were set to 100. D) HEK293APP cells were transfected with pcDNA3 or CD74 and treated as in C. Aβ40 and 42 secreted in the media, and Aβ40 in the lysates were measured with specific ELISA. The amount of Aβ was normalized to the amount of the protein in the total lysates. The asterisks (**) indicate that p values by Student's t test are less than 0.01. E) HeLa cells were transfected with pcDNA3 or CD74, and the media was replaced with fresh media containing synthetic Aβ40 and Aβ42. The media of pcDNA3 (open circles) and CD74 (closed circles) transfected cells were incubated for indicated hours, and the remaining Aβ40 and Aβ42 were measured as in D. F) HeLa cells were transfected with pcDNA3 or FLAG-CD74 together with APP. The cells were pulsed for 30 min with 35 S methionine+cysteine, and chased in fresh media for indicated hours. APP was immunoprecipitated with αAPPct antibody and analyzed by SDS-PAGE followed by autoradiography. G) HeLa cells were transfected with pcDNA3 (-) or FLAG-CD74 (+) together with wild type APP (wt APP) or the indicated mutants. The total lysates and FLAG IPs were processed as in Figure 1B. Note that APP Ncas lacks C-terminal 31 amino acids of APP and has therefore a smaller in size.
    Hela Cells, supplied by Boster Bio, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore hela cells hela dh cells
    <t>Cx26</t> immunostaining performed in <t>Hela</t> DH transfectants overexpressing Cx26WT (top), and Cx26C169Y (bottom) proteins. Through focus confocal image sequence (z-stack) taken at 0.5 µm intervals of Hela DH cells expressing Cx26WT ( A – D ) and Cx26C169Y ( F – I ) proteins and their respective maximal projection rendering ( E and J ). Yellow arrow points to representative gap junction plaque, whereas red arrows indicate immunoreaction signals at the cell plasma membrane level, which are most likely due to unpaired connexons. Scale bar, 10 µm.
    Hela Cells Hela Dh Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Active Motif hela cells
    A marine blocking agent <t>(MAXblock</t> ™ ) improves staining quality relative to standard blocking agents <t>HeLa</t> cells were fixed with methanol (−20 °C, 8 min) and blocked overnight in PBS containing (A) MAXblock ™ . MAXblock ™ was developed by Dr. Brian Bennett.
    Hela Cells, supplied by Active Motif, used in various techniques. Bioz Stars score: 92/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 131 article reviews
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    93
    Biovest hela cells
    A marine blocking agent <t>(MAXblock</t> ™ ) improves staining quality relative to standard blocking agents <t>HeLa</t> cells were fixed with methanol (−20 °C, 8 min) and blocked overnight in PBS containing (A) MAXblock ™ . MAXblock ™ was developed by Dr. Brian Bennett.
    Hela Cells, supplied by Biovest, used in various techniques. Bioz Stars score: 93/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DSMZ hela cells
    Deregulation of PLK4 expression after expression of HPV-16 E7 depends on loss of DREAM binding to the CDE and CHR elements. ( A ) <t>HCT116</t> wt and HCT116 stably transfected with a plasmid encoding HPV-16 E7 wt or its ΔDLYC mutant. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( B ) Luciferase reporter assays from lysates of HCT116 wt; p53 −/− or p21 −/− cells transfected with plasmids expressing wt or CDE/CHR mutant Plk4 promoter constructs. Plasmids expressing HPV-16 E7 wt or its ΔDLYC mutant were cotransfected. Results are given as relative light units. ( C ) Nuclear extracts from HCT116 E7 wt and E7 ΔDLYC cells were analyzed by DNA affinity purification with wt and CDE/CHR mutant Plk4 promoter probes followed by western blot. Band intensities were quantified by densitometric analyses. Relative intensities are given below the bands. Intensities of input bands were normalized to E7 ΔDLYC. Binding intensities to Plk4 promoters were normalized to CDE/CHR mutant probes from E7 ΔDLYC extracts. ( D ) Protein binding to the PLK4 promoter in <t>HeLa</t> cells was assessed by ChIP. Protein binding to the GAPDHS promoter served as a negative control. ( E ) HCT116 E7 wt and HeLa cells were treated with doxorubicin and roscovitine for 24 h. Cells without treatment served as control. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( F ) Luciferase reporter assays with wt and CDE/CHR mutant Plk4 promoter reporter constructs in HCT116 p53 −/− cells. Cells were cotransfected with p53mut, p53wt, p21mut or p21wt expression vectors and HPV-16 E7 wt or ΔDLYC mutant. Results are given as relative light units. ( G ) Venn diagram from DREAM-bound genes ( 12 , 15 ), genes upregulated by HPV E7 ( 64 ) and genes downregulated by HPV E2 proteins repressing E7 ( 65 , 66 ).
    Hela Cells, supplied by DSMZ, used in various techniques. Bioz Stars score: 92/100, based on 451 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem hela cells
    Deregulation of PLK4 expression after expression of HPV-16 E7 depends on loss of DREAM binding to the CDE and CHR elements. ( A ) <t>HCT116</t> wt and HCT116 stably transfected with a plasmid encoding HPV-16 E7 wt or its ΔDLYC mutant. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( B ) Luciferase reporter assays from lysates of HCT116 wt; p53 −/− or p21 −/− cells transfected with plasmids expressing wt or CDE/CHR mutant Plk4 promoter constructs. Plasmids expressing HPV-16 E7 wt or its ΔDLYC mutant were cotransfected. Results are given as relative light units. ( C ) Nuclear extracts from HCT116 E7 wt and E7 ΔDLYC cells were analyzed by DNA affinity purification with wt and CDE/CHR mutant Plk4 promoter probes followed by western blot. Band intensities were quantified by densitometric analyses. Relative intensities are given below the bands. Intensities of input bands were normalized to E7 ΔDLYC. Binding intensities to Plk4 promoters were normalized to CDE/CHR mutant probes from E7 ΔDLYC extracts. ( D ) Protein binding to the PLK4 promoter in <t>HeLa</t> cells was assessed by ChIP. Protein binding to the GAPDHS promoter served as a negative control. ( E ) HCT116 E7 wt and HeLa cells were treated with doxorubicin and roscovitine for 24 h. Cells without treatment served as control. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( F ) Luciferase reporter assays with wt and CDE/CHR mutant Plk4 promoter reporter constructs in HCT116 p53 −/− cells. Cells were cotransfected with p53mut, p53wt, p21mut or p21wt expression vectors and HPV-16 E7 wt or ΔDLYC mutant. Results are given as relative light units. ( G ) Venn diagram from DREAM-bound genes ( 12 , 15 ), genes upregulated by HPV E7 ( 64 ) and genes downregulated by HPV E2 proteins repressing E7 ( 65 , 66 ).
    Hela Cells, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare hela cells
    FMRP docking to chromatin is essential for FMRP-dependent modulation of γH2A.X levels in response to replication stress (A) Diagram of Agenet FMRP . (B) <t>GST-FMRP</t> or GST-FMRP carrying mutations in Agenet FMRP . (C) . (D) FMRP RNAi in <t>HeLa</t> cells abolished γH2A.X induction in response to APH as compared to control RNAi (compare lanes 1,2 to lanes 3,4). Co-transfection with constructs expressing wild type but not mutant forms of FMRP restored the induction of γH2A.X in FMRP RNAi cells in response to APH (compare lanes 5,6 to lanes 7,8 and 9,10). The slower migrating band (in lanes 5–10) is Flag-HA-FMRP (indicated by an arrowhead).
    Hela Cells, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 2135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega hela cells
    ATR is the major kinase responsible for cellular <t>XPA</t> phosphorylation after UV irradiation. A, A549 cells were mock treated (lane 1) or treated with 20 J/m 2 UV irradiation, and then further incubated for 4 hours in the presence of 100 Amol/L wortmannin (Wort; lane 4) or 10 mmol/L caffeine (Caff; lane 3) before harvesting. Total cell lysates were used for Western blot analysis with anti-XPA, anti-ATR, and anti-ATM, respectively. B, A549 cells were transfected with ATR, ATM, or green fluorescent protein (GFP) siRNA as described in Materials and Methods. Total cell lysates were harvested 72 hours after transfection and probed with the indicated antibodies, respectively. C, A549 or <t>HeLa</t> cells were transfected with indicated siRNA and then treated with 20 J/m 2 UV irradiation at 72 hours after transfection. Total cell lysates were immunoblotted with anti-XPA and antiactin antibodies, respectively. D, ATR- and ATM-deficient cells were treated with the indicated doses of UV and total cell lysates were prepared at 4 hours after treatment for Western blotting with anti-XPA and antiactin, respectively.
    Hela Cells, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 5006 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Texcell SA hela cells
    ATR is the major kinase responsible for cellular <t>XPA</t> phosphorylation after UV irradiation. A, A549 cells were mock treated (lane 1) or treated with 20 J/m 2 UV irradiation, and then further incubated for 4 hours in the presence of 100 Amol/L wortmannin (Wort; lane 4) or 10 mmol/L caffeine (Caff; lane 3) before harvesting. Total cell lysates were used for Western blot analysis with anti-XPA, anti-ATR, and anti-ATM, respectively. B, A549 cells were transfected with ATR, ATM, or green fluorescent protein (GFP) siRNA as described in Materials and Methods. Total cell lysates were harvested 72 hours after transfection and probed with the indicated antibodies, respectively. C, A549 or <t>HeLa</t> cells were transfected with indicated siRNA and then treated with 20 J/m 2 UV irradiation at 72 hours after transfection. Total cell lysates were immunoblotted with anti-XPA and antiactin antibodies, respectively. D, ATR- and ATM-deficient cells were treated with the indicated doses of UV and total cell lysates were prepared at 4 hours after treatment for Western blotting with anti-XPA and antiactin, respectively.
    Hela Cells, supplied by Texcell SA, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A) HeLa cells were transfected with pcDNA3 (-) or CD74 (+), together with Notch . The total cell lysates (TL) and FLAG IPs were analyzed as in Figure 1B. Notch was detected with αmyc antibody. B) HeLa cells were transfected with pcDNA3 (-) or CD74 (+), together with APLP2, and were analyzed as in A. C) Left panels: HEK293APP cells were transfected with the indicated plasmids. The transfected cells were further incubated for 24 hours with fresh media, and secreted sAPPα/β were measured by Westerm blot. The cell lysates were analyzed as in Figure 1B. Right panels: the quantification of sAPPα/β. The levels in pcDNA3 transfected cells were set to 100. D) HEK293APP cells were transfected with pcDNA3 or CD74 and treated as in C. Aβ40 and 42 secreted in the media, and Aβ40 in the lysates were measured with specific ELISA. The amount of Aβ was normalized to the amount of the protein in the total lysates. The asterisks (**) indicate that p values by Student's t test are less than 0.01. E) HeLa cells were transfected with pcDNA3 or CD74, and the media was replaced with fresh media containing synthetic Aβ40 and Aβ42. The media of pcDNA3 (open circles) and CD74 (closed circles) transfected cells were incubated for indicated hours, and the remaining Aβ40 and Aβ42 were measured as in D. F) HeLa cells were transfected with pcDNA3 or FLAG-CD74 together with APP. The cells were pulsed for 30 min with 35 S methionine+cysteine, and chased in fresh media for indicated hours. APP was immunoprecipitated with αAPPct antibody and analyzed by SDS-PAGE followed by autoradiography. G) HeLa cells were transfected with pcDNA3 (-) or FLAG-CD74 (+) together with wild type APP (wt APP) or the indicated mutants. The total lysates and FLAG IPs were processed as in Figure 1B. Note that APP Ncas lacks C-terminal 31 amino acids of APP and has therefore a smaller in size.

    Journal: Molecular Neurodegeneration

    Article Title: CD74 interacts with APP and suppresses the production of A?

    doi: 10.1186/1750-1326-4-41

    Figure Lengend Snippet: A) HeLa cells were transfected with pcDNA3 (-) or CD74 (+), together with Notch . The total cell lysates (TL) and FLAG IPs were analyzed as in Figure 1B. Notch was detected with αmyc antibody. B) HeLa cells were transfected with pcDNA3 (-) or CD74 (+), together with APLP2, and were analyzed as in A. C) Left panels: HEK293APP cells were transfected with the indicated plasmids. The transfected cells were further incubated for 24 hours with fresh media, and secreted sAPPα/β were measured by Westerm blot. The cell lysates were analyzed as in Figure 1B. Right panels: the quantification of sAPPα/β. The levels in pcDNA3 transfected cells were set to 100. D) HEK293APP cells were transfected with pcDNA3 or CD74 and treated as in C. Aβ40 and 42 secreted in the media, and Aβ40 in the lysates were measured with specific ELISA. The amount of Aβ was normalized to the amount of the protein in the total lysates. The asterisks (**) indicate that p values by Student's t test are less than 0.01. E) HeLa cells were transfected with pcDNA3 or CD74, and the media was replaced with fresh media containing synthetic Aβ40 and Aβ42. The media of pcDNA3 (open circles) and CD74 (closed circles) transfected cells were incubated for indicated hours, and the remaining Aβ40 and Aβ42 were measured as in D. F) HeLa cells were transfected with pcDNA3 or FLAG-CD74 together with APP. The cells were pulsed for 30 min with 35 S methionine+cysteine, and chased in fresh media for indicated hours. APP was immunoprecipitated with αAPPct antibody and analyzed by SDS-PAGE followed by autoradiography. G) HeLa cells were transfected with pcDNA3 (-) or FLAG-CD74 (+) together with wild type APP (wt APP) or the indicated mutants. The total lysates and FLAG IPs were processed as in Figure 1B. Note that APP Ncas lacks C-terminal 31 amino acids of APP and has therefore a smaller in size.

    Article Snippet: Staining of transfected HeLa cells HeLa cells were plated on coverslips coated with poly L-lysine (Sigma), and were transfected with indicated plasmids.

    Techniques: Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, SDS Page, Autoradiography

    A) A cartoon of various FLAG-tagged CD74 constructs: the yeast split-ubiquitin (TH) clone, full length CD74, two deletions constructs of CD74, and two splicing variants of mouse CD74 . The cytoplasmic and luminal parts, the locations of the FLAG tag, the endosomal-sorting signal in the cytoplasmic domain (LL), the CLIP domain (C), and the trimeric domain (T) are indicated. B) HeLa cells were transfected with pcDNA3 (-) or FLAG-CD74 (+) together with APP, and the total cell lysates (TL) and FLAG immunoprecipitants (IP) were analyzed by Westerm blot. Full length APP, APP CTF (C99 and C83), and CD74 were detected with 22C11, αAPPct, and αFLAG antibodies, respectively. The 33 and 14 kDa N-terminal fragments of CD74 were designated as CD74 NTF33 and NTF14. C) HeLa cells were transfected with FLAG-CD74 and APP, and the total lysates of the transfected cells were immunoprecipitated with either rabbit polyclonal antibody (RP) or αAPPct, and analyzed as in B. D) HeLa cells were transfected with various CD74 constructs and APP. The total lysates and FLAG IPs were analyzed as in B. Mature APP and immature APP (mAPP/imAPP) are indicated. The bands marked with asterisks (*) were attributed to the FLAG antibody used in the immunoprecipitation.

    Journal: Molecular Neurodegeneration

    Article Title: CD74 interacts with APP and suppresses the production of A?

    doi: 10.1186/1750-1326-4-41

    Figure Lengend Snippet: A) A cartoon of various FLAG-tagged CD74 constructs: the yeast split-ubiquitin (TH) clone, full length CD74, two deletions constructs of CD74, and two splicing variants of mouse CD74 . The cytoplasmic and luminal parts, the locations of the FLAG tag, the endosomal-sorting signal in the cytoplasmic domain (LL), the CLIP domain (C), and the trimeric domain (T) are indicated. B) HeLa cells were transfected with pcDNA3 (-) or FLAG-CD74 (+) together with APP, and the total cell lysates (TL) and FLAG immunoprecipitants (IP) were analyzed by Westerm blot. Full length APP, APP CTF (C99 and C83), and CD74 were detected with 22C11, αAPPct, and αFLAG antibodies, respectively. The 33 and 14 kDa N-terminal fragments of CD74 were designated as CD74 NTF33 and NTF14. C) HeLa cells were transfected with FLAG-CD74 and APP, and the total lysates of the transfected cells were immunoprecipitated with either rabbit polyclonal antibody (RP) or αAPPct, and analyzed as in B. D) HeLa cells were transfected with various CD74 constructs and APP. The total lysates and FLAG IPs were analyzed as in B. Mature APP and immature APP (mAPP/imAPP) are indicated. The bands marked with asterisks (*) were attributed to the FLAG antibody used in the immunoprecipitation.

    Article Snippet: Staining of transfected HeLa cells HeLa cells were plated on coverslips coated with poly L-lysine (Sigma), and were transfected with indicated plasmids.

    Techniques: Construct, FLAG-tag, Cross-linking Immunoprecipitation, Transfection, Immunoprecipitation

    A) HeLa cells were transfected with APP-YFP or FLAG-CD74 . APP and CD74 were detected by YFP fluorescence and αFLAG antibody staining with Alexa594 anti-mouse secondary antibody, respectively. Corresponding phase contrast views were shown. B) HeLa cells were transfected with APP-YFP or Notch-GFP together with FLAG-CD74. Notch was detected by GFP fluorescence. APP and CD74 were detected as in A. Digitally merged pictures of Green and Red channels and phase contrast view are also displayed. Colocalization Coefficient (CC) and Intensity Correlation Quotient (ICQ) were indicated. C) HeLa cells were transfected with APP, C99 or C83 together with FLAG-CD74. APP, C99, and C83 were detected with the αAPPct antibody, and FLAG-CD74 was detected as above. Merged pictures and phase contrast views were shown as in B. Bar = 10 μm. CC and ICQ are as in B.

    Journal: Molecular Neurodegeneration

    Article Title: CD74 interacts with APP and suppresses the production of A?

    doi: 10.1186/1750-1326-4-41

    Figure Lengend Snippet: A) HeLa cells were transfected with APP-YFP or FLAG-CD74 . APP and CD74 were detected by YFP fluorescence and αFLAG antibody staining with Alexa594 anti-mouse secondary antibody, respectively. Corresponding phase contrast views were shown. B) HeLa cells were transfected with APP-YFP or Notch-GFP together with FLAG-CD74. Notch was detected by GFP fluorescence. APP and CD74 were detected as in A. Digitally merged pictures of Green and Red channels and phase contrast view are also displayed. Colocalization Coefficient (CC) and Intensity Correlation Quotient (ICQ) were indicated. C) HeLa cells were transfected with APP, C99 or C83 together with FLAG-CD74. APP, C99, and C83 were detected with the αAPPct antibody, and FLAG-CD74 was detected as above. Merged pictures and phase contrast views were shown as in B. Bar = 10 μm. CC and ICQ are as in B.

    Article Snippet: Staining of transfected HeLa cells HeLa cells were plated on coverslips coated with poly L-lysine (Sigma), and were transfected with indicated plasmids.

    Techniques: Transfection, Fluorescence, Staining

    Endosomal tubulation in spastin-depleted cells requires intact MTs. (a) HeLa cells depleted of spastin by transfection with an siRNA pool were labeled with antibodies to endogenous SNX1 and α-tubulin (MT). In this and subsequent color images, the color of the lettering in the black and white images indicates the color of that image in the corresponding merged image. Arrowheads in the magnified images of the boxed areas indicate an aligned SNX1 tubule and MT. (b–d) Mock-transfected cells or cells depleted of spastin by transfection with an siRNA pool were treated with vehicle (DMSO) or the MT-depolymerizing agent nocodazole (b), the MT stabilizing agent taxol (c), or the actin-depolymerizing agent latrunculin (d), and then, the number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three (or four for latrunculin treatment) independent experiments were plotted. Representative immunofluorescence images from these experiments are shown in Fig. S2 . KD, knockdown. Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: Endosomal tubulation in spastin-depleted cells requires intact MTs. (a) HeLa cells depleted of spastin by transfection with an siRNA pool were labeled with antibodies to endogenous SNX1 and α-tubulin (MT). In this and subsequent color images, the color of the lettering in the black and white images indicates the color of that image in the corresponding merged image. Arrowheads in the magnified images of the boxed areas indicate an aligned SNX1 tubule and MT. (b–d) Mock-transfected cells or cells depleted of spastin by transfection with an siRNA pool were treated with vehicle (DMSO) or the MT-depolymerizing agent nocodazole (b), the MT stabilizing agent taxol (c), or the actin-depolymerizing agent latrunculin (d), and then, the number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three (or four for latrunculin treatment) independent experiments were plotted. Representative immunofluorescence images from these experiments are shown in Fig. S2 . KD, knockdown. Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Transfection, Labeling, Immunofluorescence

    Ist1 is required for sorting TfnR away from degradation. (a and b) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 (IST1 knockdown [KD]) were immunoblotted for the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in b. GAPDH immunoblotting serves as a loading control. (c and d) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 were treated with vehicle (DMSO) or DMSO + bafilomycin and then immunoblotted versus the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in d. (e) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 were labeled for TfnR. Increased TfnR tubulation is seen in the IST1-depleted cells (see arrowheads in inset magnified region of the boxed area). The exposure settings in the IST1 knockdown image have been increased to compensate for the reduced TfnR levels in these cells. (f–h) Confocal micrographs of HeLa cells subjected to mock transfection (f) or transfected with IST1 siRNA1 (g and h) and then labeled with the markers shown. Confocal micrograph gain settings were identical in f and g, but in h, which shows higher magnification images of the dashed areas indicated in g, gain settings were increased to reveal that the remaining TfnR signal strongly colocalized with M6PR. Insets are magnified areas of the boxed regions. (i–k) Uptake of Alexa Fluor Tfn 647 was measured by FACS over a 20-min time course in mock-transfected HeLa cells, cells transfected with spastin siRNA1 (as a positive control), and IST1 siRNA1 (i) or IST1 siRNA3 (j). Mean Tfn uptake at the 20-min time point is shown ( n = 3 experiments plotted for each histogram). A representative time course experiment is shown in k. Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: Ist1 is required for sorting TfnR away from degradation. (a and b) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 (IST1 knockdown [KD]) were immunoblotted for the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in b. GAPDH immunoblotting serves as a loading control. (c and d) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 were treated with vehicle (DMSO) or DMSO + bafilomycin and then immunoblotted versus the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in d. (e) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 were labeled for TfnR. Increased TfnR tubulation is seen in the IST1-depleted cells (see arrowheads in inset magnified region of the boxed area). The exposure settings in the IST1 knockdown image have been increased to compensate for the reduced TfnR levels in these cells. (f–h) Confocal micrographs of HeLa cells subjected to mock transfection (f) or transfected with IST1 siRNA1 (g and h) and then labeled with the markers shown. Confocal micrograph gain settings were identical in f and g, but in h, which shows higher magnification images of the dashed areas indicated in g, gain settings were increased to reveal that the remaining TfnR signal strongly colocalized with M6PR. Insets are magnified areas of the boxed regions. (i–k) Uptake of Alexa Fluor Tfn 647 was measured by FACS over a 20-min time course in mock-transfected HeLa cells, cells transfected with spastin siRNA1 (as a positive control), and IST1 siRNA1 (i) or IST1 siRNA3 (j). Mean Tfn uptake at the 20-min time point is shown ( n = 3 experiments plotted for each histogram). A representative time course experiment is shown in k. Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Transfection, Labeling, FACS, Positive Control

    IST1 regulates endosomal tubulation. (a and b) Wild-type HeLa cells were subjected to mock transfection or transfected with either of the two IST1 siRNA oligonucleotides indicated and then labeled with SNX1. The number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three independent experiments were plotted in b. (c) Depletion of IST1 was verified by immunoblotting. (d–g) Wild-type HeLa cells (d) or HeLa cells stably expressing myc-tagged siRNA-resistant IST1 (e) were subjected to mock transfection or were transfected with an siRNA targeting endogenous IST1. The number of SNX1 tubules per cell was counted as in a. To control for any variation in the baseline number of tubules per cell in the two cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in f; n = 3 independent experiments. wt, wild type. (g) Depletion of endogenous IST1 was verified by immunoblotting. (h) HeLa cells depleted of IST1 by transfection with IST1 siRNA1 were labeled with antibodies to endogenous SNX1 and α-tubulin (MT). Arrowheads in the magnified images of the boxed areas indicate an aligned SNX1 tubule and MT. (i–k) Mock-transfected cells or cells depleted of IST1 by transfection with siRNA1 were treated with vehicle (DMSO), nocodazole (i), taxol (j), or latrunculin A (k), and then, the number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three independent experiments were plotted. KD, knockdown. Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: IST1 regulates endosomal tubulation. (a and b) Wild-type HeLa cells were subjected to mock transfection or transfected with either of the two IST1 siRNA oligonucleotides indicated and then labeled with SNX1. The number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three independent experiments were plotted in b. (c) Depletion of IST1 was verified by immunoblotting. (d–g) Wild-type HeLa cells (d) or HeLa cells stably expressing myc-tagged siRNA-resistant IST1 (e) were subjected to mock transfection or were transfected with an siRNA targeting endogenous IST1. The number of SNX1 tubules per cell was counted as in a. To control for any variation in the baseline number of tubules per cell in the two cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in f; n = 3 independent experiments. wt, wild type. (g) Depletion of endogenous IST1 was verified by immunoblotting. (h) HeLa cells depleted of IST1 by transfection with IST1 siRNA1 were labeled with antibodies to endogenous SNX1 and α-tubulin (MT). Arrowheads in the magnified images of the boxed areas indicate an aligned SNX1 tubule and MT. (i–k) Mock-transfected cells or cells depleted of IST1 by transfection with siRNA1 were treated with vehicle (DMSO), nocodazole (i), taxol (j), or latrunculin A (k), and then, the number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three independent experiments were plotted. KD, knockdown. Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Transfection, Labeling, Stable Transfection, Expressing

    M1 and M87 spastin are recruited to endosomes and regulate endosomal tubulation. (a and b) GFP-VPS4-E235Q was transiently transfected into cell lines stably expressing myc-tagged M87 spastin (a) or myc-tagged M1 spastin M87A (b), and then, the cells were labeled with an anti-myc antibody. (c–f) HeLa cells (c), HeLa cells stably expressing myc-tagged M87 spastin (d), or HeLa cells expressing myc-tagged M1 spastinM87A (e) were subjected to mock transfection, transfected with an siRNA oligonucleotide directed against endogenous spastin (spastin 1, to which the myc-tagged transcripts were resistant), or with a combination of two siRNA oligonucleotides that together targeted endogenous and transfected spastin (spastin 1 and 6). The cells were labeled with an antibody to endogenous SNX1, and the number of SNX1 tubules per cell was counted (30 cells per condition). To control for any variation in the baseline number of tubules per cell in different cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in f; n = 3 independent experiments. Cellular depletion of exogenous and/or endogenous spastin in these experiments was verified by immunofluorescence and immunoblotting ( Fig. S3 ). Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: M1 and M87 spastin are recruited to endosomes and regulate endosomal tubulation. (a and b) GFP-VPS4-E235Q was transiently transfected into cell lines stably expressing myc-tagged M87 spastin (a) or myc-tagged M1 spastin M87A (b), and then, the cells were labeled with an anti-myc antibody. (c–f) HeLa cells (c), HeLa cells stably expressing myc-tagged M87 spastin (d), or HeLa cells expressing myc-tagged M1 spastinM87A (e) were subjected to mock transfection, transfected with an siRNA oligonucleotide directed against endogenous spastin (spastin 1, to which the myc-tagged transcripts were resistant), or with a combination of two siRNA oligonucleotides that together targeted endogenous and transfected spastin (spastin 1 and 6). The cells were labeled with an antibody to endogenous SNX1, and the number of SNX1 tubules per cell was counted (30 cells per condition). To control for any variation in the baseline number of tubules per cell in different cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in f; n = 3 independent experiments. Cellular depletion of exogenous and/or endogenous spastin in these experiments was verified by immunofluorescence and immunoblotting ( Fig. S3 ). Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Transfection, Stable Transfection, Expressing, Labeling, Immunofluorescence

    ATPase activity and interaction with ESCRT-III are required for spastin to regulate endosomal tubulation. (a–d) Wild-type HeLa cells (a), HeLa cell lines stably expressing myc-tagged M87 spastin (b), myc-tagged M87 spastinK388R (c), or myc-tagged M87 spastinF124D (d) were subjected to mock transfection or were transfected with an siRNA oligonucleotide directed against endogenous spastin (spastin 1, to which the myc-tagged transcripts were resistant) or with siRNA oligonucleotides directed against endogenous and exogenous spastin (spastin 1 and 6). Cellular depletion of exogenous and/or endogenous spastin in these experiments was verified by immunofluorescence and immunoblotting ( Fig. S3 ). The number of SNX1 tubules per cell was counted (30 cells per condition). To control for any variation in the baseline number of tubules per cell in different cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in e; n = 3 independent experiments. (f–h) GFP-tagged VPS4-E235Q was transiently transfected into HeLa cells stably expressing myc-tagged wild-type (wt) M87 spastin (f) or myc-tagged M87 spastinF124D (g), which has dramatically reduced binding to CHMP1B and IST1. The cells were labeled with anti-myc antibodies. The extent of colocalization between GFP-VPS4-E235Q and the spastin proteins was estimated by calculating the Pearson’s correlation coefficient for red and green pixels in each cell, using Volocity software (h; n = 3 experiments, 20 cells per condition quantified in each experiment). Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: ATPase activity and interaction with ESCRT-III are required for spastin to regulate endosomal tubulation. (a–d) Wild-type HeLa cells (a), HeLa cell lines stably expressing myc-tagged M87 spastin (b), myc-tagged M87 spastinK388R (c), or myc-tagged M87 spastinF124D (d) were subjected to mock transfection or were transfected with an siRNA oligonucleotide directed against endogenous spastin (spastin 1, to which the myc-tagged transcripts were resistant) or with siRNA oligonucleotides directed against endogenous and exogenous spastin (spastin 1 and 6). Cellular depletion of exogenous and/or endogenous spastin in these experiments was verified by immunofluorescence and immunoblotting ( Fig. S3 ). The number of SNX1 tubules per cell was counted (30 cells per condition). To control for any variation in the baseline number of tubules per cell in different cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in e; n = 3 independent experiments. (f–h) GFP-tagged VPS4-E235Q was transiently transfected into HeLa cells stably expressing myc-tagged wild-type (wt) M87 spastin (f) or myc-tagged M87 spastinF124D (g), which has dramatically reduced binding to CHMP1B and IST1. The cells were labeled with anti-myc antibodies. The extent of colocalization between GFP-VPS4-E235Q and the spastin proteins was estimated by calculating the Pearson’s correlation coefficient for red and green pixels in each cell, using Volocity software (h; n = 3 experiments, 20 cells per condition quantified in each experiment). Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Activity Assay, Stable Transfection, Expressing, Transfection, Immunofluorescence, Binding Assay, Labeling, Software

    Spastin regulates endosomal tubulation. (a and b) HeLa cells were subject to mock transfection or transfection with a spastin siRNA pool (spastin knockdown [KD]) and labeled versus endogenous SNX1 (a) or transfected SNX4-mCherry (b). The insets in a and b are magnified images of the boxed regions indicated. (c) The mean number of SNX1 and SNX4 tubules per cell was quantified ( n = 5 independent experiments for SNX1, and n = 3 for SNX4, 30 cells counted per experimental condition in each experiment). (d) The length of the longest tubule per cell was measured in 30 cells, and the mean values were plotted. (e) Depletion of spastin was confirmed by immunoblotting. Note that M1 spastin is not routinely seen in immunoblots such as this, in which the exposure is optimized to show the much stronger M87 band. Actin labeling is shown to verify equal sample loading. (f) Complex structures, defined as having at least three tubules emanating from a central punctum (arrowheads), were commonly seen in spastin-depleted cells. (g) The mean number of complex structures per cell was quantified ( n = 3, 30 cells counted per condition in each experiment). Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: Spastin regulates endosomal tubulation. (a and b) HeLa cells were subject to mock transfection or transfection with a spastin siRNA pool (spastin knockdown [KD]) and labeled versus endogenous SNX1 (a) or transfected SNX4-mCherry (b). The insets in a and b are magnified images of the boxed regions indicated. (c) The mean number of SNX1 and SNX4 tubules per cell was quantified ( n = 5 independent experiments for SNX1, and n = 3 for SNX4, 30 cells counted per experimental condition in each experiment). (d) The length of the longest tubule per cell was measured in 30 cells, and the mean values were plotted. (e) Depletion of spastin was confirmed by immunoblotting. Note that M1 spastin is not routinely seen in immunoblots such as this, in which the exposure is optimized to show the much stronger M87 band. Actin labeling is shown to verify equal sample loading. (f) Complex structures, defined as having at least three tubules emanating from a central punctum (arrowheads), were commonly seen in spastin-depleted cells. (g) The mean number of complex structures per cell was quantified ( n = 3, 30 cells counted per condition in each experiment). Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Transfection, Labeling, Western Blot

    Spastin is required for sorting TfnR away from degradation. (a and b) Mock-transfected HeLa cells or HeLa cells transfected with a pool of spastin siRNA (spastin knockdown [KD]) were immunoblotted for the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in b. (c and d) Mock-transfected HeLa cells or HeLa cells transfected with a pool of spastin siRNA were treated with vehicle (DMSO) or DMSO + bafilomycin and then immunoblotted versus the antibodies indicated. TfnR band density was quantified, and the mean density in eight independent experiments was plotted in d. (e–g) Confocal micrographs of HeLa cells subjected to mock transfection (e) or transfected with a spastin siRNA pool (f and g) and then labeled with the markers shown. Confocal micrograph gain settings were identical in e and f, but in g, which shows a higher magnification image of the dashed area indicated in f, gain settings were increased to reveal that the remaining TfnR signal strongly colocalized with M6PR. Note that tubular TfnR structures are not readily seen under confocal microscopy, as the tubules tend to leave the plane of section. Insets are magnified regions of the boxed areas. (h) Mock-transfected HeLa cells or HeLa cells transfected with a pool of spastin siRNA were fixed and labeled (without permeabilization) with a FITC-conjugated antibody against TfnR, and then, the cell-associated fluorescent signal was quantified by FACS analysis. The mean fluorescence values for three independent experiments were plotted. (i–k) Uptake of Alexa Fluor 647–conjugated Tfn was measured by FACS over a 20-min time course in mock-transfected HeLa cells, cells transfected with siRNA targeting clathrin heavy chain (CHC), or cells transfected with one of two siRNAs directed against spastin (spas1, spastin 1; spas3, spastin 3). Mean Tfn uptake at the 20-min time point ( n = 3 experiments) is shown in i, and a representative time course experiment is shown in j. Depletion of the relevant proteins targeted by siRNA was confirmed by immunoblotting, and a representative example is shown in k. (l and m) Recycling of internalized fluorescently labeled Tfn was measured over a 20-min time course, and the mean cell-associated Tfn at the 20-min time point ( n = 3 experiments) is shown in l, with a representative time course experiment shown in m. Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: Spastin is required for sorting TfnR away from degradation. (a and b) Mock-transfected HeLa cells or HeLa cells transfected with a pool of spastin siRNA (spastin knockdown [KD]) were immunoblotted for the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in b. (c and d) Mock-transfected HeLa cells or HeLa cells transfected with a pool of spastin siRNA were treated with vehicle (DMSO) or DMSO + bafilomycin and then immunoblotted versus the antibodies indicated. TfnR band density was quantified, and the mean density in eight independent experiments was plotted in d. (e–g) Confocal micrographs of HeLa cells subjected to mock transfection (e) or transfected with a spastin siRNA pool (f and g) and then labeled with the markers shown. Confocal micrograph gain settings were identical in e and f, but in g, which shows a higher magnification image of the dashed area indicated in f, gain settings were increased to reveal that the remaining TfnR signal strongly colocalized with M6PR. Note that tubular TfnR structures are not readily seen under confocal microscopy, as the tubules tend to leave the plane of section. Insets are magnified regions of the boxed areas. (h) Mock-transfected HeLa cells or HeLa cells transfected with a pool of spastin siRNA were fixed and labeled (without permeabilization) with a FITC-conjugated antibody against TfnR, and then, the cell-associated fluorescent signal was quantified by FACS analysis. The mean fluorescence values for three independent experiments were plotted. (i–k) Uptake of Alexa Fluor 647–conjugated Tfn was measured by FACS over a 20-min time course in mock-transfected HeLa cells, cells transfected with siRNA targeting clathrin heavy chain (CHC), or cells transfected with one of two siRNAs directed against spastin (spas1, spastin 1; spas3, spastin 3). Mean Tfn uptake at the 20-min time point ( n = 3 experiments) is shown in i, and a representative time course experiment is shown in j. Depletion of the relevant proteins targeted by siRNA was confirmed by immunoblotting, and a representative example is shown in k. (l and m) Recycling of internalized fluorescently labeled Tfn was measured over a 20-min time course, and the mean cell-associated Tfn at the 20-min time point ( n = 3 experiments) is shown in l, with a representative time course experiment shown in m. Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Transfection, Labeling, Confocal Microscopy, FACS, Fluorescence

    GFP as a tag in immunoaffinity experiments. Although a commercial monoclonal anti-GFP antibody is capable of isolating significant amounts of free GFP from a stable HeLa cell line, the GFP binder is more efficient, as demonstrated both by Coomassie staining of protein eluted from the affinity matrices (A) and Western blotting using anti-GFP antibodies (B). Whether the mAb or GFP binder is used to purify GFP, there are very few proteins that bind nonspecifically to this tag (C). Four independent experiments were performed to identify proteins that may copurify with GFP, as indicated by SILAC ratios greater than 1 (IP1: whole cell extract, GFP binder; IP2: whole cell extract, monoclonal anti-GFP antibody; IP3: cytoplasmic extract, monoclonal anti-GFP antibody; IP4: nuclear extract, monoclonal anti-GFP antibody). No one protein was identified in every experiment, and most of them (in bold) have been identified as binding nonspecifically to the Sepharose bead matrix. This list was then screened against a set of 18 independent GFP protein immunoaffinity experiments performed using the GFP binder for purification and parental cells as the negative control. Proteins were scored for the percentage of experiments in which they were detected (yellow), and for the percentage of experiments in which they were detected and showed a SILAC ratio greater than 1 (green). Six proteins, representing three protein classes (heat shock/chaperone, cytokeratin, and ubiquitin), have been highlighted in green as the most frequently detected and potentially able to bind GFP.

    Journal: The Journal of Cell Biology

    Article Title: Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

    doi: 10.1083/jcb.200805092

    Figure Lengend Snippet: GFP as a tag in immunoaffinity experiments. Although a commercial monoclonal anti-GFP antibody is capable of isolating significant amounts of free GFP from a stable HeLa cell line, the GFP binder is more efficient, as demonstrated both by Coomassie staining of protein eluted from the affinity matrices (A) and Western blotting using anti-GFP antibodies (B). Whether the mAb or GFP binder is used to purify GFP, there are very few proteins that bind nonspecifically to this tag (C). Four independent experiments were performed to identify proteins that may copurify with GFP, as indicated by SILAC ratios greater than 1 (IP1: whole cell extract, GFP binder; IP2: whole cell extract, monoclonal anti-GFP antibody; IP3: cytoplasmic extract, monoclonal anti-GFP antibody; IP4: nuclear extract, monoclonal anti-GFP antibody). No one protein was identified in every experiment, and most of them (in bold) have been identified as binding nonspecifically to the Sepharose bead matrix. This list was then screened against a set of 18 independent GFP protein immunoaffinity experiments performed using the GFP binder for purification and parental cells as the negative control. Proteins were scored for the percentage of experiments in which they were detected (yellow), and for the percentage of experiments in which they were detected and showed a SILAC ratio greater than 1 (green). Six proteins, representing three protein classes (heat shock/chaperone, cytokeratin, and ubiquitin), have been highlighted in green as the most frequently detected and potentially able to bind GFP.

    Article Snippet: For fixed cell imaging, a mix of parental HeLa cells and HeLa cells stably expressing GFP-SMN were paraformaldehyde fixed on glass coverslips, permeabilized with Triton X-100, stained with both anti-USP9X (detected by TRITC-anti–mouse secondary antibodies) and the DNA stain DAPI, and mounted in FluorSave mounting media (Calbiochem).

    Techniques: Staining, Western Blot, Binding Assay, Purification, Negative Control

    Identification of proteins that interact with SMN and the SMN complex. The GFP binder was used to immunopurify GFP-SMN from a stable HeLa cell line as compared with the nonexpressing parental cell line. Like endogenous SMN, GFP-SMN is found in both cytoplasmic and nucleoplasmic pools and accumulates in gems within nuclei (A). Bar, 15 μM. Detailed biochemical and proteomic studies have revealed that the core SMN complex is composed of SMN itself and Gemins 2–8 (B). The stoichiometry is not known and, although not depicted here, the complex can oligomerize. Also listed are several other proteins that have been shown to interact with the SMN complex by similar experimental approaches. In the study presented here, separate experiments were performed for cytoplasmic and nuclear extracts to independently assess interacting partners and compare these two pools. The log SILAC (i.e., heavy/light arginine and/or lysine) ratio calculated for each protein identified in the cytoplasmic GFP-SMN immunoprecipitation experiment is plotted versus total peptide intensity in C. The nucleoplasmic GFP-SMN immunoprecipitation data are plotted in a similar fashion (D).

    Journal: The Journal of Cell Biology

    Article Title: Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

    doi: 10.1083/jcb.200805092

    Figure Lengend Snippet: Identification of proteins that interact with SMN and the SMN complex. The GFP binder was used to immunopurify GFP-SMN from a stable HeLa cell line as compared with the nonexpressing parental cell line. Like endogenous SMN, GFP-SMN is found in both cytoplasmic and nucleoplasmic pools and accumulates in gems within nuclei (A). Bar, 15 μM. Detailed biochemical and proteomic studies have revealed that the core SMN complex is composed of SMN itself and Gemins 2–8 (B). The stoichiometry is not known and, although not depicted here, the complex can oligomerize. Also listed are several other proteins that have been shown to interact with the SMN complex by similar experimental approaches. In the study presented here, separate experiments were performed for cytoplasmic and nuclear extracts to independently assess interacting partners and compare these two pools. The log SILAC (i.e., heavy/light arginine and/or lysine) ratio calculated for each protein identified in the cytoplasmic GFP-SMN immunoprecipitation experiment is plotted versus total peptide intensity in C. The nucleoplasmic GFP-SMN immunoprecipitation data are plotted in a similar fashion (D).

    Article Snippet: For fixed cell imaging, a mix of parental HeLa cells and HeLa cells stably expressing GFP-SMN were paraformaldehyde fixed on glass coverslips, permeabilized with Triton X-100, stained with both anti-USP9X (detected by TRITC-anti–mouse secondary antibodies) and the DNA stain DAPI, and mounted in FluorSave mounting media (Calbiochem).

    Techniques: Immunoprecipitation

    Protocols used for SILAC-based analysis of protein interaction partners in pull-down experiments. (A) HeLa cells expressing a GFP-tagged protein are metabolically labeled by culturing in “heavy” media containing 13 C-isotopes of arginine and lysine, while the parental HeLa cells are grown in “light” media containing the 12 C-isotopes of arginine and lysine. Whole cell extracts can be prepared or, as shown here, cells can be fractionated for preparation of separate cytoplasmic and nuclear extracts. In this case, extracts are pre-cleared on Sepharose beads and then mixed in equal amounts before affinity purification of the GFP-tagged protein using the GFP binder (1 h incubation). Proteins are eluted from the beads and separated by 1D SDS-PAGE for digestion and LC-MS/MS analysis. (B) For SILAC analysis of an endogenous protein, two populations of HeLa cells are grown in light and heavy media, respectively, before harvesting and preparation of cellular extracts. Equal total protein amounts of each extract are subjected to separate immunoaffinity experiments, either using an antibody to the protein of interest or a control antibody covalently bound to beads at an equivalent concentration. The separate immunoprecipitates are mixed carefully to minimize variability and the proteins eluted and analyzed as described above.

    Journal: The Journal of Cell Biology

    Article Title: Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

    doi: 10.1083/jcb.200805092

    Figure Lengend Snippet: Protocols used for SILAC-based analysis of protein interaction partners in pull-down experiments. (A) HeLa cells expressing a GFP-tagged protein are metabolically labeled by culturing in “heavy” media containing 13 C-isotopes of arginine and lysine, while the parental HeLa cells are grown in “light” media containing the 12 C-isotopes of arginine and lysine. Whole cell extracts can be prepared or, as shown here, cells can be fractionated for preparation of separate cytoplasmic and nuclear extracts. In this case, extracts are pre-cleared on Sepharose beads and then mixed in equal amounts before affinity purification of the GFP-tagged protein using the GFP binder (1 h incubation). Proteins are eluted from the beads and separated by 1D SDS-PAGE for digestion and LC-MS/MS analysis. (B) For SILAC analysis of an endogenous protein, two populations of HeLa cells are grown in light and heavy media, respectively, before harvesting and preparation of cellular extracts. Equal total protein amounts of each extract are subjected to separate immunoaffinity experiments, either using an antibody to the protein of interest or a control antibody covalently bound to beads at an equivalent concentration. The separate immunoprecipitates are mixed carefully to minimize variability and the proteins eluted and analyzed as described above.

    Article Snippet: For fixed cell imaging, a mix of parental HeLa cells and HeLa cells stably expressing GFP-SMN were paraformaldehyde fixed on glass coverslips, permeabilized with Triton X-100, stained with both anti-USP9X (detected by TRITC-anti–mouse secondary antibodies) and the DNA stain DAPI, and mounted in FluorSave mounting media (Calbiochem).

    Techniques: Expressing, Metabolic Labelling, Labeling, Affinity Purification, Incubation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Concentration Assay

    Validation of mass spectrometric results. Cytoplasmic-specific copurification of the novel protein USP9X with GFP-SMN was confirmed by Western blotting (A). Two peptides, each with a SILAC ratio > 1, were found for USP9X in the SILAC analysis of a GFP-SMN pull-down from cytoplasmic extracts. The mass spectra of one of them is shown here for comparison (B). The quantifiable arginine is highlighted in red. This cytoplasmic enrichment of USP9X is consistent with immunostaining results using a monoclonal anti-USP9X antibody (C). Although predominantly cytoplasmic, there is a pool of USP9X in the nucleus (arrowhead), although it does not accumulate in gems (arrow). There is no difference in localization of USP9X in parental HeLa cells (top cell) versus HeLa cells stably expressing GFP-SMN (bottom cell). Bar, 5 μM. As a control, Western blotting was also used to confirm the enrichment of both endogenous SMN and GFP-SMN, and of the U1 snRNP protein U1A, from both cytoplasmic and nuclear extracts using the GFP binder, and the nuclear-specific enrichment of p80 coilin (D). For comparison, representative peptide spectra for these proteins from the SILAC analysis are shown (E). Quantifiable amino acids are highlighted in red, with the SILAC ratio in parentheses.

    Journal: The Journal of Cell Biology

    Article Title: Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

    doi: 10.1083/jcb.200805092

    Figure Lengend Snippet: Validation of mass spectrometric results. Cytoplasmic-specific copurification of the novel protein USP9X with GFP-SMN was confirmed by Western blotting (A). Two peptides, each with a SILAC ratio > 1, were found for USP9X in the SILAC analysis of a GFP-SMN pull-down from cytoplasmic extracts. The mass spectra of one of them is shown here for comparison (B). The quantifiable arginine is highlighted in red. This cytoplasmic enrichment of USP9X is consistent with immunostaining results using a monoclonal anti-USP9X antibody (C). Although predominantly cytoplasmic, there is a pool of USP9X in the nucleus (arrowhead), although it does not accumulate in gems (arrow). There is no difference in localization of USP9X in parental HeLa cells (top cell) versus HeLa cells stably expressing GFP-SMN (bottom cell). Bar, 5 μM. As a control, Western blotting was also used to confirm the enrichment of both endogenous SMN and GFP-SMN, and of the U1 snRNP protein U1A, from both cytoplasmic and nuclear extracts using the GFP binder, and the nuclear-specific enrichment of p80 coilin (D). For comparison, representative peptide spectra for these proteins from the SILAC analysis are shown (E). Quantifiable amino acids are highlighted in red, with the SILAC ratio in parentheses.

    Article Snippet: For fixed cell imaging, a mix of parental HeLa cells and HeLa cells stably expressing GFP-SMN were paraformaldehyde fixed on glass coverslips, permeabilized with Triton X-100, stained with both anti-USP9X (detected by TRITC-anti–mouse secondary antibodies) and the DNA stain DAPI, and mounted in FluorSave mounting media (Calbiochem).

    Techniques: Copurification, Western Blot, Immunostaining, Stable Transfection, Expressing

    HERV-K CA-NTD and MHR are not required for reduction of HIV-1 infectivity. HeLa cells were cotransfected with pNL4-3 and indicated plasmids. a Virus in the supernatant from cotransfected HeLa cells were collected and fractionated in rate-zonal gradient analysis. The amount of HIV-1 Gag were measured by ELISA. b Virus stocks were prepared from each fraction after rate-zonal gradient analysis. The viruses were purified and normalized by p24 ELISA. TZM-bl cells, which harbor an HIV-1 LTR-driven luciferase-reporter gene, were infected with the purified viruses. At 2 days post-infection, luciferase activities were measured by luminometor. Data from three independent experiments are shown as means ± standard deviations. P values were determined using a Student’s t test. * P

    Journal: Retrovirology

    Article Title: Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity

    doi: 10.1186/s12977-017-0351-8

    Figure Lengend Snippet: HERV-K CA-NTD and MHR are not required for reduction of HIV-1 infectivity. HeLa cells were cotransfected with pNL4-3 and indicated plasmids. a Virus in the supernatant from cotransfected HeLa cells were collected and fractionated in rate-zonal gradient analysis. The amount of HIV-1 Gag were measured by ELISA. b Virus stocks were prepared from each fraction after rate-zonal gradient analysis. The viruses were purified and normalized by p24 ELISA. TZM-bl cells, which harbor an HIV-1 LTR-driven luciferase-reporter gene, were infected with the purified viruses. At 2 days post-infection, luciferase activities were measured by luminometor. Data from three independent experiments are shown as means ± standard deviations. P values were determined using a Student’s t test. * P

    Article Snippet: Cells HeLa cells and TZM-bl cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) supplemented with 5% FBS (DMEM-10).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Purification, Luciferase

    TIN2 is phosphorylated on S295 and S330 during mitosis. ( A ) Detection of S295 and S330 phosphorylation of TIN2 during mitosis by the Phos-tag reagent after release from a double thymidine block. HeLa cells stably infected with a retrovirus encoding Flag-TIN2 in the WT, S330A, or S295A configuration were collected from asynchronous populations (A), populations arrested with a double thymidine block corresponding to the G1/S phase of the cell cycle, or populations at the points corresponding to S, G2, M and early or middle G1 (EG1 or MG1) after release from the double thymidine block. Derived lysates were then either subjected to ( top ) immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody or ( bottom ) resolved by normal SDS-PAGE and immunoblotted with either an anti-Phos-HH3 antibody to monitor cell cycle progression or an anti-Tubulin antibody as a loading control. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP) are denoted on the left of the upper panels. Left and right panels are different exposures. Representative of two experiments. ( B ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in cells arrested with nocodazole. HeLa cells stably infected with a retrovirus encoding no transgene (vector, V) or Flag-TIN2 in the WT, S330A, S295A, or AA configuration were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc). Derived lysates were then subjected to either ( top ) immunoprecipitation (IP) with αFlag and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody or ( bottom ) resolved by normal SDS-PAGE and immunoblotted with either an anti-Phos-HH3 antibody to monitor cell cycle progression or an anti-Tubulin antibody as a loading control. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left of the upper panel. Representative of three experiments. ( C ) Detection of S295 phosphorylation of endogenous TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole. Lysates from HeLa cells were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc), resolved by SDS-PAGE and immunoblotted (IB) with an anti-Phos-S295, anti-TIN2, anti-Phos-HH3, or anti-HH3 (loading control) antibody. Representative of two experiments. ( D ) Detection of S330 phosphorylation of endogenous TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole. HeLa cells were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc). Derived lysates were then either subjected to ( top ) immunoprecipitation (IP) with an anti-TIN2 antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with either an anti-Phos-S330 or anti-TIN2 antibody, or ( bottom ) resolved by normal SDS-PAGE and immunoblotted with an anti-Phos-HH3 antibody, to monitor cell cycle progression, or an anti-Tubulin antibody as a loading control. Representative of one experiment.

    Journal: PLoS ONE

    Article Title: Cell Cycle Regulated Phosphorylation of the Telomere-Associated Protein TIN2

    doi: 10.1371/journal.pone.0071697

    Figure Lengend Snippet: TIN2 is phosphorylated on S295 and S330 during mitosis. ( A ) Detection of S295 and S330 phosphorylation of TIN2 during mitosis by the Phos-tag reagent after release from a double thymidine block. HeLa cells stably infected with a retrovirus encoding Flag-TIN2 in the WT, S330A, or S295A configuration were collected from asynchronous populations (A), populations arrested with a double thymidine block corresponding to the G1/S phase of the cell cycle, or populations at the points corresponding to S, G2, M and early or middle G1 (EG1 or MG1) after release from the double thymidine block. Derived lysates were then either subjected to ( top ) immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody or ( bottom ) resolved by normal SDS-PAGE and immunoblotted with either an anti-Phos-HH3 antibody to monitor cell cycle progression or an anti-Tubulin antibody as a loading control. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP) are denoted on the left of the upper panels. Left and right panels are different exposures. Representative of two experiments. ( B ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in cells arrested with nocodazole. HeLa cells stably infected with a retrovirus encoding no transgene (vector, V) or Flag-TIN2 in the WT, S330A, S295A, or AA configuration were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc). Derived lysates were then subjected to either ( top ) immunoprecipitation (IP) with αFlag and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody or ( bottom ) resolved by normal SDS-PAGE and immunoblotted with either an anti-Phos-HH3 antibody to monitor cell cycle progression or an anti-Tubulin antibody as a loading control. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left of the upper panel. Representative of three experiments. ( C ) Detection of S295 phosphorylation of endogenous TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole. Lysates from HeLa cells were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc), resolved by SDS-PAGE and immunoblotted (IB) with an anti-Phos-S295, anti-TIN2, anti-Phos-HH3, or anti-HH3 (loading control) antibody. Representative of two experiments. ( D ) Detection of S330 phosphorylation of endogenous TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole. HeLa cells were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc). Derived lysates were then either subjected to ( top ) immunoprecipitation (IP) with an anti-TIN2 antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with either an anti-Phos-S330 or anti-TIN2 antibody, or ( bottom ) resolved by normal SDS-PAGE and immunoblotted with an anti-Phos-HH3 antibody, to monitor cell cycle progression, or an anti-Tubulin antibody as a loading control. Representative of one experiment.

    Article Snippet: Cell Synchronization by Nocodazole Arrest and Double Thymidine Block 106 of the described stably infected HeLa cells were either left untreated, arrested in G2/M by overnight treatment with 0.6 µg/ml nocodazole (catalogue # M1404, Sigma), or synchronized at G1/S by the double thymidine block, as outlined below.

    Techniques: Blocking Assay, Stable Transfection, Infection, Derivative Assay, Immunoprecipitation, SDS Page, Plasmid Preparation

    TIN2 is phosphorylated by the mitotic kinase RSK2. ( A ) Detection of S330 phosphorylation of TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole and treated with kinase inhibitors. HeLa cells stably expressing wild-type Flag-TIN2 were treated with DMSO, H-89, BI-D1870, BI 2536 or VX-680 in the presence of either nocodazole (Noc) or vehicle (DMSO). Derived lysates were immunoprecipitated (IP) with an anti-Flag antibody, resolved by SDS-PAGE, and immunoblotted (IB) with an anti-Phos-S330 antibody or, as a loading control, an anti-TIN2 antibody. Representative of two experiments. ( B ) DNA profiles of HeLa cells treated with BI-D1870. HeLa cells treated with DMSO, nocodazole (Noc), or nocodazole+ BI-D1870 were harvested, stained with propidium iodide, and subjected to fluorescence-activated cell sorting (FACS) analysis. Representative of two experiments. ( C ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous or nocodazole arrested cells with or without the RSK2 inhibitor BI-D1870. 293T cells were either untreated or treated with nocodazole (Noc), BI-D1870, or both compounds. Derived lysates were then subjected to immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left. Representative of two experiments. ( D ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous cells with ectopic RSK2 and/or the RSK2 inhibitor BI-D1870. 293T cells transiently transfected with Flag-TIN2 and the Y707A constitutively active mutant form of RSK2 (Flag-RSK2 Y707A ) were either left untreated or treated with RSK kinase inhibitor BI-D1870. Derived lysates were split into two portions. The first portions were subjected to immunoprecipitation (IP) with an anti-Flag antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left ( top ). The second portions were resolved by normal SDS-PAGE and immunoblotted with either an anti-Phospho-S6 antibody to monitor RSK2 kinase activity, or an anti-Tubulin antibody as a loading control ( bottom ). Representative of two experiments. ( E ) Detection of TIN2 phosphorylation by RSK2 in vitro . Recombinant maltose-binding protein (MBP) or N-terminal MBP-tagged TIN2 (MBP-TIN2) in the WT, S295A, S330A, or AA mutant configuration were captured with amylose resin and eluted with maltose. No protein (-) or equal amounts of the aforementioned purified MBP-TIN2 proteins were incubated with recombinant N-terminal 6His-tagged RSK2 (6His-RSK2) in the presence of ATP 32 , after which the reaction products were resolved by SDS-PAGE and either ( top ) exposed to autographic film or ( bottom ) stained with Coomassie Brilliant Blue (CBB staining). Phosphorylated (P 32 ) MBP-TIN2 and a non-specific band (*) are denoted on the left top panel. MBP-TIN2 and MBP are denoted on the left bottom panel. Representative of two experiments.

    Journal: PLoS ONE

    Article Title: Cell Cycle Regulated Phosphorylation of the Telomere-Associated Protein TIN2

    doi: 10.1371/journal.pone.0071697

    Figure Lengend Snippet: TIN2 is phosphorylated by the mitotic kinase RSK2. ( A ) Detection of S330 phosphorylation of TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole and treated with kinase inhibitors. HeLa cells stably expressing wild-type Flag-TIN2 were treated with DMSO, H-89, BI-D1870, BI 2536 or VX-680 in the presence of either nocodazole (Noc) or vehicle (DMSO). Derived lysates were immunoprecipitated (IP) with an anti-Flag antibody, resolved by SDS-PAGE, and immunoblotted (IB) with an anti-Phos-S330 antibody or, as a loading control, an anti-TIN2 antibody. Representative of two experiments. ( B ) DNA profiles of HeLa cells treated with BI-D1870. HeLa cells treated with DMSO, nocodazole (Noc), or nocodazole+ BI-D1870 were harvested, stained with propidium iodide, and subjected to fluorescence-activated cell sorting (FACS) analysis. Representative of two experiments. ( C ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous or nocodazole arrested cells with or without the RSK2 inhibitor BI-D1870. 293T cells were either untreated or treated with nocodazole (Noc), BI-D1870, or both compounds. Derived lysates were then subjected to immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left. Representative of two experiments. ( D ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous cells with ectopic RSK2 and/or the RSK2 inhibitor BI-D1870. 293T cells transiently transfected with Flag-TIN2 and the Y707A constitutively active mutant form of RSK2 (Flag-RSK2 Y707A ) were either left untreated or treated with RSK kinase inhibitor BI-D1870. Derived lysates were split into two portions. The first portions were subjected to immunoprecipitation (IP) with an anti-Flag antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left ( top ). The second portions were resolved by normal SDS-PAGE and immunoblotted with either an anti-Phospho-S6 antibody to monitor RSK2 kinase activity, or an anti-Tubulin antibody as a loading control ( bottom ). Representative of two experiments. ( E ) Detection of TIN2 phosphorylation by RSK2 in vitro . Recombinant maltose-binding protein (MBP) or N-terminal MBP-tagged TIN2 (MBP-TIN2) in the WT, S295A, S330A, or AA mutant configuration were captured with amylose resin and eluted with maltose. No protein (-) or equal amounts of the aforementioned purified MBP-TIN2 proteins were incubated with recombinant N-terminal 6His-tagged RSK2 (6His-RSK2) in the presence of ATP 32 , after which the reaction products were resolved by SDS-PAGE and either ( top ) exposed to autographic film or ( bottom ) stained with Coomassie Brilliant Blue (CBB staining). Phosphorylated (P 32 ) MBP-TIN2 and a non-specific band (*) are denoted on the left top panel. MBP-TIN2 and MBP are denoted on the left bottom panel. Representative of two experiments.

    Article Snippet: Cell Synchronization by Nocodazole Arrest and Double Thymidine Block 106 of the described stably infected HeLa cells were either left untreated, arrested in G2/M by overnight treatment with 0.6 µg/ml nocodazole (catalogue # M1404, Sigma), or synchronized at G1/S by the double thymidine block, as outlined below.

    Techniques: Stable Transfection, Expressing, Derivative Assay, Immunoprecipitation, SDS Page, Staining, Fluorescence, FACS, Transfection, Mutagenesis, Activity Assay, In Vitro, Recombinant, Binding Assay, Purification, Incubation

    Influence of the protein corona on the interactions between RGD-targeted TMV nanoparticles (of three different RGD coverages) and different cell lines A, TMV–HeLa cell interactions in serum-free (sfMEM) and complete (cMEM) media as a function of surface chemistry studied by FACS. Statistical significant differences are indicated by p-values. B, Hypothesis for the promotion of VNP uptake in HeLa cells by coronal proteins. The energy of nonspecific interactions between coronal proteins and cell surface receptors is not sufficient for effective binding of the VNP. However, the RGD-targeted VNPs can “dock”, and uptake is enhanced by nonspecific interactions between coronal proteins and cell surface receptors. C, TMV–RAW264.7 cell interactions. a. D, TMV–SC cell interactions. E, Schematic diagram showing the specificity of RGD-targeted TMV VNPs. Red arrows indicate uptake in cMEM (a protein corona is present); blue arrows indicate uptake in serum-free medium (a protein corona is absent).

    Journal: Small (Weinheim an der Bergstrasse, Germany)

    Article Title: The protein corona of plant virus nanoparticles influences their dispersion properties, cellular interactions and in vivo fates

    doi: 10.1002/smll.201502458

    Figure Lengend Snippet: Influence of the protein corona on the interactions between RGD-targeted TMV nanoparticles (of three different RGD coverages) and different cell lines A, TMV–HeLa cell interactions in serum-free (sfMEM) and complete (cMEM) media as a function of surface chemistry studied by FACS. Statistical significant differences are indicated by p-values. B, Hypothesis for the promotion of VNP uptake in HeLa cells by coronal proteins. The energy of nonspecific interactions between coronal proteins and cell surface receptors is not sufficient for effective binding of the VNP. However, the RGD-targeted VNPs can “dock”, and uptake is enhanced by nonspecific interactions between coronal proteins and cell surface receptors. C, TMV–RAW264.7 cell interactions. a. D, TMV–SC cell interactions. E, Schematic diagram showing the specificity of RGD-targeted TMV VNPs. Red arrows indicate uptake in cMEM (a protein corona is present); blue arrows indicate uptake in serum-free medium (a protein corona is absent).

    Article Snippet: For additional experiments on HeLa cells ( ) cells were resuspended in either (A) cMEM and heat inactivated cMEM (30 minutes incubation in 56°C), or (B) sfMEM and sfMEM supplemented with 1.5 mg/ml total human IgG (I4506 Sigma Aldrich).

    Techniques: FACS, Binding Assay

    Ataxin-3 regulates starvation-induced autophagy. a-b, HeLa cells stably expressing GFP-LC3 were treated with control siRNA or ataxin-3 siRNA and incubated for 1 hr with carrier alone or carrier with 1 µM of PI3P phospholipid. Then, the control cells and the ataxin-3 KD cells with the different treatments were shifted to starvation condition (HBSS, 4 hr) or kept in full-media. a, The number of LC3 dots was analysed for each of the conditions and presented as mean LC3 dots per cell ± s.e.m. S.e.m. was determined from n=5 fields from a single representative experiment out of three independent experiments. Two-way ANOVA (column factor siRNA *** P

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Ataxin-3 regulates starvation-induced autophagy. a-b, HeLa cells stably expressing GFP-LC3 were treated with control siRNA or ataxin-3 siRNA and incubated for 1 hr with carrier alone or carrier with 1 µM of PI3P phospholipid. Then, the control cells and the ataxin-3 KD cells with the different treatments were shifted to starvation condition (HBSS, 4 hr) or kept in full-media. a, The number of LC3 dots was analysed for each of the conditions and presented as mean LC3 dots per cell ± s.e.m. S.e.m. was determined from n=5 fields from a single representative experiment out of three independent experiments. Two-way ANOVA (column factor siRNA *** P

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Stable Transfection, Expressing, Incubation

    Ataxin-3 contributes to autophagosome formation. a, Primary cultures of mouse cortical neurons were transduced with control or ataxin-3 lentiviral shRNAs and analysed for the levels of LC3-I under starvation condition (HBSS, 4 hr) or together with BafA1 (400 nM, 4 hr). Results are normalised to control cells (HBSS+BafA1). Mean ± s.e.m, n=5 replicates from two independent cultures. Two-way ANOVA (N.S not significant). b, HeLa cells were transfected with different ataxin-3 siRNA and scrambled siRNA used as a control. Ataxin-3 knockdown (KD) efficiency is presented as well as basal LC3-II levels. LC3-II levels in ataxin-3-depleted HeLa cells were normalised to control cells, n=4 independent experiments. One-way ANOVA (** P

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Ataxin-3 contributes to autophagosome formation. a, Primary cultures of mouse cortical neurons were transduced with control or ataxin-3 lentiviral shRNAs and analysed for the levels of LC3-I under starvation condition (HBSS, 4 hr) or together with BafA1 (400 nM, 4 hr). Results are normalised to control cells (HBSS+BafA1). Mean ± s.e.m, n=5 replicates from two independent cultures. Two-way ANOVA (N.S not significant). b, HeLa cells were transfected with different ataxin-3 siRNA and scrambled siRNA used as a control. Ataxin-3 knockdown (KD) efficiency is presented as well as basal LC3-II levels. LC3-II levels in ataxin-3-depleted HeLa cells were normalised to control cells, n=4 independent experiments. One-way ANOVA (** P

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Transduction, Transfection

    Expansion of the polyQ domain in ataxin-3 decreased deubiquitinase activity and increased its interaction with beclin 1. a , Beclin 1 was purified from proteasome inhibitor-treated cells that co-expressed HA-Ub and was incubated in-vitro with recombinant ataxin-3 Q22 or ataxin-3 Q80 for 30 min in deubiquitination buffer and samples were analysed for beclin 1 ubiquitination using anti-HA antibodies. b, Number of LC3 dots in ataxin-3 KD HeLa cells that were transfected with GFP ataxin-3 Q28, GFP ataxin-3 Q84 and GFP ataxin-3 ΔQ and starved with HBSS in the last 4 hr. Results are normalised to control siRNA-treated cells from n=4 independent experiments. One-way ANOVA (*** P

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Expansion of the polyQ domain in ataxin-3 decreased deubiquitinase activity and increased its interaction with beclin 1. a , Beclin 1 was purified from proteasome inhibitor-treated cells that co-expressed HA-Ub and was incubated in-vitro with recombinant ataxin-3 Q22 or ataxin-3 Q80 for 30 min in deubiquitination buffer and samples were analysed for beclin 1 ubiquitination using anti-HA antibodies. b, Number of LC3 dots in ataxin-3 KD HeLa cells that were transfected with GFP ataxin-3 Q28, GFP ataxin-3 Q84 and GFP ataxin-3 ΔQ and starved with HBSS in the last 4 hr. Results are normalised to control siRNA-treated cells from n=4 independent experiments. One-way ANOVA (*** P

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Activity Assay, Purification, Incubation, In Vitro, Recombinant, Transfection

    Analysis of the interaction of the polyQ domain with beclin 1. a, Empty FLAG, FLAG beclin 1 evolutionary conserved domain (ECD) alone, FLAG beclin 1 ΔECD, FLAG beclin 1 full length and GFP Q35 were transfected in HeLa cells for 24 hr and the cell lysates were immunoprecipitated with anti-FLAG antibody. Immunocomplexes were analysed using anti-GFP antibody. b, Superimposition of human beclin 1 ECD (pdb 4DDP) and Vps30 (pdb 5DFZ), the yeast orthologue of beclin 1. Structures reveal that the N-terminal helix (dark blue helix) of the human structure is displaced, most likely due to protein truncation for crystallographic purposes. The yeast structure suggests that this helix is part of the coiled-coil CC2 of beclin 1 instead of the ECD. c, Two binding-sites in human beclin 1 ECD reveal high docking scores for polyQ 7 (the docking scores for site 1 and site 2 are -10.394 and -10.721, respectively). Sites comprising the region adjacent to the N-terminal helix (dark blue) were not considered for the docking. d-e, Surface charge illustrations of human beclin 1 ECD with the two sites of polyQ interaction. Site 2 is in close proximity to a protruding hydrophobic loop (aromatic finger) composed by Phe359, Phe360 and Trp361 (top right e - cartoon view), which are thought to be implicated in beclin 1 anchorage to lipid membranes.

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Analysis of the interaction of the polyQ domain with beclin 1. a, Empty FLAG, FLAG beclin 1 evolutionary conserved domain (ECD) alone, FLAG beclin 1 ΔECD, FLAG beclin 1 full length and GFP Q35 were transfected in HeLa cells for 24 hr and the cell lysates were immunoprecipitated with anti-FLAG antibody. Immunocomplexes were analysed using anti-GFP antibody. b, Superimposition of human beclin 1 ECD (pdb 4DDP) and Vps30 (pdb 5DFZ), the yeast orthologue of beclin 1. Structures reveal that the N-terminal helix (dark blue helix) of the human structure is displaced, most likely due to protein truncation for crystallographic purposes. The yeast structure suggests that this helix is part of the coiled-coil CC2 of beclin 1 instead of the ECD. c, Two binding-sites in human beclin 1 ECD reveal high docking scores for polyQ 7 (the docking scores for site 1 and site 2 are -10.394 and -10.721, respectively). Sites comprising the region adjacent to the N-terminal helix (dark blue) were not considered for the docking. d-e, Surface charge illustrations of human beclin 1 ECD with the two sites of polyQ interaction. Site 2 is in close proximity to a protruding hydrophobic loop (aromatic finger) composed by Phe359, Phe360 and Trp361 (top right e - cartoon view), which are thought to be implicated in beclin 1 anchorage to lipid membranes.

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Transfection, Immunoprecipitation, Binding Assay

    Impaired starvation-induced autophagy and reduced beclin 1 levels in cells expressing expanded polyQ forms of huntingtin. a, Empty FLAG, FLAG huntingtin (HTT) N-terminal fragment (1-350) Q17, FLAG HTT (1-350) ΔQ and beclin 1 were transfected in HeLa cells for 24 hr and cell lysates were immunoprecipitated with anti-beclin 1 antibody. Immunocomplexes were analysed using anti-FLAG antibody. b , GFP ataxin-3 Q28 and FLAG full-length HTT Q138 were transfected in HeLa cells for 24 hr and endogenous beclin 1 was immunoprecipitated. Immunocomplexes were analysed using anti-ataxin-3 antibody (detect GFP-ataxin-3) and anti-FLAG antibody (detect HTT). The ratio of the bound ataxin-3 to beclin 1 is presented. c, stable-inducible HEK293 cells were switched on for 48 hr with doxycycline (Dox) to express GFP-HTT wild-type exon 1 (GFP-HTT Q23) or mutant GFP HTT exon 1 (GFP-HTT Q74). In the last 4 hr cells were starved (HBSS) and beclin 1 levels were analysed in each cell type. Results are normalised to control HTT Q23 cells no Dox (n=4 independent experiments). Two-way ANOVA (column factor Dox ** P

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Impaired starvation-induced autophagy and reduced beclin 1 levels in cells expressing expanded polyQ forms of huntingtin. a, Empty FLAG, FLAG huntingtin (HTT) N-terminal fragment (1-350) Q17, FLAG HTT (1-350) ΔQ and beclin 1 were transfected in HeLa cells for 24 hr and cell lysates were immunoprecipitated with anti-beclin 1 antibody. Immunocomplexes were analysed using anti-FLAG antibody. b , GFP ataxin-3 Q28 and FLAG full-length HTT Q138 were transfected in HeLa cells for 24 hr and endogenous beclin 1 was immunoprecipitated. Immunocomplexes were analysed using anti-ataxin-3 antibody (detect GFP-ataxin-3) and anti-FLAG antibody (detect HTT). The ratio of the bound ataxin-3 to beclin 1 is presented. c, stable-inducible HEK293 cells were switched on for 48 hr with doxycycline (Dox) to express GFP-HTT wild-type exon 1 (GFP-HTT Q23) or mutant GFP HTT exon 1 (GFP-HTT Q74). In the last 4 hr cells were starved (HBSS) and beclin 1 levels were analysed in each cell type. Results are normalised to control HTT Q23 cells no Dox (n=4 independent experiments). Two-way ANOVA (column factor Dox ** P

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Expressing, Transfection, Immunoprecipitation, Mutagenesis

    Expression of polyQ tracts impairs beclin 1-dependent starvation-induced autophagy. a, HeLa cells were transfected with empty GFP or GFP Q35 with or without FLAG ataxin-3 Q22 for 24 hr and were shifted to starvation condition (HBSS) in the last 4 hr. LC3-II and beclin 1 levels were analysed from the cell lysates. Results are mean ± s.e.m normalised to control (empty GFP), n=5 independent experiments, One-way ANOVA (for LC3-II ** P

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Expression of polyQ tracts impairs beclin 1-dependent starvation-induced autophagy. a, HeLa cells were transfected with empty GFP or GFP Q35 with or without FLAG ataxin-3 Q22 for 24 hr and were shifted to starvation condition (HBSS) in the last 4 hr. LC3-II and beclin 1 levels were analysed from the cell lysates. Results are mean ± s.e.m normalised to control (empty GFP), n=5 independent experiments, One-way ANOVA (for LC3-II ** P

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Expressing, Transfection

    Effect of different disease proteins with polyQ expansion on beclin 1 ubiquitination, beclin 1 levels and starvation-induced autophagy. a, HeLa cells were transfected with empty vector, GFP atrophin-1 (ATN-1) Q19, GFP ATN-1 Q71 and HA Ub for 24 hr, treated in the last 6 hr with proteasome inhibitor (MG132 10 µM). Endogenous beclin 1 was immunoprecipitated from the lysates for ubiquitination analysis and for detection of bound ATN-1 using anti-ATN-1 antibody. b, HeLa cells were transfected with empty vector, HA androgen receptor (AR) Q25, HA AR Q120 and HA Ub for 24 hr, treated in the last 6 hr with proteasome inhibitor (MG132 10 µM). Endogenous beclin 1 was immunoprecipitated from the lysates for ubiquitination analysis and for detection of bound AR using anti-AR antibody. c , Primary fibroblasts derived from healthy controls (n=5) and from HD patients (n=7) were starved with HBSS together with bafA1 (400 nM) for 4 hr and analysed for LC3-II levels. Results are mean ± s.e.m. * P

    Journal: Nature

    Article Title: Polyglutamine tracts regulate beclin 1-dependent autophagy

    doi: 10.1038/nature22078

    Figure Lengend Snippet: Effect of different disease proteins with polyQ expansion on beclin 1 ubiquitination, beclin 1 levels and starvation-induced autophagy. a, HeLa cells were transfected with empty vector, GFP atrophin-1 (ATN-1) Q19, GFP ATN-1 Q71 and HA Ub for 24 hr, treated in the last 6 hr with proteasome inhibitor (MG132 10 µM). Endogenous beclin 1 was immunoprecipitated from the lysates for ubiquitination analysis and for detection of bound ATN-1 using anti-ATN-1 antibody. b, HeLa cells were transfected with empty vector, HA androgen receptor (AR) Q25, HA AR Q120 and HA Ub for 24 hr, treated in the last 6 hr with proteasome inhibitor (MG132 10 µM). Endogenous beclin 1 was immunoprecipitated from the lysates for ubiquitination analysis and for detection of bound AR using anti-AR antibody. c , Primary fibroblasts derived from healthy controls (n=5) and from HD patients (n=7) were starved with HBSS together with bafA1 (400 nM) for 4 hr and analysed for LC3-II levels. Results are mean ± s.e.m. * P

    Article Snippet: HeLa cells stably expressing GFP-LC3 and HeLa cells stably expressing mTagRFP-mWasabi-LC3 reporter were cultured in basal media supplemented with 500 μg/ml G418 (Sigma).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Derivative Assay

    Importin α accumulates in the nucleus in response to either the oxidative stress or the heat shock stress. After HeLa cells were treated with hydrogen peroxide or incubated at 42°C at the indicated time, the cells were stained by specific antibodies for importin α (mRch1) and Alexa488-conjugated secondary antibodies. (A) HeLa cells were cultured with a medium containing 200 μM H 2 O 2 . (B) After HeLa cells were treated with 200 μM H 2 O 2 for 1 h, the cells were incubated with fresh medium in the absence of H 2 O 2 . (C) HeLa cells were incubated at 42°C. (D) After HeLa cells were incubated for 1 h at 42°C, they were incubated at 37°C for the indicated times.

    Journal: The Journal of Cell Biology

    Article Title: Cellular stresses induce the nuclear accumulation of importin ? and cause a conventional nuclear import block

    doi: 10.1083/jcb.200312008

    Figure Lengend Snippet: Importin α accumulates in the nucleus in response to either the oxidative stress or the heat shock stress. After HeLa cells were treated with hydrogen peroxide or incubated at 42°C at the indicated time, the cells were stained by specific antibodies for importin α (mRch1) and Alexa488-conjugated secondary antibodies. (A) HeLa cells were cultured with a medium containing 200 μM H 2 O 2 . (B) After HeLa cells were treated with 200 μM H 2 O 2 for 1 h, the cells were incubated with fresh medium in the absence of H 2 O 2 . (C) HeLa cells were incubated at 42°C. (D) After HeLa cells were incubated for 1 h at 42°C, they were incubated at 37°C for the indicated times.

    Article Snippet: Cell culture HeLa cells were incubated in Dulbecco's modified MEM (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS at 37°C in a 5% CO2 atmosphere.

    Techniques: Incubation, Staining, Cell Culture

    Clonal variations in CRISPRi and their associated off-target transcriptional effects. ( A ) Expression in counts-per-million (CPM) of Cas9 in CRISPRi clonal, CRISPRi non-clonal and untransduced HeLa cells. Clone 2 was used for showing Cas9 expression in CRISPRi clonal cells. ( B) Comparison of the transcriptional differences between parental HeLa untreated cells, CRISPRi clones expressing only dCas9–KRAB (clone 2) and clones treated with two negative guide RNAs (negative guide 1 and 2). The number of genes differing between each pair of treatments is labelled and shown as connecting lines of proportional thickness. ( C ) Comparison of the transcriptional differences between parental HeLa untreated cells, non-clonal CRISPRi cells expressing dCas9–KRAB and non-clonal cells treated with two negative guide RNAs (negative guide 1 and 2). The number of genes differing between each pair of treatments is labelled as described in B. ( D ) Expression of dCas9–KRAB in three different CRISPRi clones derived from single cell cloning, confirmed by immunoblot using a Cas9 antibody. β-tubulin was used as a loading control. A Venn diagram of DEGs detected in the three different clones against untransduced cells in the absence of any guide RNAs identified 37 genes as a common transcriptional signature of cloning. The total number of genes in this analysis was 18224 and DEGs were detected at a FDR of 5% with a log 2 -fold change threshold of 0.5. ( E ) Heat map of DEGs from three different CRISPRi clones compared to non-clonal cells and parental untransduced HeLa cells in the absence of any guide RNAs. 33 out of 37 genes were downregulated in clonal cells compared to the parental population. ( F ) Downregulation of two randomly selected DEGs from E ( SCIN and CDH2 ) was validated by qPCR in clonal cells (clone 2) and in non-clonal populations. Expression levels were normalized to the geometric mean of GAPDH and RPS18 . Error bars, s.e.m. ( n = 4 biological replicates). Statistical significance by two-tailed Student's t -test: * P

    Journal: Nucleic Acids Research

    Article Title: Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

    doi: 10.1093/nar/gky437

    Figure Lengend Snippet: Clonal variations in CRISPRi and their associated off-target transcriptional effects. ( A ) Expression in counts-per-million (CPM) of Cas9 in CRISPRi clonal, CRISPRi non-clonal and untransduced HeLa cells. Clone 2 was used for showing Cas9 expression in CRISPRi clonal cells. ( B) Comparison of the transcriptional differences between parental HeLa untreated cells, CRISPRi clones expressing only dCas9–KRAB (clone 2) and clones treated with two negative guide RNAs (negative guide 1 and 2). The number of genes differing between each pair of treatments is labelled and shown as connecting lines of proportional thickness. ( C ) Comparison of the transcriptional differences between parental HeLa untreated cells, non-clonal CRISPRi cells expressing dCas9–KRAB and non-clonal cells treated with two negative guide RNAs (negative guide 1 and 2). The number of genes differing between each pair of treatments is labelled as described in B. ( D ) Expression of dCas9–KRAB in three different CRISPRi clones derived from single cell cloning, confirmed by immunoblot using a Cas9 antibody. β-tubulin was used as a loading control. A Venn diagram of DEGs detected in the three different clones against untransduced cells in the absence of any guide RNAs identified 37 genes as a common transcriptional signature of cloning. The total number of genes in this analysis was 18224 and DEGs were detected at a FDR of 5% with a log 2 -fold change threshold of 0.5. ( E ) Heat map of DEGs from three different CRISPRi clones compared to non-clonal cells and parental untransduced HeLa cells in the absence of any guide RNAs. 33 out of 37 genes were downregulated in clonal cells compared to the parental population. ( F ) Downregulation of two randomly selected DEGs from E ( SCIN and CDH2 ) was validated by qPCR in clonal cells (clone 2) and in non-clonal populations. Expression levels were normalized to the geometric mean of GAPDH and RPS18 . Error bars, s.e.m. ( n = 4 biological replicates). Statistical significance by two-tailed Student's t -test: * P

    Article Snippet: FACS analysis and cell sorting HeLa cells were transduced with lentivirus containing the pHR-SFFV-dCAS9-BFP-KRAB vector together with polybrene (5 μg/ml, Sigma).

    Techniques: Expressing, Clone Assay, Derivative Assay, Real-time Polymerase Chain Reaction, Two Tailed Test

    Cx26 immunostaining performed in Hela DH transfectants overexpressing Cx26WT (top), and Cx26C169Y (bottom) proteins. Through focus confocal image sequence (z-stack) taken at 0.5 µm intervals of Hela DH cells expressing Cx26WT ( A – D ) and Cx26C169Y ( F – I ) proteins and their respective maximal projection rendering ( E and J ). Yellow arrow points to representative gap junction plaque, whereas red arrows indicate immunoreaction signals at the cell plasma membrane level, which are most likely due to unpaired connexons. Scale bar, 10 µm.

    Journal: Human Molecular Genetics

    Article Title: The p.Cys169Tyr variant of connexin 26 is not a polymorphism

    doi: 10.1093/hmg/ddv026

    Figure Lengend Snippet: Cx26 immunostaining performed in Hela DH transfectants overexpressing Cx26WT (top), and Cx26C169Y (bottom) proteins. Through focus confocal image sequence (z-stack) taken at 0.5 µm intervals of Hela DH cells expressing Cx26WT ( A – D ) and Cx26C169Y ( F – I ) proteins and their respective maximal projection rendering ( E and J ). Yellow arrow points to representative gap junction plaque, whereas red arrows indicate immunoreaction signals at the cell plasma membrane level, which are most likely due to unpaired connexons. Scale bar, 10 µm.

    Article Snippet: Cx26 immunoreactivity in Hela cells Hela DH cells (Sigma, cat. 96112022) were grown in DMEM (Gibco, catalog no. 41965-039) supplemented with 10% FBS (Gibco, catalog no. 10106169), plated on glass coverslips.

    Techniques: Immunostaining, Sequencing, Expressing

    A marine blocking agent (MAXblock ™ ) improves staining quality relative to standard blocking agents HeLa cells were fixed with methanol (−20 °C, 8 min) and blocked overnight in PBS containing (A) MAXblock ™ . MAXblock ™ was developed by Dr. Brian Bennett.

    Journal: Methods (San Diego, Calif.)

    Article Title: Immunofluorescence Imaging of DNA Damage Response Proteins

    doi: 10.1016/j.ymeth.2009.02.009

    Figure Lengend Snippet: A marine blocking agent (MAXblock ™ ) improves staining quality relative to standard blocking agents HeLa cells were fixed with methanol (−20 °C, 8 min) and blocked overnight in PBS containing (A) MAXblock ™ . MAXblock ™ was developed by Dr. Brian Bennett.

    Article Snippet: In we compare the background fluorescence when fixed HeLa cells are blocked with a marine agent (MAXblock™ , Active Motif) , 5% non-fat dry milk ( ) or 5% BSA IV , followed by incubation with an anti-rabbit secondary Alexa 647 conjugate at a 1:250 dilution.

    Techniques: Blocking Assay, Staining

    Deregulation of PLK4 expression after expression of HPV-16 E7 depends on loss of DREAM binding to the CDE and CHR elements. ( A ) HCT116 wt and HCT116 stably transfected with a plasmid encoding HPV-16 E7 wt or its ΔDLYC mutant. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( B ) Luciferase reporter assays from lysates of HCT116 wt; p53 −/− or p21 −/− cells transfected with plasmids expressing wt or CDE/CHR mutant Plk4 promoter constructs. Plasmids expressing HPV-16 E7 wt or its ΔDLYC mutant were cotransfected. Results are given as relative light units. ( C ) Nuclear extracts from HCT116 E7 wt and E7 ΔDLYC cells were analyzed by DNA affinity purification with wt and CDE/CHR mutant Plk4 promoter probes followed by western blot. Band intensities were quantified by densitometric analyses. Relative intensities are given below the bands. Intensities of input bands were normalized to E7 ΔDLYC. Binding intensities to Plk4 promoters were normalized to CDE/CHR mutant probes from E7 ΔDLYC extracts. ( D ) Protein binding to the PLK4 promoter in HeLa cells was assessed by ChIP. Protein binding to the GAPDHS promoter served as a negative control. ( E ) HCT116 E7 wt and HeLa cells were treated with doxorubicin and roscovitine for 24 h. Cells without treatment served as control. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( F ) Luciferase reporter assays with wt and CDE/CHR mutant Plk4 promoter reporter constructs in HCT116 p53 −/− cells. Cells were cotransfected with p53mut, p53wt, p21mut or p21wt expression vectors and HPV-16 E7 wt or ΔDLYC mutant. Results are given as relative light units. ( G ) Venn diagram from DREAM-bound genes ( 12 , 15 ), genes upregulated by HPV E7 ( 64 ) and genes downregulated by HPV E2 proteins repressing E7 ( 65 , 66 ).

    Journal: Nucleic Acids Research

    Article Title: Polo-like kinase 4 transcription is activated via CRE and NRF1 elements, repressed by DREAM through CDE/CHR sites and deregulated by HPV E7 protein

    doi: 10.1093/nar/gkt849

    Figure Lengend Snippet: Deregulation of PLK4 expression after expression of HPV-16 E7 depends on loss of DREAM binding to the CDE and CHR elements. ( A ) HCT116 wt and HCT116 stably transfected with a plasmid encoding HPV-16 E7 wt or its ΔDLYC mutant. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( B ) Luciferase reporter assays from lysates of HCT116 wt; p53 −/− or p21 −/− cells transfected with plasmids expressing wt or CDE/CHR mutant Plk4 promoter constructs. Plasmids expressing HPV-16 E7 wt or its ΔDLYC mutant were cotransfected. Results are given as relative light units. ( C ) Nuclear extracts from HCT116 E7 wt and E7 ΔDLYC cells were analyzed by DNA affinity purification with wt and CDE/CHR mutant Plk4 promoter probes followed by western blot. Band intensities were quantified by densitometric analyses. Relative intensities are given below the bands. Intensities of input bands were normalized to E7 ΔDLYC. Binding intensities to Plk4 promoters were normalized to CDE/CHR mutant probes from E7 ΔDLYC extracts. ( D ) Protein binding to the PLK4 promoter in HeLa cells was assessed by ChIP. Protein binding to the GAPDHS promoter served as a negative control. ( E ) HCT116 E7 wt and HeLa cells were treated with doxorubicin and roscovitine for 24 h. Cells without treatment served as control. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( F ) Luciferase reporter assays with wt and CDE/CHR mutant Plk4 promoter reporter constructs in HCT116 p53 −/− cells. Cells were cotransfected with p53mut, p53wt, p21mut or p21wt expression vectors and HPV-16 E7 wt or ΔDLYC mutant. Results are given as relative light units. ( G ) Venn diagram from DREAM-bound genes ( 12 , 15 ), genes upregulated by HPV E7 ( 64 ) and genes downregulated by HPV E2 proteins repressing E7 ( 65 , 66 ).

    Article Snippet: HCT116 E7 wt cells and HeLa cells were treated with doxorubicin and roscovitine yielding no significant repression of Plk4 mRNA ( E, Supplementary Figure S4C ).

    Techniques: Expressing, Binding Assay, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Luciferase, Construct, Affinity Purification, Western Blot, Protein Binding, Chromatin Immunoprecipitation, Negative Control

    FMRP docking to chromatin is essential for FMRP-dependent modulation of γH2A.X levels in response to replication stress (A) Diagram of Agenet FMRP . (B) GST-FMRP or GST-FMRP carrying mutations in Agenet FMRP . (C) . (D) FMRP RNAi in HeLa cells abolished γH2A.X induction in response to APH as compared to control RNAi (compare lanes 1,2 to lanes 3,4). Co-transfection with constructs expressing wild type but not mutant forms of FMRP restored the induction of γH2A.X in FMRP RNAi cells in response to APH (compare lanes 5,6 to lanes 7,8 and 9,10). The slower migrating band (in lanes 5–10) is Flag-HA-FMRP (indicated by an arrowhead).

    Journal: Cell

    Article Title: A chromatin-dependent role of the fragile X mental retardation protein FMRP in the DNA damage response

    doi: 10.1016/j.cell.2014.03.040

    Figure Lengend Snippet: FMRP docking to chromatin is essential for FMRP-dependent modulation of γH2A.X levels in response to replication stress (A) Diagram of Agenet FMRP . (B) GST-FMRP or GST-FMRP carrying mutations in Agenet FMRP . (C) . (D) FMRP RNAi in HeLa cells abolished γH2A.X induction in response to APH as compared to control RNAi (compare lanes 1,2 to lanes 3,4). Co-transfection with constructs expressing wild type but not mutant forms of FMRP restored the induction of γH2A.X in FMRP RNAi cells in response to APH (compare lanes 5,6 to lanes 7,8 and 9,10). The slower migrating band (in lanes 5–10) is Flag-HA-FMRP (indicated by an arrowhead).

    Article Snippet: Reactions were performed in the presence of binding buffer (50mM Tris pH 7.5, 150mM NaCl, 2mM MgCl2 , 0.1% Triton-X100) using 100 ng of GST fusion proteins and 5 μg of native nucleosomes isolated from HeLa cells at +4C°, and rotated for 2 hours before addition of glutathione agarose beads (GE Healthcare).

    Techniques: Cotransfection, Construct, Expressing, Mutagenesis

    ATR is the major kinase responsible for cellular XPA phosphorylation after UV irradiation. A, A549 cells were mock treated (lane 1) or treated with 20 J/m 2 UV irradiation, and then further incubated for 4 hours in the presence of 100 Amol/L wortmannin (Wort; lane 4) or 10 mmol/L caffeine (Caff; lane 3) before harvesting. Total cell lysates were used for Western blot analysis with anti-XPA, anti-ATR, and anti-ATM, respectively. B, A549 cells were transfected with ATR, ATM, or green fluorescent protein (GFP) siRNA as described in Materials and Methods. Total cell lysates were harvested 72 hours after transfection and probed with the indicated antibodies, respectively. C, A549 or HeLa cells were transfected with indicated siRNA and then treated with 20 J/m 2 UV irradiation at 72 hours after transfection. Total cell lysates were immunoblotted with anti-XPA and antiactin antibodies, respectively. D, ATR- and ATM-deficient cells were treated with the indicated doses of UV and total cell lysates were prepared at 4 hours after treatment for Western blotting with anti-XPA and antiactin, respectively.

    Journal: Cancer research

    Article Title: Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related-Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

    doi: 10.1158/0008-5472.CAN-05-3403

    Figure Lengend Snippet: ATR is the major kinase responsible for cellular XPA phosphorylation after UV irradiation. A, A549 cells were mock treated (lane 1) or treated with 20 J/m 2 UV irradiation, and then further incubated for 4 hours in the presence of 100 Amol/L wortmannin (Wort; lane 4) or 10 mmol/L caffeine (Caff; lane 3) before harvesting. Total cell lysates were used for Western blot analysis with anti-XPA, anti-ATR, and anti-ATM, respectively. B, A549 cells were transfected with ATR, ATM, or green fluorescent protein (GFP) siRNA as described in Materials and Methods. Total cell lysates were harvested 72 hours after transfection and probed with the indicated antibodies, respectively. C, A549 or HeLa cells were transfected with indicated siRNA and then treated with 20 J/m 2 UV irradiation at 72 hours after transfection. Total cell lysates were immunoblotted with anti-XPA and antiactin antibodies, respectively. D, ATR- and ATM-deficient cells were treated with the indicated doses of UV and total cell lysates were prepared at 4 hours after treatment for Western blotting with anti-XPA and antiactin, respectively.

    Article Snippet: Thus, in vitro kinase assays were done for recombinant XPA with DNA-PK isolated from HeLa cells (Promega) and ATR/ATM immunoprecipitated from cell lysates.

    Techniques: Irradiation, Incubation, Western Blot, Transfection