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    Millipore adherent hela cells
    Endosomal tubulation in spastin-depleted cells requires intact MTs. (a) <t>HeLa</t> 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) <t>Mock-transfected</t> 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.
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    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

    Gain-of-function and rescue studies of linc00899 and its effect on TPPP expression. a Schematic diagram of ectopic overexpression of linc00899. Linc00899 and TPPP expression were analysed by qPCR after lentiviral overexpression using lincXpress vector encoding linc00899 cDNA in HeLa (left) and RPE1 cells (right). The expression was normalised to the scrambled linc00899 vector (negative control). n = 3–4 biological replicates, * P

    Journal: Nature Communications

    Article Title: A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division

    doi: 10.1038/s41467-020-14978-7

    Figure Lengend Snippet: Gain-of-function and rescue studies of linc00899 and its effect on TPPP expression. a Schematic diagram of ectopic overexpression of linc00899. Linc00899 and TPPP expression were analysed by qPCR after lentiviral overexpression using lincXpress vector encoding linc00899 cDNA in HeLa (left) and RPE1 cells (right). The expression was normalised to the scrambled linc00899 vector (negative control). n = 3–4 biological replicates, * P

    Article Snippet: HeLa and RPE1 cells were transduced with lentivirus containing dCAS9-VP64 and two gRNA targeting linc00899 or with lentivirus contacting negative gRNA (NC2) in the presence of polybrene (5 µg/ml, Sigma).

    Techniques: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Plasmid Preparation, Negative Control

    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

    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

    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

    The STR analysis to confirm the identity of HeLa cells. Left and right panels show the data on HeLa-N and HeLa-R cells, respectively. Graphs were images generated by GeneMapper ver. 3.5 (Applied Biosystems). The allele data were presented as a table on the right side of each image. Locus indicates the name of gene analyzed. It is indicated that the gene presented by one datum was homo and the gene presented by two data was hetero.

    Journal: PLoS ONE

    Article Title: Saffold Virus Type 3 (SAFV-3) Persists in HeLa Cells

    doi: 10.1371/journal.pone.0053194

    Figure Lengend Snippet: The STR analysis to confirm the identity of HeLa cells. Left and right panels show the data on HeLa-N and HeLa-R cells, respectively. Graphs were images generated by GeneMapper ver. 3.5 (Applied Biosystems). The allele data were presented as a table on the right side of each image. Locus indicates the name of gene analyzed. It is indicated that the gene presented by one datum was homo and the gene presented by two data was hetero.

    Article Snippet: On the other hand, HeLa-N was maintained in Dulbecco’s modified Eagle’s medium (DMEM, SIGMA) supplemented with 0.03% L-glutamine and 10% fetal calf serum (FCS) containing 50 U/ml of penicillin and 50 µg/ml of streptomycin .

    Techniques: Generated

    Immunofluorescent detection of the virus binding. The photos show the virus binding to the cell surface molecule(s) of each cell line. The cells fixed by 10% formalin were incubated with virus (MOI of 100). Then the viruses binding to the cell surface molecule(s) were detected by anti-SAFV-3 antiserum pre-absorbed by the homogenates of HeLa-R cells and Alexa Fluor 594-conjugated anti-rabbit IgG antibody. Left panels: HeLa-N cells, Right panels: HeLa-R cells. Upper and lower panels show Nomarski and fluorescent images, respectively. The viruses binding to cell surface of HeLa-R cells was significantly few, suggesting that the expression of receptor for SAFV infection is low in HeLa-R cells. Magnification: ×400.

    Journal: PLoS ONE

    Article Title: Saffold Virus Type 3 (SAFV-3) Persists in HeLa Cells

    doi: 10.1371/journal.pone.0053194

    Figure Lengend Snippet: Immunofluorescent detection of the virus binding. The photos show the virus binding to the cell surface molecule(s) of each cell line. The cells fixed by 10% formalin were incubated with virus (MOI of 100). Then the viruses binding to the cell surface molecule(s) were detected by anti-SAFV-3 antiserum pre-absorbed by the homogenates of HeLa-R cells and Alexa Fluor 594-conjugated anti-rabbit IgG antibody. Left panels: HeLa-N cells, Right panels: HeLa-R cells. Upper and lower panels show Nomarski and fluorescent images, respectively. The viruses binding to cell surface of HeLa-R cells was significantly few, suggesting that the expression of receptor for SAFV infection is low in HeLa-R cells. Magnification: ×400.

    Article Snippet: On the other hand, HeLa-N was maintained in Dulbecco’s modified Eagle’s medium (DMEM, SIGMA) supplemented with 0.03% L-glutamine and 10% fetal calf serum (FCS) containing 50 U/ml of penicillin and 50 µg/ml of streptomycin .

    Techniques: Binding Assay, Incubation, Expressing, Infection

    The detection of virus antigen in PSAF/HeLa-R cells by Western blotting. The anti-SAFV-3 antiserum detected the viral antigen of about 28∼30 kDa (arrow) in the lysates of PSAF/HeLa-R cells cultured for 30 days (5 passages) (lane 2) and HeLa-N cells infected with SAFV-3 (18 hours p.i.) used for a positive control (lane 3). The band of viral antigen was not detected in the lysate of HeLa-R cells used for a negative control (lane 1).

    Journal: PLoS ONE

    Article Title: Saffold Virus Type 3 (SAFV-3) Persists in HeLa Cells

    doi: 10.1371/journal.pone.0053194

    Figure Lengend Snippet: The detection of virus antigen in PSAF/HeLa-R cells by Western blotting. The anti-SAFV-3 antiserum detected the viral antigen of about 28∼30 kDa (arrow) in the lysates of PSAF/HeLa-R cells cultured for 30 days (5 passages) (lane 2) and HeLa-N cells infected with SAFV-3 (18 hours p.i.) used for a positive control (lane 3). The band of viral antigen was not detected in the lysate of HeLa-R cells used for a negative control (lane 1).

    Article Snippet: On the other hand, HeLa-N was maintained in Dulbecco’s modified Eagle’s medium (DMEM, SIGMA) supplemented with 0.03% L-glutamine and 10% fetal calf serum (FCS) containing 50 U/ml of penicillin and 50 µg/ml of streptomycin .

    Techniques: Western Blot, Cell Culture, Infection, Positive Control, Negative Control

    Growth kinetics of SAFV-3 and TMEV-DA on HeLa-N and HeLa-R cells. A: Growth kinetics of SAFV-3. Solid and broken lines indicate the growth curves of SAFV-3 on HeLa-N and HeLa-R cells, respectively. The viruses (as a mixture of cell-free and cell-associated viruses) were harvested at several time points indicated and assayed for titers by a standard plaque assay on HeLa-N cells. Titers shown are the means ± S.D. in three independent experiments. B: Growth kinetics of TMEV-DA. Solid and broken lines indicate the growth curves of TMEV-DA on HeLa-N and HeLa-R cells, respectively. The viruses (as a mixture of cell-free and cell-associated viruses) were harvested at several time points indicated and assayed for titers by a standard plaque assay on BHK-21 cells. Titers shown are the means ± S.D. in three independent experiments.

    Journal: PLoS ONE

    Article Title: Saffold Virus Type 3 (SAFV-3) Persists in HeLa Cells

    doi: 10.1371/journal.pone.0053194

    Figure Lengend Snippet: Growth kinetics of SAFV-3 and TMEV-DA on HeLa-N and HeLa-R cells. A: Growth kinetics of SAFV-3. Solid and broken lines indicate the growth curves of SAFV-3 on HeLa-N and HeLa-R cells, respectively. The viruses (as a mixture of cell-free and cell-associated viruses) were harvested at several time points indicated and assayed for titers by a standard plaque assay on HeLa-N cells. Titers shown are the means ± S.D. in three independent experiments. B: Growth kinetics of TMEV-DA. Solid and broken lines indicate the growth curves of TMEV-DA on HeLa-N and HeLa-R cells, respectively. The viruses (as a mixture of cell-free and cell-associated viruses) were harvested at several time points indicated and assayed for titers by a standard plaque assay on BHK-21 cells. Titers shown are the means ± S.D. in three independent experiments.

    Article Snippet: On the other hand, HeLa-N was maintained in Dulbecco’s modified Eagle’s medium (DMEM, SIGMA) supplemented with 0.03% L-glutamine and 10% fetal calf serum (FCS) containing 50 U/ml of penicillin and 50 µg/ml of streptomycin .

    Techniques: Plaque Assay

    Intracellular localisation of TMEM176A and TMEM176B. ( a ) Human Th17 polarised cells from naive CD4 + T cells were coated on microscopy slides, fixed, permeabilised and co-stained for TMEM176B (red) and the indicated markers (green). DAPI was used for nuclear staining (blue). Arrows indicate TMEM176B colocalisation with the cis -Golgi protein GM130. Bar, 10 μm. ( b ) Pearson’s correlation coefficients of TMEM176B and the indicated markers (n = 10–15 in each group). ( c ) HeLa cells were treated or not with nocodazole for 4 hr and subsequently fixed, permeabilised and co-stained for TMEM176B (red), GM130 ( cis -Golgi, green) and TGN46 ( trans -Golgi and trans -Golgi network (TGN), blue). Bar, 10 μm. Insets represent higher magnifications of regions of interest. Linescans show fluorescence intensity along the lines overlaying the images. ( d ) HeLa cells were co-transfected with plasmids encoding TMEM176A-HA and TMEM176B-V5 fusion proteins and subsequently fixed, permeabilised and co-stained with HA (green) and V5 (red) monoclonal antibodies. DAPI was used for nuclear staining (blue). ( e ) Human Th17 polarised cells (as in A) were co-stained for GM130 (green), TMEM176A (purple) and TMEM176B (red). DAPI was used for nuclear staining (blue). Bar, 10 μm.

    Journal: Scientific Reports

    Article Title: RORγt+ cells selectively express redundant cation channels linked to the Golgi apparatus

    doi: 10.1038/srep23682

    Figure Lengend Snippet: Intracellular localisation of TMEM176A and TMEM176B. ( a ) Human Th17 polarised cells from naive CD4 + T cells were coated on microscopy slides, fixed, permeabilised and co-stained for TMEM176B (red) and the indicated markers (green). DAPI was used for nuclear staining (blue). Arrows indicate TMEM176B colocalisation with the cis -Golgi protein GM130. Bar, 10 μm. ( b ) Pearson’s correlation coefficients of TMEM176B and the indicated markers (n = 10–15 in each group). ( c ) HeLa cells were treated or not with nocodazole for 4 hr and subsequently fixed, permeabilised and co-stained for TMEM176B (red), GM130 ( cis -Golgi, green) and TGN46 ( trans -Golgi and trans -Golgi network (TGN), blue). Bar, 10 μm. Insets represent higher magnifications of regions of interest. Linescans show fluorescence intensity along the lines overlaying the images. ( d ) HeLa cells were co-transfected with plasmids encoding TMEM176A-HA and TMEM176B-V5 fusion proteins and subsequently fixed, permeabilised and co-stained with HA (green) and V5 (red) monoclonal antibodies. DAPI was used for nuclear staining (blue). ( e ) Human Th17 polarised cells (as in A) were co-stained for GM130 (green), TMEM176A (purple) and TMEM176B (red). DAPI was used for nuclear staining (blue). Bar, 10 μm.

    Article Snippet: HeLa cells were treated with 10 μM nocodazole (Sigma) for 4 hr prior fixation to disrupt the juxtanuclear Golgi ribbon.

    Techniques: Microscopy, Staining, Fluorescence, Transfection

    NPM/B23 serine 4 phosphorylation in S-phase is mediated by Plk2. A Flag-Plk2 WT, KD or Flag-alone were overexpressed in 293T cells. 8 h after transfections cells were arrested with 4 mM hydroxyurea (HU) for 40 h. Exponentially growing cells were harvested 48 h after transfections. Cell extracts were analyzed by SDS-PAGE and Western blot using anti-Flag, anti-NPM/B23, anti-pS4 NPM/B23 and anti-α-Tubulin (loading control) antibodies. B RNAi of Plk2 was mediated by overexpression of pSuper-RNAi-Plk2 and pSuper-RNAi-Luciferase as control in 293T cells. Cell cycle synchronization and analyses were carried out as in (A). To detect Plk2 downregulation in Western blot, membranes were probed with anti-Plk2 antibodies. C RNAi of Plk1 in HeLa cells was mediated by Lipofectamine2000-transfections of Plk1 specific siRNAs. GL2 siRNAs were used as control. Cell extracts were analyzed by Western blotting with the indicated antibodies. Loading control, α-Tubulin. D Plk4 was downregulated in U2OS cells by Lipofectamine2000 mediated siRNA transfections, as control GL2 was used. S-phase arrest was achieved as described in (A). Downregulation of Plk4 and pS4 NPM/B23 levels were examined by Western blot analysis with the indicated antibodies. Loading control, α-Tubulin.

    Journal: PLoS ONE

    Article Title: Polo-Like Kinase 2-Dependent Phosphorylation of NPM/B23 on Serine 4 Triggers Centriole Duplication

    doi: 10.1371/journal.pone.0009849

    Figure Lengend Snippet: NPM/B23 serine 4 phosphorylation in S-phase is mediated by Plk2. A Flag-Plk2 WT, KD or Flag-alone were overexpressed in 293T cells. 8 h after transfections cells were arrested with 4 mM hydroxyurea (HU) for 40 h. Exponentially growing cells were harvested 48 h after transfections. Cell extracts were analyzed by SDS-PAGE and Western blot using anti-Flag, anti-NPM/B23, anti-pS4 NPM/B23 and anti-α-Tubulin (loading control) antibodies. B RNAi of Plk2 was mediated by overexpression of pSuper-RNAi-Plk2 and pSuper-RNAi-Luciferase as control in 293T cells. Cell cycle synchronization and analyses were carried out as in (A). To detect Plk2 downregulation in Western blot, membranes were probed with anti-Plk2 antibodies. C RNAi of Plk1 in HeLa cells was mediated by Lipofectamine2000-transfections of Plk1 specific siRNAs. GL2 siRNAs were used as control. Cell extracts were analyzed by Western blotting with the indicated antibodies. Loading control, α-Tubulin. D Plk4 was downregulated in U2OS cells by Lipofectamine2000 mediated siRNA transfections, as control GL2 was used. S-phase arrest was achieved as described in (A). Downregulation of Plk4 and pS4 NPM/B23 levels were examined by Western blot analysis with the indicated antibodies. Loading control, α-Tubulin.

    Article Snippet: Cell Culture, Synchronization and Transfection HeLa, 293T, U2OS and U2OS GFP-Centrin1 (S.Duensing, Pittsburgh) cells were cultured in DMEM (Sigma) containing 10% fetal bovine serum (PPA), 5% of 200 mM L-glutamine (Invitrogen) and if necessary 5% penicillin-streptomycin solution (Invitrogen) at 37°C in 5% CO2 .

    Techniques: Transfection, SDS Page, Western Blot, Over Expression, Luciferase

    CaM binds the BAF complex. ( A ) CaM beads pulled down endogenous BAF complex in addition to affinity-tagged BAF complex from crude HeLa NEs. The integrity of the tag, fused to BAF57, was confirmed by Western blotting using an HRP-conjugated IgG that recognizes

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Induction of TLR4-target genes entails calcium/calmodulin-dependent regulation of chromatin remodeling

    doi: 10.1073/pnas.0811274106

    Figure Lengend Snippet: CaM binds the BAF complex. ( A ) CaM beads pulled down endogenous BAF complex in addition to affinity-tagged BAF complex from crude HeLa NEs. The integrity of the tag, fused to BAF57, was confirmed by Western blotting using an HRP-conjugated IgG that recognizes

    Article Snippet: We mixed 5 μL (≈0.5 μg) of the BAF complex affinity-purified from the HeLa line expressing Flag-tagged BAF47 with 1 μL (1 μg) of recombinant CaM (Millipore) in 40 μL of reaction buffer (20 mM Hepes pH7.6/1 mM MgAc/1 mM Imidazole/0.1% Nonidet P-40/1% Tween-20/0.1 M NaCl) containing either 2 mM CaCl2 or 5 mM EGTA.

    Techniques: Chick Chorioallantoic Membrane Assay, Western Blot

    Some but not all atlastin1/SPG3A variants are defective in forming a branched ER network. (A) At 48 h after transfection with the indicated HA-tagged atlastin1 variant constructs, HeLa cells were further transfected using siRNAs targeting endogenous atlastin2/3. At 72 h after knockdown, cells were fixed and stained using an antibody against the HA epitope and viewed by confocal microscopy. The negative control was Myc-tagged REEP5/DP1/TB2 stained with an antibody against the Myc epitope. Scale bar, 10 μm. (B) Quantification of the percentage of cells expressing the HA- or Myc-tagged construct and showing an abnormal unbranched ER morphology. Values represent the means of three independent experiments ( > 100 cells each) ± SD. * p

    Journal: Molecular Biology of the Cell

    Article Title: ER network formation and membrane fusion by atlastin1/SPG3A disease variants

    doi: 10.1091/mbc.E14-10-1447

    Figure Lengend Snippet: Some but not all atlastin1/SPG3A variants are defective in forming a branched ER network. (A) At 48 h after transfection with the indicated HA-tagged atlastin1 variant constructs, HeLa cells were further transfected using siRNAs targeting endogenous atlastin2/3. At 72 h after knockdown, cells were fixed and stained using an antibody against the HA epitope and viewed by confocal microscopy. The negative control was Myc-tagged REEP5/DP1/TB2 stained with an antibody against the Myc epitope. Scale bar, 10 μm. (B) Quantification of the percentage of cells expressing the HA- or Myc-tagged construct and showing an abnormal unbranched ER morphology. Values represent the means of three independent experiments ( > 100 cells each) ± SD. * p

    Article Snippet: Cell culture, constructs, transfections, and reagents HeLa and Cos-7 cells were maintained in MEM (Sigma-Aldrich, St. Louis, MO) and DMEM (Sigma-Aldrich), respectively, containing 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA) at 37°C in a 5% CO2 incubator.

    Techniques: Transfection, Variant Assay, Construct, Staining, Confocal Microscopy, Negative Control, Expressing

    Some but not all SPG3A mutations impair ER network formation when expressed in Drosophila atlastin. (A) At 48 h after transfection with the indicated eYFP-tagged Drosophila atlastin variant constructs, HeLa cells were further transfected using siRNAs targeting endogenous atlastin2 and atlastin3. At 72 h after knockdown, cells were fixed and viewed by confocal microscopy. The negative control Myc-tagged REEP5/DP1/TB2 was stained with an antibody against the Myc epitope. Scale bar, 10 μm. (B) Quantification of the percentage of cells expressing either the Myc-tagged or eYFP-tagged construct and showing an abnormal unbranched ER morphology. Values represent the means of three independent experiments ( > 100 cells each) ± SD. * p

    Journal: Molecular Biology of the Cell

    Article Title: ER network formation and membrane fusion by atlastin1/SPG3A disease variants

    doi: 10.1091/mbc.E14-10-1447

    Figure Lengend Snippet: Some but not all SPG3A mutations impair ER network formation when expressed in Drosophila atlastin. (A) At 48 h after transfection with the indicated eYFP-tagged Drosophila atlastin variant constructs, HeLa cells were further transfected using siRNAs targeting endogenous atlastin2 and atlastin3. At 72 h after knockdown, cells were fixed and viewed by confocal microscopy. The negative control Myc-tagged REEP5/DP1/TB2 was stained with an antibody against the Myc epitope. Scale bar, 10 μm. (B) Quantification of the percentage of cells expressing either the Myc-tagged or eYFP-tagged construct and showing an abnormal unbranched ER morphology. Values represent the means of three independent experiments ( > 100 cells each) ± SD. * p

    Article Snippet: Cell culture, constructs, transfections, and reagents HeLa and Cos-7 cells were maintained in MEM (Sigma-Aldrich, St. Louis, MO) and DMEM (Sigma-Aldrich), respectively, containing 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA) at 37°C in a 5% CO2 incubator.

    Techniques: Transfection, Variant Assay, Construct, Confocal Microscopy, Negative Control, Staining, Expressing

    Atlastin1 and Drosophila atlastin maintain a normal branched ER network in HeLa cells in the absence of atlastin2/3. At 48 h after transfection with Myc-tagged REEP5/DP1/TB2 (neg con; A, B), eYFP-tagged Drosophila atlastin (C), or HA-tagged atlastin1 (D), cells were either left untreated (A) or further transfected using siRNAs targeting atlastin2/3 (B–D). At 72 h after knockdown, cells were fixed and stained using an antibody against the Myc or HA epitope and viewed by confocal microscopy. Scale bars, 10 μm. The insets in A–D show magnified views of a boxed region of the peripheral ER. (E) Quantification of the average number of network branch points (±SD) in five representative 100-μm 2 boxed peripheral regions of the ER under each condition. (F) Quantification of the percentage of cells expressing Myc-REEP5/DP1/TB2 (with or without RNAi), eYFP– Drosophila atlastin (with RNAi), or HA-atlastin1 (with RNAi) that displayed an abnormal unbranched ER morphology. Values represent the means of three independent experiments ( > 100 cells each) ± SD. * p

    Journal: Molecular Biology of the Cell

    Article Title: ER network formation and membrane fusion by atlastin1/SPG3A disease variants

    doi: 10.1091/mbc.E14-10-1447

    Figure Lengend Snippet: Atlastin1 and Drosophila atlastin maintain a normal branched ER network in HeLa cells in the absence of atlastin2/3. At 48 h after transfection with Myc-tagged REEP5/DP1/TB2 (neg con; A, B), eYFP-tagged Drosophila atlastin (C), or HA-tagged atlastin1 (D), cells were either left untreated (A) or further transfected using siRNAs targeting atlastin2/3 (B–D). At 72 h after knockdown, cells were fixed and stained using an antibody against the Myc or HA epitope and viewed by confocal microscopy. Scale bars, 10 μm. The insets in A–D show magnified views of a boxed region of the peripheral ER. (E) Quantification of the average number of network branch points (±SD) in five representative 100-μm 2 boxed peripheral regions of the ER under each condition. (F) Quantification of the percentage of cells expressing Myc-REEP5/DP1/TB2 (with or without RNAi), eYFP– Drosophila atlastin (with RNAi), or HA-atlastin1 (with RNAi) that displayed an abnormal unbranched ER morphology. Values represent the means of three independent experiments ( > 100 cells each) ± SD. * p

    Article Snippet: Cell culture, constructs, transfections, and reagents HeLa and Cos-7 cells were maintained in MEM (Sigma-Aldrich, St. Louis, MO) and DMEM (Sigma-Aldrich), respectively, containing 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA) at 37°C in a 5% CO2 incubator.

    Techniques: Transfection, Staining, Confocal Microscopy, Expressing

    Intracellular delivery of (−30)GFP-Cre protein. A) Averaged hydrodynamic diameter and polydispersity index of (−30)GFP-Cre-loaded nanoparticles characterized by DLS. B) Delivery efficacy of (−30)GFP-Cre-loaded nanoparticles tested against HeLa and DsRed-HeLa cells after 8 h of exposure. C) DsRed protein expression and D) cell viability of (−30)GFP-Cre-loaded nanoparticles treated DsRed-HeLa cells after 24 h of exposure.

    Journal: Advanced healthcare materials

    Article Title: Intracellular Delivery of His-Tagged Genome-Editing Proteins Enabled by Nitrilotriacetic Acid-Containing Lipidoid Nanoparticles

    doi: 10.1002/adhm.201800996

    Figure Lengend Snippet: Intracellular delivery of (−30)GFP-Cre protein. A) Averaged hydrodynamic diameter and polydispersity index of (−30)GFP-Cre-loaded nanoparticles characterized by DLS. B) Delivery efficacy of (−30)GFP-Cre-loaded nanoparticles tested against HeLa and DsRed-HeLa cells after 8 h of exposure. C) DsRed protein expression and D) cell viability of (−30)GFP-Cre-loaded nanoparticles treated DsRed-HeLa cells after 24 h of exposure.

    Article Snippet: HeLa, HEK, DsRed-HeLa and GFP-HEK cells were cultured in Dulbecco’s modified eagle’s medium (DMEM, Sigma-Aldrich) with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco).

    Techniques: Expressing

    Analysis of biological effects of BA and OA. ( a ) Survival rates at T3 as a function of dose (10–30 μM). ( b ) At T1 with DMSO (control) and triterpenoids (20 μM), HaCaT keratinocytes were stained with the dyes Acridine Orange (AO) and Propidium Iodide (PI), and evaluated under microscope. Arrows indicated nuclear PI positive dead cells with acidic vacuoles accumulation. ( c ) Alternatively, at T2 with DMSO (control), triterpenoids (20 μM) or TEM (15 μM) the acidic vacuoles AO-stained were quantified by FACS. ( d ) At T2 HaCaT keratinocytes treated with DMSO (control) and triterpenoids (20 μM) following staining with Neutral Red (30 μg/mL) were evaluated under microscope ( i ). At indicative times, the cell survival was assayed by MTT and linearly correlated to lysosomal content measured in terms of autophagy arbitrary units – AAU ( ii ). ( e ) At T3 with DMSO (control) and triterpenoids (20 μM) the cell survival of HaCaT and HeLa treated cells was assayed by MTT reduction following AAU calculation. ( f ) At T1 and T2, HaCaT keratinocytes treated with DMSO (control) and triterpenoids (20 μM) following FACS were gated according the Side Scatter (SSC) and Forward Scatter (FSC) parameters ( i ). After gating, bars showed the ponderation of cell size (FSC) and granularity (SSC) compared to control and represented as arbitrary units ( ii ). (g) After the same experimental condition ( f ), a pseudo-color scatter-plots showed gating of HaCaT treated cells according to two parameters (mitochondrial mass and cell death), following staining with MitoTracker green FM and PI dye inclusion ( i ). Regarding these parameters, subpopulations of treated-cells were identified, weigthed by control and represented as arbitrary units ( ii ). All results were obtained from at least three independent experiments and expressed as mean values ± standard error. Multiple statistical comparisons were calculated by ANOVA test, and the p-value for each pairwise group was determined by Dunnett’s T3 (high variance between groups) or Bonferroni (low variance between groups) post-hoc test. The analysis of correlation was done using Pearson’s coefficient (r). Significance difference (p

    Journal: Scientific Reports

    Article Title: Parallel damage in mitochondrial and lysosomal compartments promotes efficient cell death with autophagy: The case of the pentacyclic triterpenoids

    doi: 10.1038/srep12425

    Figure Lengend Snippet: Analysis of biological effects of BA and OA. ( a ) Survival rates at T3 as a function of dose (10–30 μM). ( b ) At T1 with DMSO (control) and triterpenoids (20 μM), HaCaT keratinocytes were stained with the dyes Acridine Orange (AO) and Propidium Iodide (PI), and evaluated under microscope. Arrows indicated nuclear PI positive dead cells with acidic vacuoles accumulation. ( c ) Alternatively, at T2 with DMSO (control), triterpenoids (20 μM) or TEM (15 μM) the acidic vacuoles AO-stained were quantified by FACS. ( d ) At T2 HaCaT keratinocytes treated with DMSO (control) and triterpenoids (20 μM) following staining with Neutral Red (30 μg/mL) were evaluated under microscope ( i ). At indicative times, the cell survival was assayed by MTT and linearly correlated to lysosomal content measured in terms of autophagy arbitrary units – AAU ( ii ). ( e ) At T3 with DMSO (control) and triterpenoids (20 μM) the cell survival of HaCaT and HeLa treated cells was assayed by MTT reduction following AAU calculation. ( f ) At T1 and T2, HaCaT keratinocytes treated with DMSO (control) and triterpenoids (20 μM) following FACS were gated according the Side Scatter (SSC) and Forward Scatter (FSC) parameters ( i ). After gating, bars showed the ponderation of cell size (FSC) and granularity (SSC) compared to control and represented as arbitrary units ( ii ). (g) After the same experimental condition ( f ), a pseudo-color scatter-plots showed gating of HaCaT treated cells according to two parameters (mitochondrial mass and cell death), following staining with MitoTracker green FM and PI dye inclusion ( i ). Regarding these parameters, subpopulations of treated-cells were identified, weigthed by control and represented as arbitrary units ( ii ). All results were obtained from at least three independent experiments and expressed as mean values ± standard error. Multiple statistical comparisons were calculated by ANOVA test, and the p-value for each pairwise group was determined by Dunnett’s T3 (high variance between groups) or Bonferroni (low variance between groups) post-hoc test. The analysis of correlation was done using Pearson’s coefficient (r). Significance difference (p

    Article Snippet: Cell lines and cell culture We cultured immortalized human skin keratinocytes (HaCaT) and epidermoid carcinoma of the cervix (HeLa) in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL of penicillin, 25 μg/mL of amphotericin B and 100 μg/mL of streptomycin in a 37 °C incubator at a moist atmosphere of 5% carbon dioxide.

    Techniques: Staining, Microscopy, Transmission Electron Microscopy, FACS, MTT Assay