Figure 6The expression levels of the indicated proteins were detected by immunoblotting analysis in CIP2A - BP KO , LINC00665 ORF overexpressed ( OE ) and respective controls of TNBC cells . PP2A activity was measured using a protein phosphatase assay kit . PP2A activity assay was performed to evaluate PP2A activity in CIP2A - BP KO , LINC00665 ORF overexpressed ( OE ) and respective control TNBC cells . Immunohistochemistry ( IHC ) staining of p - AKT from TNBC primary tumor ( n = 20 ) with high or low micropeptide CIP2A - BP expression . Lower panels show higher - magnification images of insets in upper panels . Right panel is the quantification of IHC staining p - AKT . MDA - MB - 231 cells were transfected with anti - CIP2A siRNAs . Whole - cell lysates of these cells were subjected to immunoblot analysis with CIP2A , CIP2A - BP , PP2Ac , p - AKT , AKT and β - actin antibodies . CIP2A - BP KO , LINC00665 ORF overexpressed ( OE ) and respective control MDA - MB - 231 cells were transfected with anti - CIP2A siRNAs . PP2A activity assay was performed to evaluate PP2A activity in the indicated cells . The indicated cells were pretreated with specific antagonist against AKT ( MK - 2206 ， 3μM ) for 1h . Then whole lysates of these cells were subjected to immunoblot analysis .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6The expression levels of the indicated proteins were detected by immunoblotting analysis in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective controls of TNBC cells.PP2A activity was measured using a protein phosphatase assay kit. PP2A activity assay was performed to evaluate PP2A activity in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective control TNBC cells.Immunohistochemistry (IHC) staining of p-AKT from TNBC primary tumor (n = 20) with high or low micropeptide CIP2A-BP expression. Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT.MDA-MB-231 cells were transfected with anti-CIP2A siRNAs. Whole-cell lysates of these cells were subjected to immunoblot analysis with CIP2A, CIP2A-BP, PP2Ac, p-AKT, AKT and β-actin antibodies.CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective control MDA-MB-231 cells were transfected with anti-CIP2A siRNAs. PP2A activity assay was performed to evaluate PP2A activity in the indicated cells.The indicated cells were pretreated with specific antagonist against AKT (MK-2206，3μM) for 1h. Then whole lysates of these cells were subjected to immunoblot analysis.


Figure 7Macroscopic and histological analysis of the lungs of 140 days old MMTV - PyMT mice ; lung metastatic nodules were visualized 8 weeks post - transplantation ( 5 mice per group ) . Right panel is the quantification of pulmonary metastases . Immunohistochemistry ( IHC ) staining of p - AKT of mammary tumors from MMTV - PyMT ; CIP2A - BP + / + or MMTV - PyMT ; CIP2A - BP - / - mice . Lower panels show higher - magnification images of insets in upper panels . Right panel is the quantification of IHC staining p - AKT . MMTV - PyMT mice were injected with CIP2A - BP or svCIP2A - BP via mammary fat pad . Macroscopic and histological analysis of the lungs of 140 days old MMTV - PyMT mice ; lung metastatic nodules were visualized 8 weeks post - transplantation ( 5 mice per group ) . Right panel is the quantification of pulmonary metastases . Representative IHC images of p - AKT of mammary tumors from MMTV - PyMT mice that were injected with CIP2A - BP or svCIP2A - BP via mammary fat pad ; Lower panels show higher - magnification images of insets in upper panels . Right panel is the quantification of IHC staining p - AKT . Kaplan - Meier survival curves of nude mice transplanted with MDA - MB - 231 cells and injected with CIP2A - BP or svCIP2A - BP ( 10 mice per group ) . The horizontal line indicates the time after the mice were injected with the cells via the tail vein . Macroscopic and histological analysis of the lungs of nude mice injected with MDA - MB - 231 cells and CIP2A - BP or svCIP2A - BP via tail vein ; lung metastatic nodules were visualized 8 weeks post - transplantation ( 5 mice per group ) . Right panel is the quantification of pulmonary metastases .

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Figure 7Macroscopic and histological analysis of the lungs of 140 days old MMTV-PyMT mice; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.Immunohistochemistry (IHC) staining of p-AKT of mammary tumors from MMTV-PyMT;CIP2A-BP+/+ or MMTV-PyMT;CIP2A-BP-/- mice. Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT .MMTV-PyMT mice were injected with CIP2A-BP or svCIP2A-BP via mammary fat pad. Macroscopic and histological analysis of the lungs of 140 days old MMTV-PyMT mice; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.Representative IHC images of p-AKT of mammary tumors from MMTV-PyMT mice that were injected with CIP2A-BP or svCIP2A-BP via mammary fat pad; Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT.Kaplan-Meier survival curves of nude mice transplanted with MDA-MB-231 cells and injected with CIP2A-BP or svCIP2A-BP (10 mice per group). The horizontal line indicates the time after the mice were injected with the cells via the tail vein.Macroscopic and histological analysis of the lungs of nude mice injected with MDA-MB-231 cells and CIP2A-BP or svCIP2A-BP via tail vein; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.


Figure 1Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence of H676 cells treated with the indicated compounds for 24h at 1 μM . Scale bars , 5μm . Data are representative of n = 3 biological replicates . ( C ) Western blot images ( top ) and TRF1 protein levels ( bottom ) of h676 cells treated with the indicated compounds for 24h at 1 μM . Data are representative of n = 3 ( PLKi , HSP90i and RTKi ) and n = 4 ( AuroraI , mTOR , CDKi , Docetaxel , Gemcitabine , ERKi , MEKi ) biological replicates .

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Figure 1Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence of H676 cells treated with the indicated compounds for 24h at 1 μM. Scale bars, 5μm. Data are representative of n=3 biological replicates. (C) Western blot images (top) and TRF1 protein levels (bottom) of h676 cells treated with the indicated compounds for 24h at 1 μM. Data are representative of n=3 (PLKi, HSP90i and RTKi) and n=4 (AuroraI, mTOR, CDKi, Docetaxel, Gemcitabine, ERKi, MEKi) biological replicates.


Figure 2Representative images ( left ) and percentage ( right ) of cells presenting 1 or more γH2AX and RAP1 colocalizing foci ( TIFs ) upon treatment of CHA9 - 3 lung cancer cells with the indicated compounds . White arrowheads point to colocalization of γH2AX and RAP1 . Scale bars , 5μm . Data are representative of n = 6 ( DMSO ) and n = 3 ( mTORi , PI3Ki , RTKi , MEKi , ERKi , HSPO90i , CDKi , Docetaxel ) biological replicatesRepresentative images ( left ) and percentage ( right ) of γH2AX - positive cells per field in DMSO or compound treated patient - derived h676 GSCs . Scale bars , 50μm . Data are representative of 6 ( DMSO ) and 3 ( mTORi , Docetaxel , ERKi , MEKi , RTKi , HSP90i , Gemcitabine , CDKi ) biological replicatesQuantification of multitelomeric signals ( MTS ) in h676 GSC metaphases upon treatment with the indicated compounds . Representative images of the qFISH in the metaphases ( left ) . Multitelomeric signals are indicated by arrowheads . Scale bars , 1μm . Data are representative of n = 31 ( DMSO ) , n = 18 ( mTORi ) , n = 11 ( MEKi ) and n = 24 ( RTKi ) biological replicates . Data are represented as mean ± SEM . Significant differences using unpaired t ‐ test are indicated by * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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Figure 2Representative images (left) and percentage (right) of cells presenting 1 or more γH2AX and RAP1 colocalizing foci (TIFs) upon treatment of CHA9-3 lung cancer cells with the indicated compounds. White arrowheads point to colocalization of γH2AX and RAP1. Scale bars, 5μm. Data are representative of n=6 (DMSO) and n=3 (mTORi, PI3Ki, RTKi, MEKi, ERKi, HSPO90i, CDKi, Docetaxel) biological replicatesRepresentative images (left) and percentage (right) of γH2AX-positive cells per field in DMSO or compound treated patient-derived h676 GSCs. Scale bars, 50μm. Data are representative of 6 (DMSO) and 3 (mTORi, Docetaxel, ERKi, MEKi, RTKi, HSP90i, Gemcitabine, CDKi) biological replicatesQuantification of multitelomeric signals (MTS) in h676 GSC metaphases upon treatment with the indicated compounds. Representative images of the qFISH in the metaphases (left). Multitelomeric signals are indicated by arrowheads. Scale bars, 1μm. Data are representative of n=31 (DMSO), n=18 (mTORi), n=11 (MEKi) and n=24 (RTKi) biological replicates. Data are represented as mean ± SEM. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001.


Figure 3Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.


Figure 41 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of mouse ERK2 kinase in the presence of 5μCi [ γ - 32 - P ] ATP . The mixture was resolve by SDS - PAGE followed by autoradiography . 1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of human b - Raf kinase ( WT or V600E ) in the presence of 5μCi [ γ - 32 - P ] ATP . The mixture was resolve by SDS - PAGE followed by autoradiography . 1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of mouse MEK1 kinase in the presence of 5μCi [ γ - 32 - P ] ATP . The mixture was resolve by SDS - PAGE followed by autoradiography . 1 μM of GST - TRF1 and 0 . 2 μM of mouse ERK2 kinase were incubated in the presence of ERK and MEK inhibitors . 2 μM of GST - TRF1 and 0 . 1 μM of human b - Raf kinase were incubated in the presence of the bRaf inhibitors dabrafenib and vemurafenib . 1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of human mTOR kinase1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of human mTOR kinase in the presence of the mTOR inhibitors rapamycin and Ku0063794Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 or TRF1 plus ERK2Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus bRAFWT or bRAFV600EPhosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus mTORRepresentative image ( down ) and quantification ( up ) of in vitro phosphorylation assays with the indicated GST - TRF1 wild - type or mutated forms in the presence of mouse ERK2 kinase .

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Figure 41 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse ERK2 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human b-Raf kinase (WT or V600E) in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse MEK1 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.1 μM of GST-TRF1 and 0.2 μM of mouse ERK2 kinase were incubated in the presence of ERK and MEK inhibitors.2 μM of GST-TRF1 and 0.1 μM of human b-Raf kinase were incubated in the presence of the bRaf inhibitors dabrafenib and vemurafenib.1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase in the presence of the mTOR inhibitors rapamycin and Ku0063794Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 or TRF1 plus ERK2Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus bRAFWT or bRAFV600EPhosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus mTORRepresentative image (down) and quantification (up) of in vitro phosphorylation assays with the indicated GST-TRF1 wild-type or mutated forms in the presence of mouse ERK2 kinase.


Figure 5Western blot images of Trf1lox / lox MEFS with or without overexpression of eGFP - Trf1 WT or mutant alleles followed by Cre recombinase transduction . Quantification of eGFP - TRF1 inhibition in Trf1Δ / Δ MEFs transduced with eGFP - Trf1 WT or mutant alleles as indicated . Data are representative of n = 15 biological replicatesGrowth curves of Trf1Δ / Δ MEFs transduced with eGFP - Trf1 WT or mutant alleles as indicated . Data are representative of n = 5 biological replicatesWestern blot images of Trf1lox / lox MEFS with or without overexpression of eGFP - Trf1 WT or mutant alleles followed by Cre recombinase transduction . Quantification of eGFP - TRF1 inhibition in Trf1Δ / Δ MEFs transduced with eGFP - Trf1 WT or mutant alleles as indicated . Data are representative of n = 5 biological replicatesWestern blot images of p53 - / - MEFS with or without overexpression of eGFP - Trf1 WT or mutant alleles followed treatment with ERKi . Data are representative of n = 2 biological replicatesRepresentative images ( above ) and percentage ( bottom ) of Telomeric and 53BP1 colocalizing foci ( TIFs ) per cells of p53 - / - MEFS with or without overexpression of eGFP - Trf1 WT and the indicated mutants upon treatment with the ERKi . White arrowheads : colocalization of telomeric and 53BP1 . Scale bars , 10μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Western blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction.Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=15 biological replicatesGrowth curves of Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicatesWestern blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction. Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicatesWestern blot images of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed treatment with ERKi. Data are representative of n=2 biological replicatesRepresentative images (above) and percentage (bottom) of Telomeric and 53BP1 colocalizing foci (TIFs) per cells of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT and the indicated mutants upon treatment with the ERKi. White arrowheads: colocalization of telomeric and 53BP1. Scale bars, 10μm.


Figure 6Western blot image ( above ) and quantification ( bottom ) of TRF1 protein levels upon genetic depletion of ERK1 / 2 in p53 - / - MEF line . Data are representative of n = 3 independent experiments . Representative images ( above ) and quantification ( bottom ) of TRF1 telomeric foci in ERK1 / 2 RNA interfered p53 - / - MEFs . Scale bars , 5μm . Data are representative of n = 2 independent experiments . Western blot image of eGFP - tagged and endogenous TRF1 protein levels upon genetic depletion of ERK1 / 2 in p53 - / - MEF line . Representative images ( above ) and percentage ( bottom ) of telomeric and 53BP1 colocalizing foci ( TIFs ) per cell in p53 - / - MEF with or without overexpression of eGFP - Trf1 WT and indicated mutants upon genetic depletion of ERK1 / 2 . White arrowheads : colocalization of telomeric and 53BP1 . Scale bars , 10μm .

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Figure 6Western blot image (above) and quantification (bottom) of TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line. Data are representative of n=3 independent experiments.Representative images (above) and quantification (bottom) of TRF1 telomeric foci in ERK1/2 RNA interfered p53-/- MEFs. Scale bars, 5μm. Data are representative of n=2 independent experiments.Western blot image of eGFP-tagged and endogenous TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line.Representative images (above) and percentage (bottom) of telomeric and 53BP1 colocalizing foci (TIFs) per cell in p53-/- MEF with or without overexpression of eGFP-Trf1 WT and indicated mutants upon genetic depletion of ERK1/2. White arrowheads: colocalization of telomeric and 53BP1.Scale bars, 10μm.


Figure 7Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates . Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates . Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates . Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates . Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates . Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates . Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of n = 3 ( RTKi in G ( DMSO in GWestern blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of ( DMSO in and ERKi in H ) Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of ( DMSO in ( PI3Ki inWestern blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of ( DMSO in ( PI3Ki inWestern blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of ( DMSOWestern blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of Docetaxel (

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of n=3 (RTKi in G (DMSO in GWestern blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in and ERKi in H)Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in (PI3Ki inWestern blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in (PI3Ki inWestern blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSOWestern blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of Docetaxel (DMSO


Figure 8Longitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in A : vehicle n = 16 , PI3Ki n = 8 , ERKi n = 16 , Combination n = 8 Pvalues represent the mean of all the time pointsLongitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in B : Vehicle n = 16 , PI3Ki n = 16 , MEKi n = 8 , Combination n = 8 Pvalues represent the mean of all the time pointsLongitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in C : Vehicle n = 8 , PI3Ki n = 16 , Docetaxel n = 16 , Combination n = 8 Pvalues represent the mean of all the time pointsLongitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in D : Vehicle n = 16 , PI3K n = 16 , Gemcitabine n = 4 , Combination n = 4 . Pvalues represent the mean of all the time pointsRepresentative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors : in E : vehicle n = 15 , PI3Ki n = 10 , ERKi n = 8 , Combination n = 2Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors in F : Vehicle n = 15 , PI3Ki n = 10 , MEKi n = 8 , Combination n = 6Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors ; in G : Vehicle n = 15 , PI3Ki n = 10 , Docetaxel n = 8 , Combination n = 4Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors ; in H : Vehicle n = 15 , PI3K n = 10 , Gemcitabine n = 8 , Combination n = 6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in A: vehicle n=16, PI3Ki n=8, ERKi n=16, Combination n=8 Pvalues represent the mean of all the time pointsLongitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in B: Vehicle n=16, PI3Ki n=16, MEKi n=8, Combination n=8 Pvalues represent the mean of all the time pointsLongitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in C: Vehicle n=8, PI3Ki n=16, Docetaxel n=16, Combination n=8 Pvalues represent the mean of all the time pointsLongitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in D: Vehicle n=16, PI3K n=16, Gemcitabine n=4, Combination n=4. Pvalues represent the mean of all the time pointsRepresentative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors: in E: vehicle n=15, PI3Ki n=10, ERKi n=8, Combination n=2Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors in F: Vehicle n=15, PI3Ki n=10, MEKi n=8, Combination n=6Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors ; in G: Vehicle n=15, PI3Ki n=10, Docetaxel n=8, Combination n=4Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors ; in H: Vehicle n=15, PI3K n=10, Gemcitabine n=8, Combination n=6.


Figure 1 EdU - labeled viral DNA forms large foci in ThP1 cells infected by HIV - 1 . EdU images are shown in red , DAPI images in blue . ( A ) Left : nucleus of an infected cell at 48 h p . i . displays three large and bright EdU foci ( colored arrows ) , as well as small and dim punctae ( grey arrows ) . Center : Magnified views have colored borders matching the colors of the arrows . 3D images show that EdU foci are located within the nucleoplasm ( Movie EV1 , Appendix Fig . S2 ) . Right : nucleus of an uninfected cell displays no EdU signal . ( B ) Graph shows EdU intensity profiles along lines shown in A , with colors matching the corresponding arrows . ( C ) Dual - color image of an infected cell with EdU ( red ) and immunolabeled lamin ( grey ) . Curves to the right plot the intensity profile of EdU and lamin along the dashed blue line . The EdU intensity peaks do not coincide with lamin enrichment . See also Fig . EV1A , B . ( D ) Multi - color images of an infected cell showing EdU ( red ) with CA ( yellow ) and integrase ( green ) . The colocalization of nuclear EdU foci with CA and IN ( arrows ) confirms that EdU specifically labels viral DNA and shows the presence of these proteins in vDNA foci . See also Appendix Fig . S3 . ( E , F ) vDNA foci are much larger than the predicted size of single viral genomes . ( E ) Left image shows an observed , EdU - labeled vDNA focus . Right : simulations of a single linear or circular 10 Kb long chromatinized DNA polymer chain and corresponding predicted images in diffraction - limited ( ~ 300 nm resolution ) microscopy . ( F ) Boxplots show the distribution of sizes ( FWHM , full width at half maximum ) of n = 40 measured vDNA foci ( for MOI 100 ) compared to the sizes predicted for linear ( left ) and circular ( right ) chains based on n = 100 simulated configurations each . Blue lines in boxes define medians , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . All differences are highly significant ( Wilcoxon test data vs . model : * * * p ≈ 3 ⋅ 10 − 20 , circular vs . linear model : * * * p ≈ 10 − 17 ) . ( G ) Images of vDNA foci ( arrows ) in ThP1 cells infected with multiplicities of infection ( MOI ) 10 , 50 and 100 . Images for MOI 10 are shown at two different contrast levels to reveal dimmer spots . Scale bars , 3 µm . ( H ) Boxplots compare the peak intensities inside individual vDNA foci for MOIs 10 , 50 and 100 . Central lines in boxes define medians , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . Intensities increase significantly with MOI ( Wilcoxon tests : MOI 10 vs . MOI 50 : * * * p ≈ 5 ⋅ 10 − 16 ; MOI 50 vs . MOI 100 : * * * p ≈ 5 ⋅ 10 − 5 ; MOI 10 vs . MOI 100 : * * * p ≈ 2 ⋅ 10 − 23 ) . Data from one experiment . A replicate showed similar results . ( I ) Histograms show the number of foci per nucleus for MOIs of 10 ( blue ) , 50 ( green ) and 100 ( red ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 EdU-labeled viral DNA forms large foci in ThP1 cells infected by HIV-1. EdU images are shown in red, DAPI images in blue. (A) Left: nucleus of an infected cell at 48 h p.i. displays three large and bright EdU foci (colored arrows), as well as small and dim punctae (grey arrows). Center: Magnified views have colored borders matching the colors of the arrows. 3D images show that EdU foci are located within the nucleoplasm (Movie EV1, Appendix Fig. S2). Right: nucleus of an uninfected cell displays no EdU signal. (B) Graph shows EdU intensity profiles along lines shown in A, with colors matching the corresponding arrows.  (C) Dual-color image of an infected cell with EdU (red) and immunolabeled lamin (grey). Curves to the right plot the intensity profile of EdU and lamin along the dashed blue line. The EdU intensity peaks do not coincide with lamin enrichment. See also Fig. EV1A,B.  (D) Multi-color images of an infected cell showing EdU (red) with CA (yellow) and integrase (green). The colocalization of nuclear EdU foci with CA and IN (arrows) confirms that EdU specifically labels viral DNA and shows the presence of these proteins in vDNA foci. See also Appendix Fig. S3. (E,F) vDNA foci are much larger than the predicted size of single viral genomes.  (E) Left image shows an observed, EdU-labeled vDNA focus. Right: simulations of a single linear or circular 10 Kb long chromatinized DNA polymer chain and corresponding predicted images in diffraction-limited (~300 nm resolution) microscopy. (F) Boxplots show the distribution of sizes (FWHM, full width at half maximum) of n=40 measured vDNA foci (for MOI 100) compared to the sizes predicted for linear (left) and circular (right) chains based on n=100 simulated configurations each. Blue lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. All differences are highly significant (Wilcoxon test data vs. model: ***p ≈ 3 ⋅ 10 − 20, circular vs. linear model: ***p ≈ 10 − 17).  (G) Images of vDNA foci (arrows) in ThP1 cells infected with multiplicities of infection (MOI) 10, 50 and 100. Images for MOI 10 are shown at two different contrast levels to reveal dimmer spots. Scale bars, 3 µm.  (H) Boxplots compare the peak intensities inside individual vDNA foci for MOIs 10, 50 and 100. Central lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. Intensities increase significantly with MOI (Wilcoxon tests: MOI 10 vs. MOI 50: *** p ≈ 5 ⋅ 10 − 16; MOI 50 vs. MOI 100: ***p ≈ 5 ⋅ 10 − 5; MOI 10 vs. MOI 100: ***p ≈ 2 ⋅ 10 − 23). Data from one experiment. A replicate showed similar results.  (I) Histograms show the number of foci per nucleus for MOIs of 10 (blue), 50 (green) and 100 (red).


Figure 2vDNA foci are also foci of vRNA . ( A ) Dual - color image of an infected ThP1 cell showing the viral DNA ( red ) and the RNA visualized by RNA - FISH ( green ) . Arrows show foci containing both vDNA and vRNA . The bottom row shows the boxed region magnified , with vDNA and vRNA displayed separately and in combination . See also Appendix Fig . S6 . ( C ) Scatter plot shows the intensities of vDNA and vRNA in colocalizing foci with the Spearman correlation r and associated p - value . In ThP1 cells co - infected with a HIV1 - GFP virus and a HIV1 - LUC virus , nuclear foci contain mixtures of both viruses . ( D ) Dual - color image shows RNA - FISH against GFP ( red ) and RNA - FISH against LUC ( green ) . Arrows show foci containing both GFP vRNA and LUC vRNA . The bottom row shows the boxed region magnified , with GFP vRNA and LUC vRNA displayed separately and in combination . See Appendix Fig . S7 . ( F ) Scatter plot shows intensities of GFP RNA and LUC RNA in colocalizing foci with the Spearman correlation r and associated p - value .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2vDNA foci are also foci of vRNA. (A) Dual-color image of an infected ThP1 cell showing the viral DNA (red) and the RNA visualized by RNA-FISH (green). Arrows show foci containing both vDNA and vRNA. The bottom row shows the boxed region magnified, with vDNA and vRNA displayed separately and in combination. See also Appendix Fig. S6.(C) Scatter plot shows the intensities of vDNA and vRNA in colocalizing foci with the Spearman correlation r and associated p-value. In ThP1 cells co-infected with a HIV1-GFP virus and a HIV1-LUC virus, nuclear foci contain mixtures of both viruses. (D) Dual-color image shows RNA-FISH against GFP (red) and RNA-FISH against LUC (green). Arrows show foci containing both GFP vRNA and LUC vRNA. The bottom row shows the boxed region magnified, with GFP vRNA and LUC vRNA displayed separately and in combination. See Appendix Fig. S7.  (F) Scatter plot shows intensities of GFP RNA and LUC RNA in colocalizing foci with the Spearman correlation r and associated p-value.


Figure 3 Images of infected ( right ) or uninfected ( left ) ThP1 cells showing the vDNA ( EdU ) in red , the nucleus ( DAPI ) in blue , and selected nuclear body factors in green . ( A ) Green image shows immunolabeling of CPSF6 . ( B ) Green image shows RNA - FISH against NEAT1 . ( C ) Green image shows immunolabeling of SC35 . See also Appendix Figs . S9 - 14 . ( D ) Boxplots show Pearson correlations r between vDNA ( EdU ) and CPSF6 , NEAT1 or SC35 in 17 - 19 regions of interest ( ROIs ) . Central lines in boxes define medians , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . Significance of positive or negative correlations was assessed using the Costes method of random ROI shifts . Highly significant ( p < 0 . 01 ) positive correlations between vDNA and CPSF6 intensities are found in 16 out of 19 ROIs ; highly significant negative correlations between vDNA and NEAT1 are found in 19 / 19 ROIs , and highly significant positive correlations between vDNA and SC35 are found for 16 / 17 ROIs . Data are from one experiment . For CPSF6 , the experiment was repeated with similar results . ( E ) Intensity profiles of EdU and DAPI along the dotted lines in the vDNA images above .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 Images of infected (right) or uninfected (left) ThP1 cells showing the vDNA (EdU) in red, the nucleus (DAPI) in blue, and selected nuclear body factors in green. (A) Green image shows immunolabeling of CPSF6. (B) Green image shows RNA-FISH against NEAT1. (C) Green image shows immunolabeling of SC35. See also Appendix Figs. S9-14.  (D) Boxplots show Pearson correlations r between vDNA (EdU) and CPSF6, NEAT1 or SC35 in 17-19 regions of interest (ROIs). Central lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. Significance of positive or negative correlations was assessed using the Costes method of random ROI shifts. Highly significant (p<0.01) positive correlations between vDNA and CPSF6 intensities are found in 16 out of 19 ROIs; highly significant negative correlations between vDNA and NEAT1 are found in 19/19 ROIs, and highly significant positive correlations between vDNA and SC35 are found for 16/17 ROIs. Data are from one experiment. For CPSF6, the experiment was repeated with similar results.  (E) Intensity profiles of EdU and DAPI along the dotted lines in the vDNA images above.


Figure 4 ( A ) Image of a ThP1 cell infected with an integration - deficient HIV - 1 carrying the mutation D116A in the catalytic site of IN . The EdU - labeled vDNA is shown in red and the vRNA detected by RNA - FISH in green . The nucleus ( DAPI ) is shown in blue . See Appendix Fig . S12 for a larger image region . ( C ) Scatter plot shows vDNA and vRNA intensities in colocalizing vDNA and vRNA clusters with the Spearman correlation r and associated p - value . ( D ) ThP1 cells were infected with HIV - 1 at an MOI of 20 , in absence ( left ) or presence ( right ) of nevirapine ( NVP , 10 µM ) . Blue : DAPI staining . Green : vRNA ( RNA - FISH ) . Red : vDNA ( EdU ) . ( E ) Scatter plots show intensities of vDNA and vRNA in detected vRNA clusters . ( F ) Image of a ThP1 cell infected by HIV - 1 with EU labeled RNA in absence ( left ) or presence ( right ) of nevirapine ( NVP , 10 µM ) . Blue : DAPI staining . Green : vRNA ( RNA - FISH ) . Grey : EU - labeled vRNA . See Appendix Fig . S19 for a larger image region . ( G ) Intensity profiles of EU and RNA - FISH along the dotted lines in panel F ( top ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 (A) Image of a ThP1 cell infected with an integration-deficient HIV-1 carrying the mutation D116A in the catalytic site of IN. The EdU-labeled vDNA is shown in red and the vRNA detected by RNA-FISH in green. The nucleus (DAPI) is shown in blue. See Appendix Fig. S12 for a larger image region.  (C) Scatter plot shows vDNA and vRNA intensities in colocalizing vDNA and vRNA clusters with the Spearman correlation r and associated p-value.  (D) ThP1 cells were infected with HIV-1 at an MOI of 20, in absence (left) or presence (right) of nevirapine (NVP, 10 µM). Blue: DAPI staining. Green: vRNA (RNA-FISH). Red: vDNA (EdU). (E) Scatter plots show intensities of vDNA and vRNA in detected vRNA clusters.  (F) Image of a ThP1 cell infected by HIV-1 with EU labeled RNA in absence (left) or presence (right) of nevirapine (NVP, 10 µM). Blue: DAPI staining. Green: vRNA (RNA-FISH). Grey: EU-labeled vRNA. See Appendix Fig. S19 for a larger image region. (G) Intensity profiles of EU and RNA-FISH along the dotted lines in panel F (top).


Figure 5 The left panel shows the timeline of drug exposure experiments . ( A - D ) Images show vRNA and vDNA in an infected ThP1 cell ( MOI 20 ) for each of four experimental conditions . See Appendix Fig . S15 - S17 for images of larger regions . ( A , B ) ThP1 cells were exposed to NVP for 48 h ( A ) or 72 h after infection ( B ) , then NVP was washed out , and cells were fixed for click chemistry 24 h later . ( C ) Cells were exposed to PF74 for 72 h after infection , then fixed for click chemistry . ( D ) Cells were exposed to NVP for 24 h after infection , then NVP was washed out , and cells were exposed to PF74 , before being fixed for click chemistry 24 h later . ( E , F , H ) Scatter plots show vDNA and vRNA intensities in colocalizing clusters with Spearman correlation r and associated p - values . Replicates of the four experiments yielded similar results . ( G ) qPCR measures DNA synthesis ( left ) and nuclear import ( right ) at 72 h p . i . in absence of drug treatment , or after exposure to NVP or PF74 . Bars and error bars define mean and standard deviation , respectively . Samples were analyzed in triplicate and two biological replicates were performed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 The left panel shows the timeline of drug exposure experiments. (A-D) Images show vRNA and vDNA in an infected ThP1 cell (MOI 20) for each of four experimental conditions. See Appendix Fig. S15-S17 for images of larger regions. (A,B) ThP1 cells were exposed to NVP for 48 h (A) or 72 h after infection (B), then NVP was washed out, and cells were fixed for click chemistry 24 h later. (C) Cells were exposed to PF74 for 72 h after infection, then fixed for click chemistry. (D) Cells were exposed to NVP for 24 h after infection, then NVP was washed out, and cells were exposed to PF74, before being fixed for click chemistry 24 h later. (E,F,H) Scatter plots show vDNA and vRNA intensities in colocalizing clusters with Spearman correlation r and associated p-values. Replicates of the four experiments yielded similar results. (G) qPCR measures DNA synthesis (left) and nuclear import (right) at 72 h p.i. in absence of drug treatment, or after exposure to NVP or PF74. Bars and error bars define mean and standard deviation, respectively. Samples were analyzed in triplicate and two biological replicates were performed.


Figure 6 The left panel shows the timeline of infection and / or drug exposure experiments . ( A - D ) Percentage of GFP positive cells at 3 and 7 days post - infection analyzed by FACS in absence of EdU . ( A ) Uninfected control cells . ( B ) Untreated , infected ThP1 cells fixed at 3 d ( 72 h ) p . i . ( C ) Infected cells were exposed to NVP for 72 h , then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p . i . ( D ) Infected cells were cultured for 72 h p . i . , then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p . i . ( E - H ) Multicolor images of ThP1 cells infected with a GFP - reporter virus . vRNA ( RNA - FISH ) is labeled in red , GFP in green , and nuclei ( DAPI ) in blue . ( E ) Uninfected control cells . ( F ) Untreated , infected ThP1 cells fixed at 3 d ( 72 h ) p . i . ( G ) Infected cells were exposed to NVP for 72 h , then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p . i . ( H ) Infected cells were cultured for 72 h p . i . , then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p . i . The experiment was repeated three times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 The left panel shows the timeline of infection and/or drug exposure experiments. (A-D) Percentage of GFP positive cells at 3 and 7 days post-infection analyzed by FACS in absence of EdU. (A) Uninfected control cells. (B) Untreated, infected ThP1 cells fixed at 3 d (72 h) p.i. (C) Infected cells were exposed to NVP for 72 h, then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p.i. (D) Infected cells were cultured for 72 h p.i., then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p.i. (E-H) Multicolor images of ThP1 cells infected with a GFP-reporter virus. vRNA (RNA-FISH) is labeled in red, GFP in green, and nuclei (DAPI) in blue. (E) Uninfected control cells. (F) Untreated, infected ThP1 cells fixed at 3 d (72 h) p.i. (G) Infected cells were exposed to NVP for 72 h, then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p.i. (H) Infected cells were cultured for 72 h p.i., then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p.i. The experiment was repeated three times.


Figure 7 The left panel shows the timeline of infection and / or drug exposure experiments . ( A - D ) Multicolor images of vDNA , vRNA , DAPI and / or GFP in monocyte derived macrophages ( MDMs ) from two different donors , infected with HIV1 . Cells from donor 1 were infected with VSV - G - pseudotyped HIV - 1ΔEnv Vpx carrying a GFP reporter ( A ) and cells from donor 2 were infected with a VSV - G - pseudotyped HIV - 1ΔEnv without Vpx ( B - D ) . ( A , B ) Infected cells were left untreated and fixed at 6 d p . i . ( C ) Infected cells were treated with 10 µM Nevirapine ( NVP ) throughout the experiment and fixed at 6 d p . i . ( D ) Infected cells were exposed to NVP for 3 d p . i . , then NVP was washed out and cells were cultured for another 4 d and fixed at 7 d p . i . Arrows in ( A - D ) show the position of selected vRNA foci . ( E ) Boxplot shows the size of DNA foci in untreated infected cells from donor 2 imaged at 7 d p . i . ( B ) , measured by the FWHM of n = 24 foci . The red line defines the median , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . ( F ) Boxplot shows Pearson ' s r , measuring colocalization of DNA with RNA foci in the same cells ( n = 20 ) . Center line defines the median , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . ( G ) FACS analysis of Gag positive cells from donor 2 in different conditions . Red : uninfected cells . Blue : untreated infected cells at 7 d p . i . as in B . Orange : cells were treated with NVP for 7 d as in C . Green : infected cells were exposed to NVP for 3 d , then NVP was washed out and cells were cultured for another 4 d as in D . Median of fluorescence intensities ( MFI ) for each sample are shown in the table .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7 The left panel shows the timeline of infection and/or drug exposure experiments. (A-D) Multicolor images of vDNA, vRNA, DAPI and/or GFP in monocyte derived macrophages (MDMs) from two different donors, infected with HIV1. Cells from donor 1 were infected with VSV-G-pseudotyped HIV-1ΔEnv Vpx carrying a GFP reporter (A) and cells from donor 2 were infected with a VSV-G-pseudotyped HIV-1ΔEnv without Vpx (B-D). (A,B) Infected cells were left untreated and fixed at 6 d p.i. (C) Infected cells were treated with 10 µM Nevirapine (NVP) throughout the experiment and fixed at 6 d p.i. (D) Infected cells were exposed to NVP for 3 d p.i., then NVP was washed out and cells were cultured for another 4 d and fixed at 7 d p.i. Arrows in (A-D) show the position of selected vRNA foci. (E) Boxplot shows the size of DNA foci in untreated infected cells from donor 2 imaged at 7 d p.i. (B), measured by the FWHM of n=24 foci. The red line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. (F) Boxplot shows Pearson's r, measuring colocalization of DNA with RNA foci in the same cells (n=20). Center line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points.  (G) FACS analysis of Gag positive cells from donor 2 in different conditions. Red: uninfected cells. Blue: untreated infected cells at 7 d p.i. as in B. Orange: cells were treated with NVP for 7 d as in C. Green: infected cells were exposed to NVP for 3 d, then NVP was washed out and cells were cultured for another 4 d as in D. Median of fluorescence intensities (MFI) for each sample are shown in the table.


figf1a ) COS - 7 cells expressing EGFP - tagged wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin exon 1 ( green ) for 48 h stained for endogenous mTOR ( red ) show mTOR in mutant aggregates . ( b ) Double - labeling immunohistochemistry of huntingtin ( EM48 ; green ) and mTOR ( red ) in brain tissue of mice expressing mutant huntingtin . ( c ) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease or unaffected controls using antibody to mTOR . ( d ) Western blot of lysates from stable inducible PC12 cells 48 h after expression of wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin showing mTOR in the stacking gel ( mTOR insoluble ) in cells expressing mutant huntingtin . ( e ) High - molecular - mass aggregates of mTOR in both PC12 ( i ) and COS - 7 ( ii ) cells expressing mutant ( Gln74 ; Q74 ) huntingtin . Blots of cell lysates from PC12 cells were probed with antibodies to phosphorylated mTOR ( Ser2448 ; p - mTOR ; iii ) and to GFP ( iv ) ; both showed insoluble material in the stack . ( f ) Cell lysates from stable inducible PC12 cells and transiently transfected COS - 7 cells expressing wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin for 48 h were immunoprecipitated using antibody to GFP and immunoblotted with antibody to mTOR . Uninduced PC12 cells with mutant huntingtin ( UI ) and untransfected COS - 7 cells ( UT ) were controls . ( g ) Brain lysates from mice expressing mutant huntingtin ( Tg ) or control ( Cont ) littermates were immunoprecipitated with antibody to mTOR and immunodetected with an antibody recognizing the polyglutamine tract ( IC2 ) . Htt ,

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figf1a) COS-7 cells expressing EGFP-tagged wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin exon 1 (green) for 48 h stained for endogenous mTOR (red) show mTOR in mutant aggregates.(b) Double-labeling immunohistochemistry of huntingtin (EM48; green) and mTOR (red) in brain tissue of mice expressing mutant huntingtin.(c) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease or unaffected controls using antibody to mTOR.(d) Western blot of lysates from stable inducible PC12 cells 48 h after expression of wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin showing mTOR in the stacking gel (mTOR insoluble) in cells expressing mutant huntingtin.(e) High-molecular-mass aggregates of mTOR in both PC12 (i) and COS-7 (ii) cells expressing mutant (Gln74; Q74) huntingtin. Blots of cell lysates from PC12 cells were probed with antibodies to phosphorylated mTOR (Ser2448; p-mTOR; iii) and to GFP (iv); both showed insoluble material in the stack.(f) Cell lysates from stable inducible PC12 cells and transiently transfected COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin for 48 h were immunoprecipitated using antibody to GFP and immunoblotted with antibody to mTOR. Uninduced PC12 cells with mutant huntingtin (UI) and untransfected COS-7 cells (UT) were controls.(g) Brain lysates from mice expressing mutant huntingtin (Tg) or control (Cont) littermates were immunoprecipitated with antibody to mTOR and immunodetected with an antibody recognizing the polyglutamine tract (IC2). Htt, huntingtin


figf2 ( a ) Reduced levels of soluble mTOR ( as a function of tubulin ) in stable inducible PC12 cells expressing mutant ( Gln74 ; Q74 ) huntingtin compared with those expressing wild - type ( Gln23 ; Q23 ) huntingtin ( four independent experiments ; P 0 . 0001 ; unpaired t - test ) . ( b ) Reduced levels of soluble mTOR in brain lysates from mice expressing mutant huntingtin ( M1 , M2 ; n = 9 ) compared with age - matched wild - type littermates ( C1 , C2 ; n = 9 ; P 0 . 001 , unpaired t - test ) . ( c ) COS - 7 cells transiently transfected with wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin ( green ) labeled for endogenous 4E - BP1 ( red ) . ( d ) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease showed 4E - BP1 in intranuclear inclusions . No 4E - BP1 - stained inclusions were seen in controls ( data not shown ) . ( ( e ) Western blot of cell lysates from stable inducible PC12 cells transfected with wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin shows that cells expressing mutant huntingtin had 31 % ( ± 4 . 3 % s . e . ) less phosphorylated S6K1 ( top ) relative to total S6K1 ( bottom ) than cells expressing wild - type huntingtin . ( f ) Similarly , cells expressing mutant ( Gln74 ; Q74 ) huntingtin had 30 % ( ± 4 . 5 % s . d . ) less phosphorylated 4E - BP1 ( top ) relative to total 4E - BP1 ( bottom ) than cells expressing wild - type ( Gln23 ; Q23 ) huntingtin . Expression of wild - type and mutant huntingtin was induced for 48 h ( e , f ) . ( g ) Cells expressing mutant ( Gln74 ; Q74 ) huntingtin also had lower levels of phosphorylated forms of transiently transfected FLAG - tagged 4E - BP1 than cells expressing wild - type ( Gln23 ; Q23 ) huntingtin . The multiple bands in 4E - BP1 ( f , g ) are due to its different phosphorylated species .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(a) Reduced levels of soluble mTOR (as a function of tubulin) in stable inducible PC12 cells expressing mutant (Gln74; Q74) huntingtin compared with those expressing wild-type (Gln23; Q23) huntingtin (four independent experiments; P 0.0001; unpaired t-test).(b) Reduced levels of soluble mTOR in brain lysates from mice expressing mutant huntingtin (M1, M2; n = 9) compared with age-matched wild-type littermates (C1, C2; n = 9; P 0.001, unpaired t-test).(c) COS-7 cells transiently transfected with wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin (green) labeled for endogenous 4E-BP1 (red).(d) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease showed 4E-BP1 in intranuclear inclusions. No 4E-BP1-stained inclusions were seen in controls (data not shown). ((e) Western blot of cell lysates from stable inducible PC12 cells transfected with wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin shows that cells expressing mutant huntingtin had 31% (± 4.3% s.e.) less phosphorylated S6K1 (top) relative to total S6K1 (bottom) than cells expressing wild-type huntingtin. (f) Similarly, cells expressing mutant (Gln74; Q74) huntingtin had 30% (± 4.5% s.d.) less phosphorylated 4E-BP1 (top) relative to total 4E-BP1 (bottom) than cells expressing wild-type (Gln23; Q23) huntingtin. Expression of wild-type and mutant huntingtin was induced for 48 h (e,f). (g) Cells expressing mutant (Gln74; Q74) huntingtin also had lower levels of phosphorylated forms of transiently transfected FLAG-tagged 4E-BP1 than cells expressing wild-type (Gln23; Q23) huntingtin. The multiple bands in 4E-BP1 (f,g) are due to its different phosphorylated species.


figf3 ( a ) COS - 7 cells expressing wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin ( green ) were labeled for S6 phosphorylated at Ser235 and Ser236 ( red ) . Percentages of cells expressing wild - type or mutant huntingtin that did ( + agg ) or did not ( − agg ) contain aggregates were scored for negative staining for phosphorylated S6 ( p - S6 ) . ( b ) Brain sections from control ( WT ) and mice expressing mutant huntingtin ( HD ) were stained for phosphorylated S6 ( red ) and with antibody to huntingtin ( EM48 ; green ) . ( c , d ) Impaired mTOR - dependent TOP translation in cells expressing mutant huntingtin . ( c ) PC12 cells expressing wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin were transfected with luciferase constructs containing 5 ′ untranslated sequence with ( + ) or without ( − ) a TOP sequence along with β - gal . Each bar represents the difference between the luciferase activities in the presence versus the absence of rapamycin . Data shown are from one representative experiment ( of three ) done in sextuplicate ( error bars represent s . e . ; raw data are given in Supplementary Fig . 4 online ) . ( d ) COS - 7 cells were cotransfected with wild - type or mutant huntingtin , a nontypical TOP or control luciferase vector and β - gal . The experiment was done and analyzed as in c ( raw data are given in Supplementary Fig . 4 online ) ( e - g ) Relevance of mTOR sequestration to other polyglutamine expansion diseases ( e ) COS - 7 cells expressing Xpress - tagged ataxin - 1 with 92 polyglutamine repeats ( Q92 ) for 48 h were doubly labeled with antibodies to Xpress ( green ) and to mTOR ( red ) . ( f ) mTOR immunohistochemistry in brains of individuals with dentatorubral - pallidoluysian atrophy ( DRPLA ) and spinocerebellar ataxia types 2 , 3 and 7 ( SCA2 , SCA3 and SCA7 ) . ( g ) Staining for phosphorylated S6 ( red ) in COS - 7 cells transiently transfected with mutant ( ataxin Q92 ; green ) or wild - type ( ataxin Q2 ) ataxin - 1 constructs . Results were significant and similar to those in a . Arrows in a , b , e and g show cells containing aggregates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin (green) were labeled for S6 phosphorylated at Ser235 and Ser236 (red). Percentages of cells expressing wild-type or mutant huntingtin that did (+agg) or did not (−agg) contain aggregates were scored for negative staining for phosphorylated S6 (p-S6).(b) Brain sections from control (WT) and mice expressing mutant huntingtin (HD) were stained for phosphorylated S6 (red) and with antibody to huntingtin (EM48; green).(c,d) Impaired mTOR-dependent TOP translation in cells expressing mutant huntingtin. (c) PC12 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin were transfected with luciferase constructs containing 5′ untranslated sequence with (+) or without (−) a TOP sequence along with β-gal. Each bar represents the difference between the luciferase activities in the presence versus the absence of rapamycin. Data shown are from one representative experiment (of three) done in sextuplicate (error bars represent s.e.; raw data are given in Supplementary Fig. 4 online). (d) COS-7 cells were cotransfected with wild-type or mutant huntingtin, a nontypical TOP or control luciferase vector and β-gal. The experiment was done and analyzed as in c (raw data are given in Supplementary Fig. 4 online) (e-g) Relevance of mTOR sequestration to other polyglutamine expansion diseases(e) COS-7 cells expressing Xpress-tagged ataxin-1 with 92 polyglutamine repeats (Q92) for 48 h were doubly labeled with antibodies to Xpress (green) and to mTOR (red).(f) mTOR immunohistochemistry in brains of individuals with dentatorubral-pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia types 2, 3 and 7 (SCA2, SCA3 and SCA7).(g) Staining for phosphorylated S6 (red) in COS-7 cells transiently transfected with mutant (ataxin Q92; green) or wild-type (ataxin Q2) ataxin-1 constructs. Results were significant and similar to those in a. Arrows in a, b, e and g show cells containing aggregates.


figf4 ( a ) LC3 - II levels , which closely correlate with autophagic activity , were higher in mutant lines ( expressing Gln74 huntingtin ; Q74 ; lanes 3 and 4 ) than in wild - type lines ( expressing Gln23 huntingtin ; Q23 ; lanes 1 and 2 ) . Actin was used as a loading control . Data are shown for two independent clonal lines expressing mutant or wild - type huntingtin . Quantification of the band intensity is shown in the right panel . ( b ) Immunofluorescence analysis of COS - 7 cells expressing wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin with antibody to LC3 showed more autophagic vacuoles in cells expressing mutant huntingtin that had aggregates ( arrow ) . COS - 7 cells without the primary antibody ( negative ) and COS - 7 cells without any treatment ( untreated ) stained for LC3 are also shown . The numbers of EGFP - positive cells expressing wild - type or mutant huntingtin with ( + agg ) and without ( − agg ) aggregates that had higher numbers of autophagic vacuoles ( > 15 - 20 vesicles per cell ) is shown in the graph . COS - 7 cells grown in starvation medium ( starvation ) or treated with rapamycin ( Rap ) for 4 h to induce autophagy had more autophagic vacuoles ( arrows ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) LC3-II levels, which closely correlate with autophagic activity, were higher in mutant lines (expressing Gln74 huntingtin; Q74; lanes 3 and 4) than in wild-type lines (expressing Gln23 huntingtin; Q23; lanes 1 and 2). Actin was used as a loading control. Data are shown for two independent clonal lines expressing mutant or wild-type huntingtin. Quantification of the band intensity is shown in the right panel.(b) Immunofluorescence analysis of COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin with antibody to LC3 showed more autophagic vacuoles in cells expressing mutant huntingtin that had aggregates (arrow). COS-7 cells without the primary antibody (negative) and COS-7 cells without any treatment (untreated) stained for LC3 are also shown. The numbers of EGFP-positive cells expressing wild-type or mutant huntingtin with (+agg) and without (−agg) aggregates that had higher numbers of autophagic vacuoles (>15-20 vesicles per cell) is shown in the graph. COS-7 cells grown in starvation medium (starvation) or treated with rapamycin (Rap) for 4 h to induce autophagy had more autophagic vacuoles (arrows).


figf5 ( a ) Activation of mTOR activity with rheb increases aggregation and toxicity of mutant ( Gln74 ; Q74 ) huntingtin . COS - 7 cells were cotransfected with a mammalian expression vector encoding rheb ( or empty vector control ) and mutant huntingtin for 48 h at a 3 : 1 mass ratio ( rheb or empty vector to huntingtin ) to ensure that almost all cells expressing huntingtin also expressed rheb . The percentages of EGFP - positive cells with aggregates or apoptotic nuclear morphology ( as described previously ) were markedly higher when rheb was overexpressed . Control cells express mutant huntingtin ( green ) without rheb . The middle panels show images captured at the same comparatively low gain to distinguish aggregates from soluble EGFP . The left panels show the same fields at a similar higher gain to indicate the total number of EGFP - positive cells ( this shows that the effect seen in the middle panels is not due to loss of cells ) . Nuclear staining with DAPI ( blue ) is shown in the right panels . * * * P 0 . 0001 . Error bars represent s . e . for a representative triplicate experiment . ( b , c ) Treatment with rapamycin rescues huntingtin - induced degeneration in flies 2 d after eclosion . Photographs of ommatidia of flies expressing mutant ( Gln120 ; Q120 ) huntingtin treated with DMSO or rapamycin from the larval stage into adulthood . More rhabdomeres are visible in the ommatidia of flies treated with rapamycin . This effect is significant ( P 0 . 0001 ; Mann - Whitney U test ) . At 2 d after eclosion , there is no reduction in the number of visible rhabdomeres in wild - type untreated flies ( data not shown ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) Activation of mTOR activity with rheb increases aggregation and toxicity of mutant (Gln74; Q74) huntingtin. COS-7 cells were cotransfected with a mammalian expression vector encoding rheb (or empty vector control) and mutant huntingtin for 48 h at a 3:1 mass ratio (rheb or empty vector to huntingtin) to ensure that almost all cells expressing huntingtin also expressed rheb. The percentages of EGFP-positive cells with aggregates or apoptotic nuclear morphology (as described previously) were markedly higher when rheb was overexpressed. Control cells express mutant huntingtin (green) without rheb. The middle panels show images captured at the same comparatively low gain to distinguish aggregates from soluble EGFP. The left panels show the same fields at a similar higher gain to indicate the total number of EGFP-positive cells (this shows that the effect seen in the middle panels is not due to loss of cells). Nuclear staining with DAPI (blue) is shown in the right panels. ***P 0.0001. Error bars represent s.e. for a representative triplicate experiment.(b,c) Treatment with rapamycin rescues huntingtin-induced degeneration in flies 2 d after eclosion. Photographs of ommatidia of flies expressing mutant (Gln120; Q120) huntingtin treated with DMSO or rapamycin from the larval stage into adulthood. More rhabdomeres are visible in the ommatidia of flies treated with rapamycin. This effect is significant (P 0.0001; Mann-Whitney U test). At 2 d after eclosion, there is no reduction in the number of visible rhabdomeres in wild-type untreated flies (data not shown).


figf6Open bars , mice treated with a placebo ; filled bars , mice treated with CCI - 779 . ( a - c ) Behavioral tests in mice expressing mutant huntingtin after treatment at 16 weeks ( CCI - 779 , n = 14 ; placebo , n = 14 ) , 18 weeks ( CCI - 779 , n = 10 ; placebo , n = 9 ) , 20 weeks ( CCI - 779 , n = 9 ; placebo , n = 7 ) and 22 weeks ( CCI - 779 , n = 5 ; placebo , n = 6 ) of age . Scores are explained in Methods . Data for all time points are given in Supplementary Figure 13 online . ( a ) Tremors . Overall effect from all treated time points : P = 0 . 0006 , odds ratio ( OR ) = 0 . 21 , 95 % confidence interval ( 95 % c . i . ) = 0 . 098 - 0 . 45 . 16 weeks , P = 0 . 01 ; 18 weeks , P = 0 . 08 ; 20 weeks , P = 0 . 14 ; 22 weeks , P = 0 . 07 . ( b ) Wire maneuver . Overall effect from all treated time points : P = 0 . 02 , OR = 0 . 51 , 95 % c . i . = 0 . 29 - 0 . 92 . 16 weeks , P = 0 . 29 ; 18 weeks , P = 0 . 40 ; 20 weeks , P = 0 . 003 ; 22 weeks , P = 0 . 008 . ( c ) Grip strength . Overall effect from all treated time points : P 0 . 0001 OR = 12 . 73 , 95 % c . i . = 4 . 35 - 37 . 2 . 16 weeks , P = 0 . 1827 ; 18 weeks , P = 0 . 0412 ; 20 weeks , P = 0 . 0021 ; 22 weeks , P = 0 . 0285 . ( d ) Accelerating rotarod tests in mice expressing mutant huntingtin after treatment at 4 weeks , ( CCI - 779 , n = 16 ; placebo , n = 17 ) , 14 weeks ( CCI - 779 , n = 11 ; placebo , n = 10 ) , 16 weeks ( CCI - 779 , n = 14 ; placebo , n = 14 ) and 18 weeks ( CCI - 779 , n = 10 ; placebo , n = 9 ) of age . Overall effect from all treated time points : P = 0 . 035 . 4 weeks , P = 0 . 227 ; 14 weeks , P = 0 . 012 ; 16 weeks , P = 0 . 0025 ; 18 weeks , P = 0 . 057 . CCI - 779 had no discernable effects on the performance of nontransgenic mice on any of these behavioral tests ( data not shown ) . There was no difference in the male : female ratios of the groups treated with placebo or with CCI - 779 .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6Open bars, mice treated with a placebo; filled bars, mice treated with CCI-779. (a-c) Behavioral tests in mice expressing mutant huntingtin after treatment at 16 weeks (CCI-779, n = 14; placebo, n = 14), 18 weeks (CCI-779, n = 10; placebo, n = 9), 20 weeks (CCI-779, n = 9; placebo, n = 7) and 22 weeks (CCI-779, n = 5; placebo, n = 6) of age. Scores are explained in Methods. Data for all time points are given in Supplementary Figure 13 online. (a) Tremors. Overall effect from all treated time points: P = 0.0006, odds ratio (OR) = 0.21, 95% confidence interval (95% c.i.) = 0.098-0.45. 16 weeks, P = 0.01; 18 weeks, P = 0.08; 20 weeks, P = 0.14; 22 weeks, P = 0.07. (b) Wire maneuver. Overall effect from all treated time points: P = 0.02, OR = 0.51, 95% c.i. = 0.29-0.92. 16 weeks, P = 0.29; 18 weeks, P = 0.40; 20 weeks, P = 0.003; 22 weeks, P = 0.008. (c) Grip strength. Overall effect from all treated time points: P 0.0001 OR = 12.73, 95% c.i. = 4.35-37.2. 16 weeks, P = 0.1827; 18 weeks, P = 0.0412; 20 weeks, P = 0.0021; 22 weeks, P = 0.0285.(d) Accelerating rotarod tests in mice expressing mutant huntingtin after treatment at 4 weeks, (CCI-779, n = 16; placebo, n = 17), 14 weeks (CCI-779, n = 11; placebo, n = 10), 16 weeks (CCI-779, n = 14; placebo, n = 14) and 18 weeks (CCI-779, n = 10; placebo, n = 9) of age. Overall effect from all treated time points: P = 0.035. 4 weeks, P = 0.227; 14 weeks, P = 0.012; 16 weeks, P = 0.0025; 18 weeks, P = 0.057. CCI-779 had no discernable effects on the performance of nontransgenic mice on any of these behavioral tests (data not shown). There was no difference in the male:female ratios of the groups treated with placebo or with CCI-779.


figf7 ( a ) Weights of mice expressing mutant huntingtin ( Tg ) and nontransgenic ( NT ) littermates treated with CCI - 779 or with a placebo . ( b ) Brain weights of mice expressing mutant huntingtin treated with CCI - 779 ( black bars ) or with a placebo ( white bars ) and nontransgenic untreated littermates ( gray bars ) at 8 weeks ( CCI - 779 , n = 3 ; placebo , n = 3 ; nontransgenic , n = 7 ; CCI - 779 versus placebo , P = 0 . 023 ) , 12 weeks ( CCI - 779 , n = 2 ; placebo , n = 3 ; nontransgenic , n = 5 ; CCI - 779 versus placebo , P = 0 . 90 ) and 16 weeks ( CCI - 779 , n = 2 ; placebo , n = 3 ; nontransgenic , n = 2 ; CCI - 779 versus placebo , P = 0 . 22 ) of age . ( c ) Ratio of brain weight to body weight of mice expressing mutant huntingtin treated with CCI - 779 ( black bars ) or with a placebo ( white bars ) and nontransgenic untreated littermates ( gray bars ) at 8 weeks ( CCI - 779 , n = 3 ; placebo , n = 3 ; nontransgenic , n = 7 ; CCI - 779 versus placebo , P = 0 . 06 ) , 12 weeks ( CCI - 779 , n = 2 ; placebo , n = 3 ; nontransgenic , n = 5 ; CCI - 779 versus placebo , P = 0 . 38 ) and 16 weeks ( CCI - 779 , n = 2 ; placebo , n = 3 ; nontransgenic , n = 2 ; CCI - 779 versus placebo , P = 0 . 56 ) of age .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(a) Weights of mice expressing mutant huntingtin (Tg) and nontransgenic (NT) littermates treated with CCI-779 or with a placebo. (b) Brain weights of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.023), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.90) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.22) of age. (c) Ratio of brain weight to body weight of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.06), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.38) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.56) of age.


figf8 ( a ) CCI - 779 reduces aggregate load in mice expressing mutant huntingtin . Peroxidase immunohistochemistry of brains of mice expressing mutant huntingtin at 16 weeks of age . Mice treated with CCI - 779 had fewer aggregates per unit area in the striatum than mice treated with a placebo . Overall cell staining in the tissues of mice treated with CCI - 779 was weaker than in those treated with a placebo and in nontransgenic untreated littermates ( data not shown ) . Aggregates , when visible , were smaller in mice treated with CCI - 779 than in those treated with a placebo , as apparent in the higher magnification images of single cells with aggregate ( bottom panels ) . Six coronal sections per mouse were analyzed ; these corresponded to the bregma ± 1 mm . We counted aggregates in five different high - power fields ( 60 × objective ) per slide on each side of the brain , sampling fields from the dorsal to the ventral part of the striatum just underneath the external capsule . The striata of mice treated with CCI - 779 had 70 % fewer aggregates than the striata of mice treated with a placebo ( P = 0 . 0001 , two - tailed t - test ) . ( b ) CCI - 779 blocks mTOR signaling in vivo . Immunohistochemistry of mice with Huntington disease at 16 weeks of age with antibody to phosphorylated S6 , in the cortical area and striatum . Levels of phosphorylated S6 were lower in mice treated with CCI - 779 than in those treated with a placebo .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf8(a) CCI-779 reduces aggregate load in mice expressing mutant huntingtin. Peroxidase immunohistochemistry of brains of mice expressing mutant huntingtin at 16 weeks of age. Mice treated with CCI-779 had fewer aggregates per unit area in the striatum than mice treated with a placebo. Overall cell staining in the tissues of mice treated with CCI-779 was weaker than in those treated with a placebo and in nontransgenic untreated littermates (data not shown). Aggregates, when visible, were smaller in mice treated with CCI-779 than in those treated with a placebo, as apparent in the higher magnification images of single cells with aggregate (bottom panels). Six coronal sections per mouse were analyzed; these corresponded to the bregma ± 1 mm. We counted aggregates in five different high-power fields (60× objective) per slide on each side of the brain, sampling fields from the dorsal to the ventral part of the striatum just underneath the external capsule. The striata of mice treated with CCI-779 had 70% fewer aggregates than the striata of mice treated with a placebo (P = 0.0001, two-tailed t-test).(b) CCI-779 blocks mTOR signaling in vivo. Immunohistochemistry of mice with Huntington disease at 16 weeks of age with antibody to phosphorylated S6, in the cortical area and striatum. Levels of phosphorylated S6 were lower in mice treated with CCI-779 than in those treated with a placebo.


Figure 1 ( D ) Electron microscopy images from purified 40S and 60S ribosomal complexes ( 1 pmol each ) assembled in vitro in the presence of MgCl2 , and in the presence or absence of 5 pmol of ( PR ) 20 . Assembled 80S particles are indicated ( red arrows ) . Scale bar ( white ) represents 10 nm . ( E ) Quantification of 80S particles identified in ( D ) ( n = 1000 ) in non - assembly ( 1 mM MgCl2 ) or assembly ( 5 mM MgCl2 ) conditions . (

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(D) Electron microscopy images from purified 40S and 60S ribosomal complexes (1 pmol each) assembled in vitro in the presence of MgCl2, and in the presence or absence of 5 pmol of (PR)20. Assembled 80S particles are indicated (red arrows). Scale bar (white) represents 10 nm. (E) Quantification of 80S particles identified in (D) (n=1000) in non-assembly (1 mM MgCl2) or assembly (5 mM MgCl2) conditions. (


Figure 2 ( C , D ) High - throughput microscopy ( HTM ) - mediated analysis of 5 - ethynyl - uridine ( EU ) ( C ) and polyA + mRNA ( D ) levels per nucleus found in U2OS cells exposed to 10 μM ( PR ) 20 for 16 h . EU was added 30 ´ prior to fixation ( n = 3 ) . Black lines indicate median values in both panels . In D , boxes define upper and lower quartiles and whiskers mark the highest and lowest observations . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - test . Representative images from these analyses are provided in each case . Scale bar ( white ) indicates 2 - 5 μm . ( E ) Effect of ( PR ) 20 ( 10 μM , 16 h ) in the number of Cajal Bodies ( CB ) identified by immunofluorescence with anti - COILIN and anti - SMN antibodies ( n = 3 ) . Data represent mean values ± SD . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - test . Representative images from these analyses are provided in each case . Scale bar ( white ) indicates 2 - 5 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(C, D) High-throughput microscopy (HTM)-mediated analysis of 5-ethynyl-uridine (EU) (C) and polyA+ mRNA (D) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 16 h. EU was added 30´ prior to fixation (n=3). Black lines indicate median values in both panels. In D, boxes define upper and lower quartiles and whiskers mark the highest and lowest observations. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm.(E) Effect of (PR)20 (10 μM, 16 h) in the number of Cajal Bodies (CB) identified by immunofluorescence with anti-COILIN and anti-SMN antibodies (n=3). Data represent mean values ± SD. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm.