Journal: Nucleic Acids Research

Article Title: Condensin I and condensin II proteins form a LINE-1 dependent super condensin complex and cooperate to repress LINE-1

doi: 10.1093/nar/gkac802

Figure Lengend Snippet: NCAPD2 and NCAPD3 associate to form SCCs in a L1 RNA dependent manner. ( A ) Reciprocal NCAPD3 and NCAPD2 co-IP/immunoblots were conducted in HT-29 cells to detect an association between NCAPD3 and NCAPD2. Antibody only (i.e. no lysate) and IgG antibody IPs served as negative controls. Each experiment was performed three times and representative blots are shown. ( B ) NCAPG2 co-IP/immunoblot experiments were conducted in HT-29 cells to detect an association with NCAPD2 (middle panel) and with NCAPG (bottom panel). Antibody only (i.e. no lysate) and IgG antibody IPs served as negative controls. Each experiment was performed three times and representative blots are shown. ( C ) Proximity Ligation Assays (PLAs) were performed to detect an association between NCAPD2 and NCAPD3 in HT-29 cells transfected with control (siCTRL) or L1 siRNA (siL1). Single antibody controls were performed in parallel for each experiment. Images were taken using a confocal microscope with a 63x objective; maximum projections are shown. ( D ) Volocity imaging software was used to analyze confocal images and quantify the average number of nuclear and cytoplasmic PLA foci per cell. Each dot represents the average of 50–150 nuclei evaluated from a single image. Images were taken from three independent experiments. ( E ) PLAs were performed to detect associations between NCAPD3 and NCAPG2 in HT-29 cells transfected with siCTRL or siL1 and results were quantified as described in (D). P values were determined by performing unpaired t -tests. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns = not significant. Error bars indicate standard deviations from the mean.

Article Snippet: Nuclear PLA foci were identified and quantified from 3D images of individual focal planes within z-stacks, using Volocity 3D Image Analysis Software v 6.5.1 (Quorum Technologies Inc. Puslinch, Ontario).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Ligation, Transfection, Control, Microscopy, Imaging, Software

Journal: Nucleic Acids Research

Article Title: Condensin I and condensin II proteins form a LINE-1 dependent super condensin complex and cooperate to repress LINE-1

doi: 10.1093/nar/gkac802

Figure Lengend Snippet: NRTI Treatment reduces L1 transcripts and L1 retrotransposition events and decreases cytoplasmic SCC formation. ( A ) HT-29 cells were treated with either DMSO or DMSO containing Zidovudine and Didanosine at the indicated drug concentrations for 10 days then fixed and stained with crystal violet (CV) to measure cell toxicity. Shown are averages from three independent experiments. ( B ) Retrotransposition assays were performed using HT-29 cells that were transfected with pJM101/L1.3 and treated with either DMSO or a combination of the nucleoside reverse transcriptase inhibitors (NRTIs) Zidovudine and Didanosine; each drug was at a final concentration of 50 μM in tissue culture media. Quantification of retrotransposition assays from three independent experiments is shown. ( C ) qRT-PCR analysis of endogenous L1 RNA levels (using a 5′UTR primer pair; See and Methods) in HT-29 cells treated with either DMSO or NRTIs. The average relative transcript levels for three independent experiments are shown. ( D ) PLA was performed to detect an association between NCAPD2 and NCAPD3 in HT-29 cells treated with DMSO or NRTIs at a final concentration of 50μM in tissue culture media. Single antibody controls were performed in parallel for each experiment. Images were taken using a confocal microscope with a 63x objective and maximum projections are shown. Volocity imaging software was used to analyze confocal images as noted in Figure to quantify the average number of nuclear (left chart) and cytoplasmic (right chart) PLA foci per cell. P values were determined by performing unpaired t -tests. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ns = not significant. Error bars indicate standard deviations from the mean.

Article Snippet: Nuclear PLA foci were identified and quantified from 3D images of individual focal planes within z-stacks, using Volocity 3D Image Analysis Software v 6.5.1 (Quorum Technologies Inc. Puslinch, Ontario).

Techniques: Staining, Transfection, Reverse Transcription, Concentration Assay, Quantitative RT-PCR, Microscopy, Imaging, Software

Journal: Nucleic Acids Research

Article Title: Condensin I and condensin II proteins form a LINE-1 dependent super condensin complex and cooperate to repress LINE-1

doi: 10.1093/nar/gkac802

Figure Lengend Snippet: The 3′UTR of L1 RNA antagonizes SCC formation. ( A ) HT-29 cells were transfected with the pCEP4 empty vector, pJM101/L1.3 expression vector, or L1 expression vector harboring a 3′UTR deletion (pJM101/L1.3 3′UTRΔ) and Proximity Ligation Assays were performed to detect association between NCAPD2 and NCAPD3. Single antibody controls were performed in parallel for each experiment. Images shown were taken on a confocal microscope with a 63x objective and maximum projections are shown. ( B , C ) Volocity imaging software was used to analyze confocal images as noted in Figure to quantify the average number of nuclear (B) and cytoplasmic (C) PLA foci per cell. Images shown were taken from two independent experiments. P values were determined by performing unpaired t -tests. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ns = not significant. Error bars indicate standard deviations from the mean.

Article Snippet: Nuclear PLA foci were identified and quantified from 3D images of individual focal planes within z-stacks, using Volocity 3D Image Analysis Software v 6.5.1 (Quorum Technologies Inc. Puslinch, Ontario).

Techniques: Transfection, Plasmid Preparation, Expressing, Ligation, Microscopy, Imaging, Software