Journal: Frontiers in Microbiology

Article Title: Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes

doi: 10.3389/fmicb.2021.738125

Figure Lengend Snippet: Usage of the non-theta replication mechanism during transient replication is characteristic to different human papillomavirus (HPV) types. (A) Schematic representation of the expected mobility of the theta replication intermediates in neutral-neutral two-dimensional (2D) gel in case a circular replicon is linearized near the origin of replication (ori). Almost a straight diagonal line emanating from the spot of 1n linear molecules represents a doubleY structure, with two replication forks that started from the ori to opposite directions advancing as the line retreats from the spot of 1n linear molecules. A non-linear line ending in the 2D spot represents the structure arising from the non-theta type of replication. (B) Sketch of the HPV replicon at the different stages of bidirectional replication. Ori represents origin of replication; scissors refer to the position, where the HPV genome is linearized. (C) Neutral-neutral 2D analysis of the replication intermediates arising from the HPV5, HPV11, and HPV18 genome replication after transient transfection of the respective genomes into U2OS cells. Episomal DNA was extracted 72h post-transfection, linearized with a restriction enzyme cutting near ori [HPV5 – SacI (nt 702); HPV11- BstEII (nt 7,899) HPV18 – BglI (nt 7,656)] run on an agarose gel at two dimensions and analyzed by Southern blotting (SB). Black arrows refer to the theta replication intermediates, black arrowheads refer to the almost fully replicated genomes, white arrows refer to the non-theta replication intermediates, asterisks depict the linear HPV genome fragments running between the size of 1n and 2n.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC No HTB-96) and SV40 transformed African green monkey kidney cell line Cos1 (ATCC No CRL-1650) were propagated in the normal growth medium containing Iscove’s Modified Dulbecco’s Medium, 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C, and 5% CO2.

Techniques: Two-Dimensional Gel Electrophoresis, Transfection, Agarose Gel Electrophoresis, Southern Blot

Journal: Frontiers in Microbiology

Article Title: Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes

doi: 10.3389/fmicb.2021.738125

Figure Lengend Snippet: Appearance of the non-theta replication intermediates during the transient replication of the HPV18 genomes in the U2OS cells concurs with the oligomerization of the replicated viral genomes. (A) An example of different forms of the HPV18 genome arising as a result of the transient replication in U2OS cells at 3, 4, or 5days after the transfection. Genomic DNA was extracted from the cells at indicated time points, digested with HPV18 non-cutter HindIII and DpnI to separate non-replicated input DNA, resolved on 0.6% gel and analyzed by SB. Migration of the monomeric closed circular (1xCC), monomeric open circular (1XOC), dimeric closed circular (2xCC), and oligomeric forms are shown. Asterisk represents migration of non-replicated, DpnI sensitive fragment of HPV18. (B) Quantification of the replicated monomeric and oligomeric HPV18 genomes at 3, 4, and 5days after the transfection in the U2OS cells. The data are expressed as average means±the SD ( * p <0.05; ** p <0.01; and *** p <0.001) of four independent experiments. Monomeric open circular and dimeric closed circular genomes migrate together under the conditions used to resolve DNA and were therefore omitted from calculation. (C) An example of different types of the replication intermediates arising during the transient replication of the HPV18 genome in U2OS cells at 3, 4, and 5days post-transfection. Black arrows refer to the theta replication intermediates, black arrowheads refer to the almost fully replicated genomes, white arrows refer to the non-theta replication intermediates. (D) Quantification of the theta- and non-theta replication intermediates arising during the transient replication of the HPV18 genomes at 3, 4, and 5days after the transfection in the U2OS cells. The data are expressed as average means±the SD ( * p <0.05; ** p <0.01; and *** p <0.001) of four independent experiments.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC No HTB-96) and SV40 transformed African green monkey kidney cell line Cos1 (ATCC No CRL-1650) were propagated in the normal growth medium containing Iscove’s Modified Dulbecco’s Medium, 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C, and 5% CO2.

Techniques: Transfection, Migration

Journal: Frontiers in Microbiology

Article Title: Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes

doi: 10.3389/fmicb.2021.738125

Figure Lengend Snippet: The non-theta replication of the HPV18 genomes is dependent on the replicative DNA polymerase(s). (A) The cell cycle profile of the aphidicolin arrested cells (0h), followed by the release into the cell cycle at various time points (3, 7.5, and 24h) post-arrest. The U2OS cells were transfected with the HPV18 genome, 48h after the transfection the cells were arrested at G1/S phase using aphidicolin treatment (2μg/ml) for 24h, followed by the removal of the drug and release of the cells to cycling conditions for the indicated periods of time. (B) Neutral-neutral 2D analysis of the replication intermediates arising from the HPV18 genome replication after the transfection into the U2OS cells at various time points after the G1/S block using aphidicolin. Nearly all replication intermediates are absent at the end of 24-h block by aphidicolin (0h). Instead, the collapsed intermediates migrate above the 1n linear molecule spot (indicated by asterisk). At 3h post-release, when majority of the cells have entered the S-phase, both theta and non-theta type of replication intermediates are present. About 7.5h after the release majority of the cells have entered G2/M phase, and still both type of replication intermediates are detectable. At 24h post-release, the cells have restored the normal distribution of the cell cycle and both theta- and non-theta replication intermediates can be seen. Black arrows refer to the theta replication intermediates, black arrowheads refer to the almost fully replicated genomes, and white arrows refer to the non-theta replication intermediates.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC No HTB-96) and SV40 transformed African green monkey kidney cell line Cos1 (ATCC No CRL-1650) were propagated in the normal growth medium containing Iscove’s Modified Dulbecco’s Medium, 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C, and 5% CO2.

Techniques: Transfection, Blocking Assay

Journal: Frontiers in Microbiology

Article Title: Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes

doi: 10.3389/fmicb.2021.738125

Figure Lengend Snippet: Mechanism of replication initiated from the HPV18 ori depends on the size of the replicon. (A) Transient replication of the HPV18 ori harboring replicons with different sizes in the U2OS cells. pUCURR18 (4kb), pGLURR18 (6kb), or pGEXURR18 (8,2 kb) were transfected together with the HPV18 E1 and E2 expression vectors into the U2OS cells. Episomal DNA was extracted 72h after the transfection, digested with replicon non-cutter (left panel) or single cutter enzyme (right panel), combined with DpnI to digest the non-replicated plasmids, resolved in 0.8% agarose gel and analyzed by SB. Asterisks represent migration of the monomeric closed circular forms of the respective replicons. (B) Neutral-neutral 2D analysis of the replication intermediates arising from the HPV18 harboring replicons of different sizes during the transient replication in the U2OS cells and the SV40 origin containing pCDNA3 replicon in Cos1 cells. The replication intermediate structure that varies between different HPV replicons is indicated with an arrow.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC No HTB-96) and SV40 transformed African green monkey kidney cell line Cos1 (ATCC No CRL-1650) were propagated in the normal growth medium containing Iscove’s Modified Dulbecco’s Medium, 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C, and 5% CO2.

Techniques: Transfection, Expressing, Agarose Gel Electrophoresis, Migration

Journal: Frontiers in Microbiology

Article Title: Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes

doi: 10.3389/fmicb.2021.738125

Figure Lengend Snippet: Maintenance replication of the HPV31 genomes proceeds via distinct mechanisms in the CIN612 keratinocytes. (A) SB showing the status of the HPV31 genomes in CIN612 cells. HPV31 noncutter (BamHI) or single cutter (BstXI) enzymes were used to digest episomal DNA. Migration of the monomeric closed circular (1xCC), monomeric open circular (1XOC), linear (1xLin) and oligomeric forms are shown. (B) Neutral-neutral 2D analysis of the replication intermediates arising from the HPV31 maintenance replication in the CIN216 cells, HPV5 and HPV18 maintenance replication in the U2OS cells. Black arrows refer to the theta replication intermediates; white arrows refer to the non-theta replication intermediates.

Article Snippet: Human osteosarcoma cell line U2OS (ATCC No HTB-96) and SV40 transformed African green monkey kidney cell line Cos1 (ATCC No CRL-1650) were propagated in the normal growth medium containing Iscove’s Modified Dulbecco’s Medium, 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C, and 5% CO2.

Techniques: Migration

Journal: Nucleic Acids Research

Article Title: Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers

doi: 10.1093/nar/gkad441

Figure Lengend Snippet: An experimental system to study genome instability and fork stalling caused by GAA•TTC repeats. ( A ) pJC_GAA100 plasmid used in this study. The relative positions of its most relevant features are indicated inside: The centromeric sequence CEN6, the Autonomous Replication Sequence (ARS4), the ColE1 unidirectional origin (ColE1 Ori), the ampicillin resistance gene (Amp R ), the Large T antigen gene, the SV40 origin of replication (SV40 ori) and the selectable cassette for repeat expansions ( UR-GAA 100 -A3-TRP1 : depicted in B). Outside, the relative positions of sites recognized by specific restriction endonucleases are indicated. ( B ) Schematic of the system to select for repeat expansion in yeast. An artificially split URA3 gene contains 100 GAA•TTC repeats such that expansion events abrogate splicing and result in resistance to a 5-fluoroorotic acid (5-FOA-r). The addition of more than 10 repeats increased the overall length of the intron beyond the splicing threshold. The selectable cassette was cloned into the pJC_GAA100 plasmid. TRP1 - an auxotrophic marker for the selection of yeast transformants bearing the plasmids. ( C ) Schematic representation of the assay. Plasmids for the study were transfected into HEK-293T cells, and after culturing them for 48 h, DNA was isolated and digested with DpnI. Repeat expansions were detected upon DNA transformation into yeast. Single-colony PCR was performed for 5-FOA-r clones to confirm expansion events. Expansion frequency was calculated by dividing the number of colonies with repeat expansions by the total number of TRP + transformants. To calculate background expansion frequency, pJC_GAA100 plasmid isolated from E. coli was transformed directly into yeast. To study DNA replication through the repeats, DNA was digested with appropriate restriction enzymes and replication intermediates were analyzed by 2-dimensional (2D) agarose gel electrophoresis.

Article Snippet: Plasmid DNA from human cells was digested for one hour with 50 DpnI (NEB) units to eliminate unreplicated bacterial DNA, EtOH precipitated, resuspended in TE buffer and transformed into yeast SMY537 strain ( MAT a, leu2-Δ1, trp1-Δ63, ura3–52, his3–200 , bar1::HIS3 , can1::KanMX ( ) by the lithium acetate method ( ).

Techniques: Plasmid Preparation, Sequencing, Clone Assay, Marker, Selection, Transfection, Isolation, Transformation Assay, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers

doi: 10.1093/nar/gkad441

Figure Lengend Snippet: GAA•TTC repeat expansion upon candidate gene knockdown by siRNA. ( A ) Confirmation of GAA•TTC expansions in HEK-293T cells. Repeat lengths in 5-FOA-resistant colonies from independent experiments were analyzed by PCR. On the upper part, a cartoon representation of a cassette with a bar above the GAA•TTC repeats indicates the product of single-colony PCR, used to determine the repeat length. The red arrow points to the length of the PCR product carrying the starting (GAA) 100 •(TTC)100 repeat. ‘*’ marks large-scale GAA•TTC expansions. Only clones with expansions were used to calculate expansion frequencies in (D). ( B ) Distribution of repeats added to the (GAA) 100 •(TTC) 100 repeat during plasmid replication in HEK-293T cells (dark grey bars). The geometric mean of repeats added is 65 repeats (interquartile range 44.5–100.5). ( C ) Expansion frequencies for pJC_GAA100 plasmid derived from E. coli (baseline) and HEK-293T cells. Error bars indicate the standard error of the mean. ‘*’ P < 0.05 and ‘**’ indicates a P < 0.001 in two-way Welch ANOVA test. ( D ) Expansion frequencies following siRNA gene knockdown in HEK293T cells. The baseline expansion frequency upon yeast transformation by plasmid DNA isolated from E. coli (white bar) is shown for the comparison. Error bars indicate the standard error of the mean. Significance compared to the siControl frequency value was determined using a two-way Welch ANOVA test, ‘#’ P < 0.05 versus baseline, ‘*’ P < 0.05, ‘**’ indicates a P < 0.001 versus siControl. See for details.

Article Snippet: Plasmid DNA from human cells was digested for one hour with 50 DpnI (NEB) units to eliminate unreplicated bacterial DNA, EtOH precipitated, resuspended in TE buffer and transformed into yeast SMY537 strain ( MAT a, leu2-Δ1, trp1-Δ63, ura3–52, his3–200 , bar1::HIS3 , can1::KanMX ( ) by the lithium acetate method ( ).

Techniques: Knockdown, Clone Assay, Plasmid Preparation, Derivative Assay, Transformation Assay, Isolation, Comparison

Journal: Nucleic Acids Research

Article Title: Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers

doi: 10.1093/nar/gkad441

Figure Lengend Snippet: Analysis of pJC_GAA replication in HEK-293T cells by 2D gel electrophoresis. ( A ) Linear map of pJC_GAA100 restriction fragment. ( B ) Representative 2D gels of replication through zero and 100 GAA•TTC repeats are shown in the far-left column with their corresponding interpretative diagrams to their right. DNA was isolated, digested with BsrGI, XbaI, and DpnI, and analyzed by 2D gel electrophoresis. The simulation program 2D gel was used to predict the shape of twelve consecutive replication intermediates (RIs). A linear map is shown on top of each series of RIs, showing the relative positions of SV40 ori (green) UR (blue). The red arrow points to the location of the stall at the (GAA) 100 •(TTC) 100 repeat. ( C ) Quantification of the fraction of stalled forks. The ratio of radioactivity in the peak area to that corresponding area of a smooth replication arc reflects the extent of replication slowing. Quantification was done with ImageJ (NIH). N.A., non-applicable. ( D ) Quantification of spindle spot intensity compared to the whole arc. The ratio of radioactivity signal at the spindle spot was compared to the radioactivity signal of the whole arc on Image Lab®. N.A., non-applicable.

Article Snippet: Plasmid DNA from human cells was digested for one hour with 50 DpnI (NEB) units to eliminate unreplicated bacterial DNA, EtOH precipitated, resuspended in TE buffer and transformed into yeast SMY537 strain ( MAT a, leu2-Δ1, trp1-Δ63, ura3–52, his3–200 , bar1::HIS3 , can1::KanMX ( ) by the lithium acetate method ( ).

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Isolation, Radioactivity

Journal: Nucleic Acids Research

Article Title: Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers

doi: 10.1093/nar/gkad441

Figure Lengend Snippet: Analysis of replication through GAA•TTC repeats by two-dimensional (2D) agarose gel electrophoresis upon candidate gene knockdown by siRNA. ( A ) Representative 2D gels of replication through the (GAA) 100 •(TTC) 100 repeats in HEK-293T cells. DNA was isolated, digested with BsrG1, DpnI and XbaI, and analyzed by 2D gel. The red arrow points to the location of the stalling at the GAA•TTC repeats. At least three experiments were analyzed for each siRNA treatment. ( B ) Quantification of the spindle spot at the replication fork. The ratio of radioactivity signal at the spindle spot was compared to the radioactivity signal of the whole arc using Image Lab®. Error bars indicate the standard error of the mean. ‘*’ P < 0.05, ‘***’ indicates a P < 0.0001 versus siControl. See for details.

Article Snippet: Plasmid DNA from human cells was digested for one hour with 50 DpnI (NEB) units to eliminate unreplicated bacterial DNA, EtOH precipitated, resuspended in TE buffer and transformed into yeast SMY537 strain ( MAT a, leu2-Δ1, trp1-Δ63, ura3–52, his3–200 , bar1::HIS3 , can1::KanMX ( ) by the lithium acetate method ( ).

Techniques: Agarose Gel Electrophoresis, Knockdown, Isolation, Two-Dimensional Gel Electrophoresis, Radioactivity

Journal: bioRxiv

Article Title: HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

doi: 10.1101/438945

Figure Lengend Snippet: ( A ) Scheme of the lesion ICL unhooking assay. ( B ) ICL unhooking assay gel quantified in . ( C ) Scheme of the key events during ICL repair in Xenopus egg extract and the products measured in lesion bypass assay (see and for an example of a gel and quantification. ( D ) Lesion bypass assay gel quantified in . ( E ) Repeat of the experiment shown in . ( E-H ) Efficiency of ChIP with HSF2BP, XPF, REV1 and FANCD2 antibodies from the experiment shown in . ( I ) Efficiency of MMC-induced (50 ng/ml overnight) FANCD2 monoubiquitination (appearance of the slower migrating L-form) in control and HSF2BP-overproducing (human or mouse, GFP-tagged or untagged) HeLa cells. Immunoblots with two different anti-FANCD2 antibodies are shown. Asterisks indicate non-specific bands.

Article Snippet: Antibodies used in this study were against RAD51 (rabbit 2307 ( )), FANCD2 (NB 100-316, Novus Biologicals), H2B (07-371, Millipore), MSH2 (Ab-2, Oncogene), HSP90 (ab13492, Abcam), BRCA2 (Ab-1, OP95, Calbiochem), FLAG (M2 antibody, Sigma), GFP (clones 7.1 and 13.1, Roche), ORC2 (551178,BD Pharmingen), PARP-1 (C2-10, Enzo), 6xHis tag (ab18184, Abcam), and XRCC3 (ab6494, Abcam).

Techniques: Western Blot

Journal: bioRxiv

Article Title: HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

doi: 10.1101/438945

Figure Lengend Snippet: HSF2BP inhibits HR in the FA pathway ( A ) HSF2BP does not inhibit lesion unhooking in Xenopus egg extract. Prelabeled pICL was replicated in Xenopus egg extracts, replication products were isolated, digested by HincII, and separated on a denaturing agarose gel . The decline of the X-structures was quantified and plotted. See scheme in , main text and Methods for detailed description. ( B ) HSF2BP does not inhibit translesion synthesis in Xenopus egg extract. pICL was replicated in Xenopus egg extracts in the presence of 32P-lll-dCTP, replication products were isolated, digested by HincII, or HincII and SapI, and separated on a denaturing agarose gel . Extension products were quantified and plotted. Scheme of the assay in . ( C ) Inhibition of HR intermediate formation by HSF2BP revealed by 2D gel electrophoresis. pICL was replicated in Xenopus egg extracts in the presence of 32P- -dCTP, replication products were isolated, digested by HincII, and analyzed by 2D gel electrophoresis. ( D ) Stable RAD51 accumulation on pICL during repair is abrogated by HSF2BP wildtype, but not the R200T mutant. The pICL plasmid was pulled down by incubation with streptavidin beads coated with biotinylated LacI. Presence of bound RAD51 and FANCD2 was determined by immunoblotting. ( E ) Efficiency of RAD51 focus formation in HSF2BP-overproducing and control cells with or without 100 nM MMC treatment. After treatment cells were fixed, stained for immunofluorescence, mounted with DAPI and imaged using confocal microscopy. Images were scored manually for the number of foci. Data from three independent experiments is plotted, line represents average ± s.e.m., statistical significance was determined using the Kruskal-Wallis test with Dunn’s post-test. ( F ) HSF2BP inhibits BRCA2 loading at the ICL in Xenopus egg extract. pICL replication samples at the indicated time points were analyzed by BRCA2 ChIP.

Article Snippet: Antibodies used in this study were against RAD51 (rabbit 2307 ( )), FANCD2 (NB 100-316, Novus Biologicals), H2B (07-371, Millipore), MSH2 (Ab-2, Oncogene), HSP90 (ab13492, Abcam), BRCA2 (Ab-1, OP95, Calbiochem), FLAG (M2 antibody, Sigma), GFP (clones 7.1 and 13.1, Roche), ORC2 (551178,BD Pharmingen), PARP-1 (C2-10, Enzo), 6xHis tag (ab18184, Abcam), and XRCC3 (ab6494, Abcam).

Techniques: Isolation, Agarose Gel Electrophoresis, Translesion Synthesis, Inhibition, Two-Dimensional Gel Electrophoresis, Electrophoresis, Mutagenesis, Plasmid Preparation, Incubation, Western Blot, Staining, Immunofluorescence, Confocal Microscopy

Journal: Human Molecular Genetics

Article Title: Activating the synthesis of progerin, the mutant prelamin A in Hutchinson–Gilford progeria syndrome, with antisense oligonucleotides

doi: 10.1093/hmg/ddp184

Figure Lengend Snippet: Modulating the utilization of the exon 11 splice donor site with antisense oligonucleotides corresponding to sequences within exon 11 of human LMNA . All of the antisense oligonucleotides (ASOs) contained a 2′- O -methoxy-ethyl ribose backbone. ( A ) Exons 11 and 12 of LMNA , depicting both ‘wild-type splicing’ and ‘HGPS splicing’ (which uses the alternate splice donor site in exon 11). Also shown are the locations of ASOs located upstream of the alternate splice donor site (i.e. 365), ASOs downstream from the splice donor site (i.e. 324) and ASOs that straddle the site (i.e. 076 and 790). ( B ) SYTO 60-stained agarose gel showing RT–PCR products of RNA prepared from an HGPS cell line. The PCR products correspond to wild-type prelamin A and progerin transcripts. Transfection with ASO 324 increases progerin transcripts, whereas ASO 365 lowers progerin transcripts, as judged by image analysis on a LI-COR infrared scanner. ( C ) Quantification of progerin transcripts, by quantitative RT–PCR, in HGPS fibroblasts (AG03513 and GM01972) transfected with different ASOs. Results are plotted relative to progerin transcript levels in fibroblasts transfected with ASO 333. ( D ) SYTO 60-stained gel showing relative levels of wild-type prelamin A and progerin transcripts in wild-type fibroblasts, HGPS fibroblasts and wild-type fibroblasts transfected with either ASO 324 or control ASO 333. Also shown are DNA sequencing chromatograms of RT–PCR products corresponding to wild-type prelamin A transcripts in ASO 324-treated wild-type cells (upper), progerin transcripts in ASO 324-treated wild-type cells (middle) and progerin transcripts in HGPS fibroblasts (lower).

Article Snippet: Human HGPS fibroblasts (AG11498, AG06917, AG11513, AG03513, AG06297 and GM01972; all with the G608G mutation), control human fibroblasts (CRL-1474, AG07095, AG08470, AG16409, CRL-7469 and GM01651) and HeLa cells were from the American Type Culture Collection (Manassas, VA, USA) or Coriell Cell Repository ( http://locus.umdnj.edu/ccr/ ) ( ).

Techniques: Staining, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Transfection, Quantitative RT-PCR, Control, DNA Sequencing

Journal: Human Molecular Genetics

Article Title: Activating the synthesis of progerin, the mutant prelamin A in Hutchinson–Gilford progeria syndrome, with antisense oligonucleotides

doi: 10.1093/hmg/ddp184

Figure Lengend Snippet: Western blots with an antibody against lamin A/C showing that transfection of antisense oligonucleotide 074 yields higher levels of progerin expression. ( A ) Western blot of wild-type fibroblasts (GM01651) and HGPS fibroblasts (AG11513) 2 days after transfection with ASO 074, revealing progerin in the wild-type fibroblasts and increased levels of progerin in the HGPS fibroblasts. ( B ) Western blot of a wild-type fibroblast cell line (GM01651) 1–5 days after transfection with ASO 074, revealing a time-dependent increase in progerin expression (similar results were observed with cell line AG07095; data not shown). In both experiments, the levels of progerin in wild-type fibroblasts exceeded those in HGPS fibroblasts. ( C ) Western blot showing that transfection of ASO 365 into HGPS fibroblasts (AG11513) results in slightly reduced levels of progerin. ( D ) Western blot of ASO 074-transfected restrictive dermopathy (RD) fibroblasts and HeLa cells. Progerin was found in cell extracts prepared 2 days after the transfection.

Article Snippet: Human HGPS fibroblasts (AG11498, AG06917, AG11513, AG03513, AG06297 and GM01972; all with the G608G mutation), control human fibroblasts (CRL-1474, AG07095, AG08470, AG16409, CRL-7469 and GM01651) and HeLa cells were from the American Type Culture Collection (Manassas, VA, USA) or Coriell Cell Repository ( http://locus.umdnj.edu/ccr/ ) ( ).

Techniques: Western Blot, Transfection, Expressing

Journal: Human Molecular Genetics

Article Title: Activating the synthesis of progerin, the mutant prelamin A in Hutchinson–Gilford progeria syndrome, with antisense oligonucleotides

doi: 10.1093/hmg/ddp184

Figure Lengend Snippet: Western blot with an antibody against lamin A/C showing that transfection of an antisense oligonucleotide directed against the splice donor sequence at the beginning of intron 11 results in progerin expression. ( A ) Schematic showing the location of five ASOs near the splice donor sequence at the beginning of intron 11. ( B ) Western blot of extracts from wild-type fibroblasts (AG07095) showing that transfection of ASO 893 (corresponding to the first 20 nucleotides of intron 11) results in the production of progerin.

Article Snippet: Human HGPS fibroblasts (AG11498, AG06917, AG11513, AG03513, AG06297 and GM01972; all with the G608G mutation), control human fibroblasts (CRL-1474, AG07095, AG08470, AG16409, CRL-7469 and GM01651) and HeLa cells were from the American Type Culture Collection (Manassas, VA, USA) or Coriell Cell Repository ( http://locus.umdnj.edu/ccr/ ) ( ).

Techniques: Western Blot, Transfection, Sequencing, Expressing

Journal: Human Molecular Genetics

Article Title: Activating the synthesis of progerin, the mutant prelamin A in Hutchinson–Gilford progeria syndrome, with antisense oligonucleotides

doi: 10.1093/hmg/ddp184

Figure Lengend Snippet: Metabolic labeling experiments showing that the progerin that accumulates in antisense oligonucleotide-transfected wild-type fibroblasts is farnesylated. Fibroblasts were metabolically labeled with 8-anilinogeraniol (AG) in the presence or absence of a protein farnesyltransferase inhibitor (FTI) (ABT-100). After entering cells, AG is incorporated into anilinogeranyl diphosphate (AGPP), which is used as a substrate by protein farnesyltransferase. The incorporation of the AGPP into proteins can be monitored by western blotting with an antibody against AG. ( A ) Western blot of HGPS fibroblast extracts, performed with an Odyssey infrared imaging system, showing that progerin is farnesylated. Shown is the merged image of western blots with an antibody specific for lamin A/C (red) and an antibody against AG (green) before (left panel) and after (right panel) immune precipitation with a lamin A/C antibody. Incorporation of AG into progerin was blocked by the FTI. ( B ) Western blot of ASO-transfected wild-type fibroblasts showing that the progerin is farnesylated. Wild-type fibroblasts were transfected with ASO 074 to induce progerin synthesis and incubated with the AG substrate. After 2 days, extracts were prepared and the A-type lamins isolated by immune precipitation with an antibody against lamin A/C. The progerin in the wild-type cells was farnesylated (green), and this farnesylation could be blocked with an FTI.

Article Snippet: Human HGPS fibroblasts (AG11498, AG06917, AG11513, AG03513, AG06297 and GM01972; all with the G608G mutation), control human fibroblasts (CRL-1474, AG07095, AG08470, AG16409, CRL-7469 and GM01651) and HeLa cells were from the American Type Culture Collection (Manassas, VA, USA) or Coriell Cell Repository ( http://locus.umdnj.edu/ccr/ ) ( ).

Techniques: Labeling, Transfection, Metabolic Labelling, Western Blot, Imaging, Incubation, Isolation

Journal: Human Molecular Genetics

Article Title: Activating the synthesis of progerin, the mutant prelamin A in Hutchinson–Gilford progeria syndrome, with antisense oligonucleotides

doi: 10.1093/hmg/ddp184

Figure Lengend Snippet: Increased percentage of misshapen nuclei in wild-type and HGPS fibroblasts after transfection with antisense oligonucleotide 074. ( A ) Epifluorescence microscopy showing increased numbers of misshapen nuclei in wild-type fibroblasts transfected with ASO 074, which promotes alternate splicing and progerin synthesis. ASO 333, which has no effect on splicing, was used as a control. ( B ) Bar graph comparing the frequency of misshapen nuclei in wild-type and HGPS fibroblasts transfected with ASOs 074 and 333. The ratios inside the bars reflect the number of misshapen nuclei divided by the total number of nuclei that were scored. The solid black circles show the percentage of misshapen nuclei in two independent experiments. Differences in the frequency of misshapen nuclei were assessed with a χ 2 -test.

Article Snippet: Human HGPS fibroblasts (AG11498, AG06917, AG11513, AG03513, AG06297 and GM01972; all with the G608G mutation), control human fibroblasts (CRL-1474, AG07095, AG08470, AG16409, CRL-7469 and GM01651) and HeLa cells were from the American Type Culture Collection (Manassas, VA, USA) or Coriell Cell Repository ( http://locus.umdnj.edu/ccr/ ) ( ).

Techniques: Transfection, Epifluorescence Microscopy, Control

Journal: Human Molecular Genetics

Article Title: Activating the synthesis of progerin, the mutant prelamin A in Hutchinson–Gilford progeria syndrome, with antisense oligonucleotides

doi: 10.1093/hmg/ddp184

Figure Lengend Snippet: Two-dimensional gel electrophoresis experiments revealing distinct patterns of protein expression in wild-type human fibroblasts after transfection with ASO 324, an antisense oligonucleotide that promotes alternate splicing and progerin synthesis, or ASO 333, a control oligonucleotide that has no effect on splicing. Wild-type fibroblasts (CRL-1474) were transfected with ASO 324 or ASO 333, and soluble extracts were labeled with cyanine dyes (red and green). Equal amounts of the extracts were mixed together and fractionated by 2D-gel electrophoresis. The gel was imaged on a Typhoon TRIO scanner and individual protein spots were quantified with DeCyder. In the merged image (far right), red spots represent proteins that increase in ASO 324-transfected cells, whereas green spots represent proteins that decrease (relative to cells transfected with ASO 333). Two independent experiments are shown. The locations of eight proteins (identified by mass spectrometry) are shown in one of the experiments.

Article Snippet: Human HGPS fibroblasts (AG11498, AG06917, AG11513, AG03513, AG06297 and GM01972; all with the G608G mutation), control human fibroblasts (CRL-1474, AG07095, AG08470, AG16409, CRL-7469 and GM01651) and HeLa cells were from the American Type Culture Collection (Manassas, VA, USA) or Coriell Cell Repository ( http://locus.umdnj.edu/ccr/ ) ( ).

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Expressing, Transfection, Control, Labeling, Mass Spectrometry

Journal: International Journal of Molecular Sciences

Article Title: Sphingomyelin Deacylase, the Enzyme Involved in the Pathogenesis of Atopic Dermatitis, Is Identical to the β-Subunit of Acid Ceramidase

doi: 10.3390/ijms21228789

Figure Lengend Snippet: Typical purification steps of SM deacylase from rat skin. SM deacylase activity was determined by measuring the production of SPC by LC-MS/MS as described in the Materials and Methods section.

Article Snippet: For biochemical experiments, recombinant aCDase was prepared as follows: The medium containing expressed aCDase was subjected to affinity chromatography using ConA-sepharose (GE healthcare; Little Chalfont, England).

Techniques: Purification, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Sphingomyelin Deacylase, the Enzyme Involved in the Pathogenesis of Atopic Dermatitis, Is Identical to the β-Subunit of Acid Ceramidase

doi: 10.3390/ijms21228789

Figure Lengend Snippet: ( A ): SDS-PAGE of SM deacylase active fractions in each purification step. Aliquots of SM deacylase active fractions after each purification step were collected and analyzed by 12.5% SDS-PAGE, followed by staining with SYPRO Ruby and visualization of fluorescence using a Typhoon-9400 scanner. Lane 1, isoelectric focusing (Rotofor); Lane 2, ion exchange chromatography (SP Sepharose); Lane 3, first gel filtration chromatography (Superdex 200); Lane 4, second gel filtration chromatography (Superdex 200); Lane 5, affinity chromatography (RP-415 C20). ( B ): Estimation of the molecular weight of SM deacylase enzyme activity. SM deacylase purified from rat skin was separated by gel filtration chromatography using a Superdex 200 column. Eluted fractions were collected by volume and were assayed for SM deacylase activity (closed circles); estimated apparent molecular mass by protein standards (open circles); cytochrome C (124 kDa), albumin (66 kDa), ovalbumin (44 kDa), chymotrypsin (24 kDa). ( C ): Determination of SM deacylase active protein spot on 2D-PAGE gels. ( a ) Purified enzymes were isoelectrically focused at 12 W constant power for 4 h at 4 °C in native conditions. The IEF strips were cut into 40 equal parts and were then subjected to assays for SM deacylase activity. The activity in each slot was plotted along with the distance from the edge. ( b ) 2D electrophoresis was performed by mounting another IEF separated strip gel on top of an SDS-PAGE gel. After electrophoresis, the gel was stained by SYPRO Ruby and detected using a fluorescence image scanner. The pI range was from 6.5 to 10 and the molecular mass range was from 114 to 14 kDa, as indicated. The protein spot indicated by the arrow was prepared for MS/MS analysis. The two-dimensional gel image is representative of a minimum of three replicates.

Article Snippet: For biochemical experiments, recombinant aCDase was prepared as follows: The medium containing expressed aCDase was subjected to affinity chromatography using ConA-sepharose (GE healthcare; Little Chalfont, England).

Techniques: SDS Page, Purification, Staining, Fluorescence, Ion Exchange Chromatography, Filtration, Chromatography, Affinity Chromatography, Molecular Weight, Activity Assay, Two-Dimensional Gel Electrophoresis, Stripping Membranes, Electrophoresis, Tandem Mass Spectroscopy

Journal: Nucleic Acids Research

Article Title: Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers

doi: 10.1093/nar/gkad441

Figure Lengend Snippet: An experimental system to study genome instability and fork stalling caused by GAA•TTC repeats. ( A ) pJC_GAA100 plasmid used in this study. The relative positions of its most relevant features are indicated inside: The centromeric sequence CEN6, the Autonomous Replication Sequence (ARS4), the ColE1 unidirectional origin (ColE1 Ori), the ampicillin resistance gene (Amp R ), the Large T antigen gene, the SV40 origin of replication (SV40 ori) and the selectable cassette for repeat expansions ( UR-GAA 100 -A3-TRP1 : depicted in B). Outside, the relative positions of sites recognized by specific restriction endonucleases are indicated. ( B ) Schematic of the system to select for repeat expansion in yeast. An artificially split URA3 gene contains 100 GAA•TTC repeats such that expansion events abrogate splicing and result in resistance to a 5-fluoroorotic acid (5-FOA-r). The addition of more than 10 repeats increased the overall length of the intron beyond the splicing threshold. The selectable cassette was cloned into the pJC_GAA100 plasmid. TRP1 - an auxotrophic marker for the selection of yeast transformants bearing the plasmids. ( C ) Schematic representation of the assay. Plasmids for the study were transfected into HEK-293T cells, and after culturing them for 48 h, DNA was isolated and digested with DpnI. Repeat expansions were detected upon DNA transformation into yeast. Single-colony PCR was performed for 5-FOA-r clones to confirm expansion events. Expansion frequency was calculated by dividing the number of colonies with repeat expansions by the total number of TRP + transformants. To calculate background expansion frequency, pJC_GAA100 plasmid isolated from E. coli was transformed directly into yeast. To study DNA replication through the repeats, DNA was digested with appropriate restriction enzymes and replication intermediates were analyzed by 2-dimensional (2D) agarose gel electrophoresis.

Article Snippet: Plasmid replication intermediates, extracted from human cells as described above, were digested by DpnI, XbaI and BrsGI restriction endonucleases (New England Biolabs), EtOH precipitated and resuspended in TE buffer.

Techniques: Plasmid Preparation, Sequencing, Clone Assay, Marker, Selection, Transfection, Isolation, Transformation Assay, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers

doi: 10.1093/nar/gkad441

Figure Lengend Snippet: Analysis of pJC_GAA replication in HEK-293T cells by 2D gel electrophoresis. ( A ) Linear map of pJC_GAA100 restriction fragment. ( B ) Representative 2D gels of replication through zero and 100 GAA•TTC repeats are shown in the far-left column with their corresponding interpretative diagrams to their right. DNA was isolated, digested with BsrGI, XbaI, and DpnI, and analyzed by 2D gel electrophoresis. The simulation program 2D gel was used to predict the shape of twelve consecutive replication intermediates (RIs). A linear map is shown on top of each series of RIs, showing the relative positions of SV40 ori (green) UR (blue). The red arrow points to the location of the stall at the (GAA) 100 •(TTC) 100 repeat. ( C ) Quantification of the fraction of stalled forks. The ratio of radioactivity in the peak area to that corresponding area of a smooth replication arc reflects the extent of replication slowing. Quantification was done with ImageJ (NIH). N.A., non-applicable. ( D ) Quantification of spindle spot intensity compared to the whole arc. The ratio of radioactivity signal at the spindle spot was compared to the radioactivity signal of the whole arc on Image Lab®. N.A., non-applicable.

Article Snippet: Plasmid replication intermediates, extracted from human cells as described above, were digested by DpnI, XbaI and BrsGI restriction endonucleases (New England Biolabs), EtOH precipitated and resuspended in TE buffer.

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Isolation, Radioactivity

Journal: Nucleic Acids Research

Article Title: Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers

doi: 10.1093/nar/gkad441

Figure Lengend Snippet: Analysis of replication through GAA•TTC repeats by two-dimensional (2D) agarose gel electrophoresis upon candidate gene knockdown by siRNA. ( A ) Representative 2D gels of replication through the (GAA) 100 •(TTC) 100 repeats in HEK-293T cells. DNA was isolated, digested with BsrG1, DpnI and XbaI, and analyzed by 2D gel. The red arrow points to the location of the stalling at the GAA•TTC repeats. At least three experiments were analyzed for each siRNA treatment. ( B ) Quantification of the spindle spot at the replication fork. The ratio of radioactivity signal at the spindle spot was compared to the radioactivity signal of the whole arc using Image Lab®. Error bars indicate the standard error of the mean. ‘*’ P < 0.05, ‘***’ indicates a P < 0.0001 versus siControl. See for details.

Article Snippet: Plasmid replication intermediates, extracted from human cells as described above, were digested by DpnI, XbaI and BrsGI restriction endonucleases (New England Biolabs), EtOH precipitated and resuspended in TE buffer.

Techniques: Agarose Gel Electrophoresis, Knockdown, Isolation, Two-Dimensional Gel Electrophoresis, Radioactivity