Journal: PLoS ONE

Article Title: Luciferase-based reporting of suicide gene activity in murine mesenchymal stem cells

doi: 10.1371/journal.pone.0220013

Figure Lengend Snippet: (a) Nucleofection with pmaxGFP. MSCs were nucleofected with pmaxGFP and imaged at 6 and 24 hours post nucleofection. Brightfield and fluorescent images were obtained using a Nikon Eclipse TS2 microscope at 20x magnification, 100μm scale. (b) Schematic representation of the pVITRO2-Luc2 and pVITRO2-Luc2/CDUPRT mammalian expression plasmids. (c) Antibiotic sensitivity assay. Native MSCs were grown in the presence of a 2-fold serial dilution of Hygromycin B (0–500μg/ml) for 14 days. (d) Nucleofection with bioluminescent plasmids. MSCs were nucleofected with pVITRO2- Luc2 and pVITRO2- Luc2/CDUPRT , and selected for with Hygromycin B for two weeks. Images were acquired on a Leica DM Microscope at 10x magnification, 100μm scale. (e) BLI in MSC- Luc2 , MSC- Luc2/CDUPRT #1 and MSC- Luc2/CDUPRT #2 over a linear cell concentration range. The cells were incubated with 1:1 D-luciferin (300μg/ml) and imaged on the IVIS Lumina II according to the in vitro BLI acquisition settings. The images are represented at t = 30min. Lane 1: D-PBS, Lane 2–10: Nucleofected MSC vs control MSC. (f) Analysis of luciferase activity in MSC- Luc2 , MSC- Luc2/CDUPRT #1 and MSC- Luc2/CDUPRT #2 . Luminescence was captured at multiple time-points following incubation with D-luciferin was analyzed using GraphPad Prism 7. Data are represented as the mean average radiance ± SDs of triplicates.

Article Snippet: Briefly, the luciferase reporter gene Luc2 ( Photinus pyralis ), encoded within the vector pGL4.20 ( Luc2 /Puro) (Promega, USA), was digested with the restriction enzymes EcoRV-HF and BamHI-HF (New England Biolabs, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro-mcs (InvivoGen, USA), to generate the plasmid pVITRO2- Luc2 .

Techniques: Microscopy, Expressing, Sensitive Assay, Serial Dilution, Concentration Assay, Incubation, In Vitro, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: Luciferase-based reporting of suicide gene activity in murine mesenchymal stem cells

doi: 10.1371/journal.pone.0220013

Figure Lengend Snippet: (A) In vitro BLI of CDUPRT activity. BLI was assessed following the addition of 5-FC and 5-FU to MSC- Luc2 (A-D) and MSC- Luc2/CDUPRT #1 (E-H). For 5-FC BLI data: Lanes 1–12 contains 2-fold serial dilutions of 5-FC from 0–2000μg/ml. For 5-FU BLI data: Lanes 1–7 contains 10-fold serial dilutions of 5-FU from 0–1000μg/ml. (B) Analysis of BLI data. Data are represented as the mean average radiance ± SEMs (n = 3). Baseline luminescence is indicated by a dotted-line. A two-way ANOVA and Sidak’s post-hoc were performed, p<0.05.

Article Snippet: Briefly, the luciferase reporter gene Luc2 ( Photinus pyralis ), encoded within the vector pGL4.20 ( Luc2 /Puro) (Promega, USA), was digested with the restriction enzymes EcoRV-HF and BamHI-HF (New England Biolabs, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro-mcs (InvivoGen, USA), to generate the plasmid pVITRO2- Luc2 .

Techniques: In Vitro, Activity Assay

Journal: PLoS ONE

Article Title: Luciferase-based reporting of suicide gene activity in murine mesenchymal stem cells

doi: 10.1371/journal.pone.0220013

Figure Lengend Snippet: (a) Nucleofection with pmaxGFP. MSCs were nucleofected with pmaxGFP and imaged at 6 and 24 hours post nucleofection. Brightfield and fluorescent images were obtained using a Nikon Eclipse TS2 microscope at 20x magnification, 100μm scale. (b) Schematic representation of the pVITRO2-Luc2 and pVITRO2-Luc2/CDUPRT mammalian expression plasmids. (c) Antibiotic sensitivity assay. Native MSCs were grown in the presence of a 2-fold serial dilution of Hygromycin B (0–500μg/ml) for 14 days. (d) Nucleofection with bioluminescent plasmids. MSCs were nucleofected with pVITRO2- Luc2 and pVITRO2- Luc2/CDUPRT , and selected for with Hygromycin B for two weeks. Images were acquired on a Leica DM Microscope at 10x magnification, 100μm scale. (e) BLI in MSC- Luc2 , MSC- Luc2/CDUPRT #1 and MSC- Luc2/CDUPRT #2 over a linear cell concentration range. The cells were incubated with 1:1 D-luciferin (300μg/ml) and imaged on the IVIS Lumina II according to the in vitro BLI acquisition settings. The images are represented at t = 30min. Lane 1: D-PBS, Lane 2–10: Nucleofected MSC vs control MSC. (f) Analysis of luciferase activity in MSC- Luc2 , MSC- Luc2/CDUPRT #1 and MSC- Luc2/CDUPRT #2 . Luminescence was captured at multiple time-points following incubation with D-luciferin was analyzed using GraphPad Prism 7. Data are represented as the mean average radiance ± SDs of triplicates.

Article Snippet: Briefly, the luciferase reporter gene Luc2 ( Photinus pyralis ), encoded within the vector pGL4.20 ( Luc2 /Puro) (Promega, USA), was digested with the restriction enzymes EcoRV-HF and BamHI-HF (New England Biolabs, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro-mcs (InvivoGen, USA), to generate the plasmid pVITRO2- Luc2 .

Techniques: Microscopy, Expressing, Sensitive Assay, Serial Dilution, Concentration Assay, Incubation, In Vitro, Control, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: Luciferase-based reporting of suicide gene activity in murine mesenchymal stem cells

doi: 10.1371/journal.pone.0220013

Figure Lengend Snippet: (A) In vitro BLI of CDUPRT activity. BLI was assessed following the addition of 5-FC and 5-FU to MSC- Luc2 (A-D) and MSC- Luc2/CDUPRT #1 (E-H). For 5-FC BLI data: Lanes 1–12 contains 2-fold serial dilutions of 5-FC from 0–2000μg/ml. For 5-FU BLI data: Lanes 1–7 contains 10-fold serial dilutions of 5-FU from 0–1000μg/ml. (B) Analysis of BLI data. Data are represented as the mean average radiance ± SEMs (n = 3). Baseline luminescence is indicated by a dotted-line. A two-way ANOVA and Sidak’s post-hoc were performed, p<0.05.

Article Snippet: Briefly, the luciferase reporter gene Luc2 ( Photinus pyralis ), encoded within the vector pGL4.20 ( Luc2 /Puro) (Promega, USA), was digested with the restriction enzymes EcoRV-HF and BamHI-HF (New England Biolabs, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro-mcs (InvivoGen, USA), to generate the plasmid pVITRO2- Luc2 .

Techniques: In Vitro, Activity Assay

Journal: Biotechnology and bioengineering

Article Title: ‘Trim’ming PolyQ proteins with engineered PML

doi: 10.1002/bit.27220

Figure Lengend Snippet: A) Characterization of levels of Atxn1 82Q-GFP in HEK 293T cells using flow cytometry 24 hours after co-transfection with plasmids encoding Atxn1 82Q-GFP and vector control/ PML/PML-scFv fusion proteins. GFP intensity was measured as median fluorescence intensity. RMFI= MFI (SAMPLE)/MFI (VECTOR CONTROL). Values represent mean ± sd, n=3. B) Characterization of levels of Atxn1 82Q-GFP by flow cytometry, 48 hrs after co-transfection with plasmids encoding Atxn1 82Q-GFP and vector control/ PML/PML-scFv fusion proteins. Values represent mean ± sd, n=3. C) Characterization of levels of Atxn1 82Q-GFP in HEK 293T cells by Western Blotting. HEK 293T cells were co-transfected with Atxn1 82Q-GFP and 1-VC, 2-PML, 3- PML ΔSRS2,4-PML-6E, 5-PML-MW1, 6-PML-FRZ. For total cell lysate, cells were processed with 2% SDS buffer and probed by western blotting with an anti-GFP antibody (1:500). For fractionation, cells were processed in NP 40 buffer and the soluble fraction in this buffer was labelled as NS. Pellet was dissolved in SDS buffer, resulting in the SS fraction. VC: Vector Control.

Article Snippet: DNA encoding Atxn1 82Q-GFP was synthesized by Gene Universal Inc. (Newark, DE) and cloned between BglII and BstBI sites in the mcs2 of pvitro2-hygro-mcs vector (InvivoGen, San Diego, CA).

Techniques: Flow Cytometry, Cotransfection, Plasmid Preparation, Control, Fluorescence, Western Blot, Transfection, Fractionation

Journal: Biotechnology and bioengineering

Article Title: ‘Trim’ming PolyQ proteins with engineered PML

doi: 10.1002/bit.27220

Figure Lengend Snippet: A) HEK 293T cells were co-transfected with Atxn1 82Q-GFP and VC/PML/PML-scFvs (PML-MW1, PML-6E, PML-FRZ) in four-chambered glass bottom cover-glass. Cells were processed for DAPI staining and incubated with PML-specific mouse antibody, followed by further incubation with a Cy3-conjugated-Anti-mouse secondary antibody. A Spinning-Disc Confocal microscope was used to acquire images at 40×. Left column shows the DAPI staining overlaid with Bright field image in order to show nuclear staining. The images in the middle column represent an overlay of DAPI and Red channel, where Cy3-stained PML/PML-scFvs are seen as red specks localized around the nucleus (marked using yellow arrows in the middle column). The images in the right column represent an overlay of DAPI, Red, and Green channels, where along with PML/PML-scFv expression, Atxn1 82Q-GFP is seen either as intense green dots (Red arrow) for cells co-transfected with VC/ PML-FRZ (First and 5th row), as partially diffuse green dots for cells expressing PML (2nd row) and almost completely solubilized Atxn1 82Q-GFP for cells expressing PML-MW1 (4th row). Images were processed using Volocity software. B) HEK 293T cells were co-transfected with Atxn1 82Q-GFP and VC/PML/PML-scFvs and after an incubation of 24 hours, total cell lysate was probed using anti-SUMO 2/3 antibodies in western blotting. The experiment was performed with three biological repeats for each construct, as grouped by the marked lines. The samples were run on two separate gels as shown by a line between the samples but both blots were imaged together. Sumo 2/3 conjugates appear as a smear above anticipated Atxn1 82Q-GFP band at 122 KDa. VC: Vector Control.

Article Snippet: DNA encoding Atxn1 82Q-GFP was synthesized by Gene Universal Inc. (Newark, DE) and cloned between BglII and BstBI sites in the mcs2 of pvitro2-hygro-mcs vector (InvivoGen, San Diego, CA).

Techniques: Transfection, Staining, Incubation, Microscopy, Expressing, Software, Western Blot, Construct, Plasmid Preparation, Control

Journal: Biotechnology and bioengineering

Article Title: ‘Trim’ming PolyQ proteins with engineered PML

doi: 10.1002/bit.27220

Figure Lengend Snippet: HEK 293T cells were co-transfected with Atxn1 82Q-GFP and VC/ PML/ PMLΔSRS2/PML-scFv fusion proteins. After 24 hours of incubation, cells were sorted and cells with Atxn1 82Q-GFP aggregates were counted using an epifluorescence microscope. The graph shows the fraction of HEK 293T cells without visible Atxn1 82Q-GFP aggregates as a function of time (hours). Vector control (green circle); PML (Red square); PMLΔSRS2 (brown triangle); PML-6E (inverted purple triangle); PML-MW1 (orange diamond); and PML-FRZ (black circle). The values represent mean ± sd, n=3. VC: Vector Control.

Article Snippet: DNA encoding Atxn1 82Q-GFP was synthesized by Gene Universal Inc. (Newark, DE) and cloned between BglII and BstBI sites in the mcs2 of pvitro2-hygro-mcs vector (InvivoGen, San Diego, CA).

Techniques: Transfection, Incubation, Microscopy, Plasmid Preparation, Control

Journal: Biotechnology and bioengineering

Article Title: ‘Trim’ming PolyQ proteins with engineered PML

doi: 10.1002/bit.27220

Figure Lengend Snippet: A) HEK 293T cells were co-transfected with Atxn1 82Q-GFP and PML/PML-6E/MW1 encoded in Doxycycline inducible pTRE3G plasmid. PML/PML-scFv expression was induced by Doxycycline treatment, 48 hours after co-transfection in half of the wells for each construct. The remaining uninduced wells formed the control groups, denoted as UI. Total Atxn1 82Q-GFP present after 24 hours of Doxycycline treatment was measured as MFI using flow cytometry. B) Aggregation kinetics were studied for cells expressing pre-formed aggregates of Atxn1 82Q-GFP co-transfected with VC, PML, or PML-6E/MW1. As explained for (A), after an incubation of 48 hours, cells were induced with Doxycycline. Twenty four hours after Doxycycline induction, cells were sorted and the number of cells with visible protein aggregates were counted for the next 24 hours. The fraction of cells without aggregates was plotted against time post-seeding. The first data point is at 3 hours post seeding. Both plots represent mean ± sd, n=3

Article Snippet: DNA encoding Atxn1 82Q-GFP was synthesized by Gene Universal Inc. (Newark, DE) and cloned between BglII and BstBI sites in the mcs2 of pvitro2-hygro-mcs vector (InvivoGen, San Diego, CA).

Techniques: Transfection, Plasmid Preparation, Expressing, Cotransfection, Construct, Control, Flow Cytometry, Incubation