Journal: Cell Research

Article Title: Pericytes augment glioblastoma cell resistance to temozolomide through CCL5-CCR5 paracrine signaling

doi: 10.1038/s41422-021-00528-3

Figure Lengend Snippet: a Schematic diagram of Desmin promoter-driven expression of HsvTK vector and control vector. Gene therapy was achieved through administration of GCV, which is converted to a toxic metabolite to eliminate cells expressing HsvTK. b Schematic diagram of the combined treatment of TMZ and GCV in mice bearing GBM-2 (pericyte high ) xenografts expressing DesPro-TK or control DesPro. GCV (50 mg/kg, i.p.) was given for 4 days with 1-day interval since Day 15 and TMZ (5 mg/kg, i.p.) was given for 3 consecutive days since Day 16 after tumor implantation. Xenograft growth was monitored by bioluminescence imaging on Day 15 and Day 23 after tumor implantation. IVIS, In Vivo Imaging System. c , d In vivo bioluminescence images ( c ) and quantification ( d ) of tumor growth of human GBM-2 xenografts treated with GCV together with or without TMZ on Day 15 and Day 23 after tumor implantation. ns, not significant. * P < 0.05. n = 5 for each group. e Kaplan–Meier survival analysis of mice bearing GBM-2 xenografts with the indicated treatment. n = 5 for each group. f , g Immunofluorescence staining ( f ) and quantification ( g ) of pericyte marker α-SMA (green) in GBM-2 xenografts treated with GCV together with or without TMZ. ns, not significant. * P < 0.05. Scale bars, 25 μm. h , i Immunofluorescence staining ( h ) and quantification ( i ) of γ-H2AX (red)-positive cells in GBM-2 xenografts treated with GCV together with or without TMZ. ** P < 0.01. Scale bars, 25 μm. j TMZ concentration in GBM-2 xenografts and blood in mice expressing DesPro-TK or control DesPro with GCV treatment. n = 4 for each group.

Article Snippet: To determine the effect of pericyte depletion on TMZ treatment, mice were treated with vehicle (5% DMSO + 30% PEG300 + 65% ddH 2 O, i.p.), TMZ (5 mg/kg, i.p., Selleckchem, S1237), GCV (50 mg/kg, i.p., Selleckchem, S1878), or the combination of TMZ and GCV.

Techniques: Expressing, Plasmid Preparation, Control, Tumor Implantation, Imaging, In Vivo Imaging, In Vivo, Immunofluorescence, Staining, Marker, Concentration Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Antiviral Drug Ganciclovir Is a Potent Inhibitor of the Proliferation of Müller Glia–Derived Progenitors During Zebrafish Retinal Regeneration

doi: 10.1167/iovs.15-18669

Figure Lengend Snippet: Ganciclovir treatment significantly reduced the number of MGPCs after retinal injury. ( A ) Green fluorescent protein ( green ) and BrdU ( red ) immunofluorescence shows that GCV treatment decreased the number of BrdU+ MGPCs localized to the injury site at 4 dpi. DAPI (4′,6-diamidino-2-phenylindole) channel ( blue ) was added to the overlay images to show retinal layer structures. Tg(1016tuba1a:GFP) fish received a pulse of BrdU 3 hours before they were killed at 4 dpi. ( B ) Quantification of BrdU+ MGPCs in ( A ). * P < 0.05; ** P < 0.01 compared to PBS control, n = 4. ( C ) TUNEL staining and quantification of TUNEL+ cell numbers from PBS-, GCV-, or ouabain-treated retina at 3 dpi. Arrows mark TUNEL+ cells. #No significant difference, P > 0.05, n = 4. Scale bars : 100 μm. The asterisks mark the injury site (needle poke). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; dpi, days post injury; MGPCs, Müller glia–derived progenitor cells.

Article Snippet: Ganciclovir sodium (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in PBS at indicated concentrations; 1 μL PBS or GCV was then delivered at the time of injury using the same needle to poke the retina or was injected intravitreally at the indicated time.

Techniques: Immunofluorescence, Control, TUNEL Assay, Staining, Derivative Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Antiviral Drug Ganciclovir Is a Potent Inhibitor of the Proliferation of Müller Glia–Derived Progenitors During Zebrafish Retinal Regeneration

doi: 10.1167/iovs.15-18669

Figure Lengend Snippet: Ganciclovir had no effect on Müller glia dedifferentiation and the initial MGPC formation. ( A ) Confocal microscopy of retinal sections after GFP and BrdU immunofluorescence shows the presence of GFP+ Müller glia near injury site in PBS- or GCV-treated retina at 4 dpi. Tg(1016tuba1a:GFP) fish were given a pulse of BrdU 3 hours before they were killed at 4 dpi. Note that there are many fewer BrdU+ MGPCs in GCV-treated retina than in control. Arrows indicate GFP+ columns. Each GFP+ column represents an activated Müller glia and its daughter progenitor cells. ( B ) Quantification of the number of GFP+ columns at the injury site at 4 dpi. #No significant difference, P > 0.05, n = 4. ( C ) Immunofluorescence shows the initial formation of MGPCs in the INL in PBS- or GCV-treated retina at 2 dpi. Tg(1016tuba1a:GFP) fish received a pulse of BrdU 3 hours before they were killed at 2 dpi. ( D ) Quantification of the number of BrdU+ MGPCs per injury at 2, 3, and 4 dpi in PBS- or GCV-treated retina. Fish received a pulse of BrdU 3 hours prior to killing at 2, 3, or 4 dpi. #No significant difference, P > 0.05; * P < 0.05; ** P < 0.01. n = 4 for each group. Scale bars : 50 μm. The asterisks mark the injury site (needle poke).

Article Snippet: Ganciclovir sodium (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in PBS at indicated concentrations; 1 μL PBS or GCV was then delivered at the time of injury using the same needle to poke the retina or was injected intravitreally at the indicated time.

Techniques: Confocal Microscopy, Immunofluorescence, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Antiviral Drug Ganciclovir Is a Potent Inhibitor of the Proliferation of Müller Glia–Derived Progenitors During Zebrafish Retinal Regeneration

doi: 10.1167/iovs.15-18669

Figure Lengend Snippet: Ganciclovir inhibits MGPC proliferation. ( A ) Schematic illustration showing the experimental timeline. Retinas of wild-type zebrafish were injured, and all fish received a pulse of BrdU at 45 hpi. Fish then received intravitreous PBS or GCV injection once daily until 4 dpi. Retinas were collected at indicated time points (48, 72, and 96 hpi). ( B ) Bromodeoxyuridine immunofluorescence showing that GCV inhibits the expansion of MGPCs in the retina. Bromodeoxyuridine signals are shown in green . (C) Quantification of ( B ). * P < 0.05 compared to PBS-treated control. n = 3 for each group. Scale bars : 100 μm. The asterisks mark the injury site (needle poke).

Article Snippet: Ganciclovir sodium (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in PBS at indicated concentrations; 1 μL PBS or GCV was then delivered at the time of injury using the same needle to poke the retina or was injected intravitreally at the indicated time.

Techniques: Injection, Immunofluorescence, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Antiviral Drug Ganciclovir Is a Potent Inhibitor of the Proliferation of Müller Glia–Derived Progenitors During Zebrafish Retinal Regeneration

doi: 10.1167/iovs.15-18669

Figure Lengend Snippet: Ganciclovir treatment had no effect on microglia accumulation at the injury site. ( A – D ) Immunofluorescence shows the BrdU+ MGPCs ( green ) and IB4+ microglia ( red ) at the injury site at 2 and 3 dpi. Note that all the IB4+ microglia were BrdU negative. ( E ) Quantification of the number of microglia at the injury site at 2 and 3 dpi. The asterisks mark the injury site (needle poke). # P > 0.05 compared to PBS-treated control. n = 3 for each group. Scale bars : 100 μm.

Article Snippet: Ganciclovir sodium (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in PBS at indicated concentrations; 1 μL PBS or GCV was then delivered at the time of injury using the same needle to poke the retina or was injected intravitreally at the indicated time.

Techniques: Immunofluorescence, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Antiviral Drug Ganciclovir Is a Potent Inhibitor of the Proliferation of Müller Glia–Derived Progenitors During Zebrafish Retinal Regeneration

doi: 10.1167/iovs.15-18669

Figure Lengend Snippet: Induction of p53 and p21 cip1 after GCV administration. ( A ) qPCR shows that GCV treatment inhibits the expression of ccna2 , ccnb1 , ccnd1 , cdk1 , and cdk2 significantly at 3 dpi. ( B ) Ganciclovir treatment increases the expression of p21 cip1 at 2 and 3 dpi significantly. ( C ) Ganciclovir significantly increases the expression level of p53 mRNA at 2 and 3 dpi. ( D ) Western blot analysis shows elevated expression level of the p53 protein in GCV-treated retina compared to that of control at 3 dpi. n = 3 for each group. * P < 0.05 compared to PBS control; ** P < 0.01 compared to PBS control.

Article Snippet: Ganciclovir sodium (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in PBS at indicated concentrations; 1 μL PBS or GCV was then delivered at the time of injury using the same needle to poke the retina or was injected intravitreally at the indicated time.

Techniques: Expressing, Western Blot, Control

Journal: Nature Communications

Article Title: Engineering a synthetic gene circuit for high-performance inducible expression in mammalian systems

doi: 10.1038/s41467-024-47592-y

Figure Lengend Snippet: a Three alternative constructs to express the cytotoxic HSV-TK gene. pCMV-HSV-TK: positive control, with constitutive expression of HSV-TK. Tet-On3G: the constitutively expressed rtTA3G induces the cytotoxic HSV-TK gene harbouring a DR in its 3’UTR, binding to pTRE3G in the presence of doxycycline. CASwitch v.2: the same as the Tet-On3G but for the presence of the CasRx downstream of the pCMV/TO. b Viability of HEK293T cells transfected with the indicated constructs and grown in the presence of ganciclovir. Mock transfected cells represent the negative control. Cell viability is reported as a percentage of the viability of mock transfected cells in the absence of doxycycline. The error bars represent the mean and standard deviation of biological replicates across two independent experiments ( n = 9). Statistical analysis with ANOVA (one-tailed) after determining equal or unequal variances by D’Agostino & Pearson test (**** P -value < 0.0001) c Crystal violet staining of transfected HEK293T cells to highlight viable cells. d Plasmids required for AAV production. Two alternative experimental implementations for inducible expression of the Helper genes using either the Tet-On3G system or the CASwitch v.2 are also shown. e Assay for testing AAV vector inducible production yield by means of viral transduction. Created with Biorender. f , g Flow cytometry of cells transduced with cell lysates of HEK293T cells transfected with the indicated configurations. At least 10,000 cells were analysed for each point. The bar-plot in ( g ) reports, for each experimental condition, the mean value of the percentage of transduced cells across of biological replicates for two independent experiments ( n = 6) with error bars corresponding to the standard deviation. Statistical analysis by ANOVA (one-tailed), after determining equal or unequal variances by D’Agostino & Pearson test (**** P -value < 0.0001). HSV-TK: Herpes Simplex Virus Thymidine Kinase; AAV: Adeno-Associated Virus; E2A(DBP): Early 2A DNA Binding Protein gene; E4(Orf6): Early 4 Open reading frame 6 gene; VaRNA-I: Viral associated RNA-I; Rep: AAV-2 Replication genes; Cap: AAV-2 Capsid genes. Source data are provided as a Source Data file.

Article Snippet: 24 hr after cell seeding, we added doxycycline (0, 1000 ng/mL) and Ganciclovir sodium (GCV; 100 μg/mL) (MedChem Express; #HY-13637A) and immediately transfected them with a mixture of DNA and PEI (1500 ng DNA/8 μl PEI; stock concentration 0.324 mg/ml, pH 7.5).

Techniques: Construct, Positive Control, Expressing, Binding Assay, Transfection, Negative Control, Standard Deviation, One-tailed Test, Staining, Plasmid Preparation, Transduction, Flow Cytometry, Virus

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Hypoxia-directed tumor targeting of CRISPR-Cas9 and HSV-TK suicide gene therapy using lipid nanoparticles

doi: 10.1016/j.omtm.2022.03.008

Figure Lengend Snippet: Hypoxia-regulated expression and activity of HSV-tk (A) Schematic depicting the two promoters used to direct expression of HSV-tk constructs. The cytomegalovirus (CMV) promoter drives the constitutive expression of HSV-tk, thereby serving as the positive control for these experiments. The hypoxia-regulated promoter consists of a minimal CMV promoter and five copies of the human VEGF HRE. (B) Western-blot analysis of HeLa cells transfected with either the CMV or HRE-driven HSV-tk WT or mutant 30 constructs. Transfected cells were cultured in the presence of 10 μM BAY 85-3934 or hypoxia for 24 h prior to harvesting for protein analysis. (C) An Alamar assay for viability was carried out at 120 h post-transfection of the indicated HSV-tk constructs in HeLa cells cultured in 1 μM GCV with either 10 μM BAY 85-3934 or hypoxia. For hypoxia treatment, cells were treated with intermittent hypoxia. Cells were placed into hypoxic conditions for a total of 48 h, in 24 h intervals. (D) Confocal fluorescent microscopy for DNA-damage signaling marker γH2AX during HSV-tk treatment. HeLa cells were transfected with the HRE-HSV-tk mutant 30 construct and cultured in the presence of 1 μM GCV with either 10 μM BAY 85-3934 or DMSO vehicle control for 48 h. Cells were stained for HSV-tk (green), DNA-damage marker γH2AX (red), and the nuclear morphology indicator Hoescht 33342 (blue). Scale bar, 20 μm. Data are represented as mean ± SD and are representative of n = 3 biological replicates; ∗∗∗∗p < 0.0001. See also Figure S2 .

Article Snippet: For experiments involving the suicide gene HSV-tk, cells were treated with GCV (ACROS Organics 461710010) at the indicated concentrations.

Techniques: Expressing, Activity Assay, Construct, Positive Control, Western Blot, Transfection, Mutagenesis, Cell Culture, Microscopy, Marker, Control, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Hypoxia-directed tumor targeting of CRISPR-Cas9 and HSV-TK suicide gene therapy using lipid nanoparticles

doi: 10.1016/j.omtm.2022.03.008

Figure Lengend Snippet: Delivery of hypoxia-regulated HSV-tk and CRISPR-Cas9 using LNPs (A) Lipid nanoparticles encapsulating our HSV-tk and Cas9 constructs were synthesized and characterized for size, polydispersity, zeta potential, and encapsulation efficiency. Data represented as mean ± SD; for n = 13 LNPs each containing different DNA cargo. (B) Fluorescent microscopy images of EGFP induction (top panel) 48 h after LNP addition. HeLa cells received HRE-EGFP-packaged LNPs and were treated with either DMSO control or 10 μM BAY 85-3934 or were cultured in hypoxia for 24 h. LNPs used in this study were labeled with Cy3, allowing for visualization of uptake in cells (bottom panel). Scale bar, 100 μm. (C) Flow cytometry analysis of EGFP expression in HeLa cells 72 h after addition of HRE-EGFP-packaged LNPs. Cells were treated with either DMSO control or 10 μM BAY 85-3934 or were cultured in hypoxia for 24 h. (D) Representative bright-field image of BROD-0417-C71 neurosphere. (E) Alamar assay evaluating potency of LNPs delivering the indicated HSV-tk constructs to HCM-BROD-0417-C71 cells in the presence of 3 μM GCV. Cells were further treated with either 0.2% DMSO, 20 nM BAY 85-3934, or hypoxia treatment at 1 week post-LNP treatment. (F) Alamar assay comparing viability of HCM-BROD-0417-C71 cells 1 week post-addition of LNPs packaged with the indicated constructs. Cells were cultured in the presence of either 0.2% DMSO control, BAY 85-3934 at a concentration of 20 nM, or hypoxia. Data are represented as mean ± SD of n = 3 biological replicates; ∗∗∗∗p < 0.0001. See also Figures S4 and .

Article Snippet: For experiments involving the suicide gene HSV-tk, cells were treated with GCV (ACROS Organics 461710010) at the indicated concentrations.

Techniques: CRISPR, Construct, Synthesized, Zeta Potential Analyzer, Encapsulation, Microscopy, Control, Cell Culture, Labeling, Flow Cytometry, Expressing, Concentration Assay

Journal: Pathogens

Article Title: Oral Administration of Valganciclovir Reduces Clinical Signs, Virus Shedding and Cell-Associated Viremia in Ponies Experimentally Infected with the Equid Herpesvirus-1 C 2254 Variant

doi: 10.3390/pathogens11050539

Figure Lengend Snippet: Serum concentration of ganciclovir (GCV) from D − 5 to D + 20. Ponies A to D (treated group) were administered valganciclovir (VGCV) from D0 to D + 13, and ponies E to H (control group) received a placebo. Blood samples were taken just prior to the first daily VGCV administration. No GCV was detected in the sera of the control group ponies at D − 5, D + 1, D + 7 and D + 14. The half-maximal effective concentration (EC 50 ) of GCV was previously determined in vitro against the EHV-1 FR-56628 strain on equine embryonic kidney (EEK) cells . The blue shading represents the duration of VGCV treatment.

Article Snippet: Deuterium-marked ganciclovir (Toronto Research Chemicals) was used as internal standard for the quantification of GCV.

Techniques: Concentration Assay, Control, In Vitro