Journal: Cancers

Article Title: Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways

doi: 10.3390/cancers13040787

Figure Lengend Snippet: ( A ) Western blotting analyses of the indicated pro-homologous recombination (HR) factors in total cell extracts from GCT27 paired cell lines. Graph bars quantify protein level differences among GCT27 cell lines. Data are mean values ± s.d. of either three (breast cancer type 1 susceptibility protein, BRCA1) or six (C-terminal binding protein-1 interacting protein, CtIP) independent experiments. Original blots see . Statistical analyses were performed using a two-tailed t -test (* p < 0.05; ** p < 0.01). ( B ) Western blotting analyses of the indicated pro-HR factors in chromatin extracts from GCT27 paired cell lines. Cells were treated with a pulse of 3 μM cisplatin for 6 h and analyzed 16 h after culture in drug-free media. Histone H3 (H3) was used as a loading control. Original blots see . ( C ) Quantification of BRCA1 foci number in GCT27 paired cell lines before (Ctr) and after treatment with 3 μM cisplatin. Treated cells were collected both at the end of treatment (6 h) and 16 h after drug washout. Data are mean values ± s.d. of two independent experiments, each performed in duplicate. ( D ) Western blotting analyses of the indicated pro-HR factors in total cell extracts of 2102EP paired cell lines. Tubulin and cyclin A were assessed to detect differences in protein loading and cell cycle phase distribution, respectively. Original blots see . ( E ) Western blotting analyses of the indicated pro-HR factors in chromatin extracts from the indicated cell lines, before and after treatment with 3 μM cisplatin for 6 h. Cell extracts were prepared from cells collected 16 h after drug removal. H3 was used as a loading control. Original blots see . ( F , G ) Western blotting analyses of the indicated pro-non-homologous end joining (NHEJ) factors in total cell extracts of 2102EP and GCT27 cell lines. Clathrin was used as a loading control for 53BP1 and DNA-PKcs, while KU70 expression was normalized using tubulin. Graph bars quantify protein levels difference among cell lines. Original blots see . Data are mean values ± s.d. of six (53BP1) or three (DNA-PKcs) independent experiments. In all quantifications, the expression of the indicated proteins was normalized on the expression of either tubulin or clathrin. Cyclin A was assessed to detect possible differences in cell cycle phase distribution among cell lines. Statistical analyses were performed using a one-tailed t -test (* p < 0.05; ** p < 0.01; **** p < 0.0001). A.U. = arbitrary units.

Article Snippet: To stably increase the expression of 53BP1 in 2102EPcis-r cells, we infected them using retroviral particles expressing either the N-Myc-53BP1 WT pPLC-Puro vector (Addgene, Watertown, NY, USA, 19836) or pLPC-N MYC (Addgene, 12540) as a control.

Techniques: Western Blot, Homologous Recombination, Binding Assay, Two Tailed Test, Non-Homologous End Joining, Expressing, One-tailed Test

Journal: Cancers

Article Title: Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways

doi: 10.3390/cancers13040787

Figure Lengend Snippet: ( A , B ) NHEJ repair proficiency of GCT27 and 2102EP cell lines as measured by the EJ5-GFP reporter substrate. NZE-GFP indicates the transfection efficiency of each cell line. Data are mean values ± s.d. of three independent experiments. ( C , D ) Colony assay of the indicated cell lines treated with the indicated doses of X-rays. The surviving fraction was monitored by following colony formation for up to 10 to 14 days after treatment. Data are mean values ± s.d. of two independent experiments, each performed in triplicate. ( E , F ) Quantification of 53BP1 foci in S/G2 (cyclin A-positive) nuclei of the indicated cell lines after X-ray treatment. Data are mean values ± s.d. of three independent experiments. ( G , H ) Quantification of 53BP1 foci in S/G2 nuclei of the indicated cell lines after cisplatin treatment. Cells were treated with a 6 h pulse of 3 μM cisplatin and collected at the end of stimulation and 16 h after drug washout. Data are mean values ± s.d. of three independent experiments. Statistical analyses were performed using an unpaired two-tailed ( A , B , E – H ) or one-tailed ( C , D ) t -test (* p < 0.05; ** p < 0.01).

Article Snippet: To stably increase the expression of 53BP1 in 2102EPcis-r cells, we infected them using retroviral particles expressing either the N-Myc-53BP1 WT pPLC-Puro vector (Addgene, Watertown, NY, USA, 19836) or pLPC-N MYC (Addgene, 12540) as a control.

Techniques: Transfection, Colony Assay, Two Tailed Test, One-tailed Test

Journal: Cancers

Article Title: Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways

doi: 10.3390/cancers13040787

Figure Lengend Snippet: ( A ) Western blotting analyses of 53BP1 expression in 2102EP cells. Here, #1, #2, #3 are clones of 2102EPcis-r cells overexpressing 53BP1 at a level comparable to that of cis-s cells. Ctr indicates the level of 53BP1 protein in naïve 2102EPcis-r cells. Tubulin was used as loading control. Original blots see . ( B ) Colony assay of control and 53BP1-overexpressing clones of 2102EPcis-r after treatment with a pulse of 3 μM cisplatin for 6 h. Ctr = cells transfected with a control vector; Unt. = untreated; cisp. = cisplatin-treated. ( C ) Quantification of surviving colonies of the indicated 2102EPcis-r cells, treated as described in ( B ). ( D ) Western blotting analyses of 53BP1 expression in GCT27 cells. GCT27cis-s and GCT27cis-r are non-infected paired clones. GCT27cis-s ShRNACtr are cells infected with a non-specific shRNA, while GCT27cis-s ShRNAmir1 are cells infected with an shRNA specific to 53BP1. Tubulin was used as a loading control. Original blots see . ( E ) Colony assay of GCT27 cell lines infected with either ShRNACtr or ShRNAmir1. Data are mean values ± s.d. of three independent experiments. ( F , G ) Surviving fraction of the indicated cell lines after treatment with either cisplatin or cisplatin combined with DNA-PKi. Survival was evaluated by crystal violet assay. Data are mean values ± s.d. of either three (GCT27) or six (2102EP) independent experiments. Statistical analyses were performed using the unpaired two-tailed ( C , F , G ) or one-tailed ( E ) t -test (* p < 0.05; ** p < 0.01; *** p < 0.0001).

Article Snippet: To stably increase the expression of 53BP1 in 2102EPcis-r cells, we infected them using retroviral particles expressing either the N-Myc-53BP1 WT pPLC-Puro vector (Addgene, Watertown, NY, USA, 19836) or pLPC-N MYC (Addgene, 12540) as a control.

Techniques: Western Blot, Expressing, Clone Assay, Colony Assay, Transfection, Plasmid Preparation, Infection, shRNA, Crystal Violet Assay, Two Tailed Test, One-tailed Test

Journal: eLife

Article Title: Structure of the human heparan-α-glucosaminide N -acetyltransferase (HGSNAT)

doi: 10.7554/eLife.93510

Figure Lengend Snippet:

Article Snippet: The synthesized gene was then cloned into the pEG BacMam expression vector (Addgene plasmid # 160683) between EcoRI and NotI restriction sites, to be expressed via baculoviral transduction in HEK293S GnTI - cells (ATCC # CRL-3022) as a fusion protein containing an N-terminal Strep-tag-II-GFP.

Techniques: Sequencing, Expressing, Transfection, Construct, Recombinant, Plasmid Preparation, Cloning, Modification, Software, Cell Culture, Size-exclusion Chromatography, Affinity Purification, Electron Microscopy

Journal: BMC Biology

Article Title: Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

doi: 10.1186/s12915-024-01879-0

Figure Lengend Snippet: REST promotes glioblastoma growth. A Boxplot of REST mRNA expression in TCGA-LGG and TCGA-GBM samples compared to normal brain samples from TCGA and GTEx datasets (* p < 0.001). B Survival analysis using data from TCGA-GBM and TCGA-LGG projects. Analysis was done using GEPIA2 web server ( A , B ). C Basal REST protein amount in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs SVGp12 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File A. D Proliferation of GBM cell lines assessed by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File B. E Western blot confirms the lack of REST in homozygous REST-KO clones of T98G ( left ) and HEK293 ( right ). F Proliferation of WT and REST-KO T98G cells was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three to four independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post hoc tests. Individual data values are provided in Additional File C. G Proliferation of T98G WT and REST-KO C10 cells (transfected with empty vector pLPC vs REST OE) was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments. Statistical difference was tested using two-tailed paired t -test. Individual data values are provided in Additional File D. H Wound scratch assay and its quantification using ImageJ. Shown are mean ± SD from three independent biological experiments. Groups were compared using paired t -tests. Individual data values are provided in Additional File E. I Effect of REST loss on GSC marker expression. Shown are fold changes (FC) vs CRISPR Control derived from three independent biological replicates. Comparison vs control was performed using unpaired one-tailed t -tests. Dashed line indicates FC = 1. Individual data values are provided in Additional File F. *** p < 0.001; ** p < 0.01; * p < 0.05; ns—not significant

Article Snippet: On the next day, cells were transiently transfected with 500 ng either REST-WT-expressing pLPC-vector (Addgene, #41903) or empty pLPC vector (Addgene, #12521) with Fugene HD transfection reagent (Promega) following manufacturer’s instructions.

Techniques: Expressing, Western Blot, Clone Assay, Comparison, Transfection, Plasmid Preparation, Two Tailed Test, Wound Healing Assay, Marker, CRISPR, Derivative Assay, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism

doi: 10.3390/ijms17060800

Figure Lengend Snippet: Establishment of hTERT -immortalized Sca-1+ CSC lines. ( A ) Schematic structure of pLPCX- hTERT -IRES- GFP ( top ). Correct construction of pLPCX- hTERT -IRES- GFP was confirmed by digestion with restriction enzymes ( bottom ), Bgl II (Lane 2), Cla I (Lane 3), or Bgl II /Cla I (Lane 4). Lane M: λ/ Hind III marker. Lane 1: Supercoiled pLPCX- hTER T-IRES- GFP ; ( B ) retroviruses were produced in 293GPG packaging cells by transfection with a retroviral vector encoding hTERT -IRES- GFP using Lipofectamine 2000. At 48 h post transfection, expression of GFP (green), a reporter gene was monitored in 293GPG cells. Scale bars = 20 μm; ( C ) Sca-1+ CSCs transduced with retroviruses expressing hTERT -IRES- GFP (green) were selected in 96-well plates at a single cell level by limiting dilution for 12 days; and ( D ) two putative Sca-1+ CSC lines were finally selected. Nuclei were stained with DAPI (blue).

Article Snippet: We generated the pLPCX- hTERT -IRES- GFP vector, a Bgl II- Sal I fragment containing hTERT cDNA was amplified by PCR using pCI-neo-hEST2 (Addgene, Cambridge, MA, USA) as a template.

Techniques: Marker, Produced, Transfection, Retroviral, Plasmid Preparation, Expressing, Transduction, Staining

Journal: International Journal of Molecular Sciences

Article Title: Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism

doi: 10.3390/ijms17060800

Figure Lengend Snippet: Sca-1+/CD31− CSCs hTERT exhibit stem cell potency. For phenotypic characterization, Sca-1+/CD31− CSCs hTERT expressing GFP (green) were analyzed by immunostaining ( A ) and flow cytometry ( B ) with different cell surface antibodies (red); ( C ) real-time PCR was performed to detect both endogenous mTERT and transduced hTERT transcripts in primary Sca-1+ CSCs and immortalized Sca-1+/CD31− CSCs hTERT . Data represent mean ± SD from three independent experiments (* p < 0.05; ** p < 0.01). ND , not determined; ( D ) for proliferation analysis, Sca-1+/CD31− CSCs hTERT were seeded at 1 × 10 3 cells/well in 96-well microplates, cultured with Mesencult Basal Medium supplemented with cytokines for six days, and analyzed by WST-1 assay. Data represent mean ± SD from four independent experiments; and ( E ) Sca-1+/CD31− CSCs hTERT exhibit multi-differentiation potential. Differentiation was analyzed by immunostaining with a cardiomyocyte marker (cTnT, red), an endothelial marker (vWF, red), and by Oil-Red O staining (red) or Alizarin Red S staining (red). Nuclei were stained with DAPI (blue). Scale bars = 20 μm.

Article Snippet: We generated the pLPCX- hTERT -IRES- GFP vector, a Bgl II- Sal I fragment containing hTERT cDNA was amplified by PCR using pCI-neo-hEST2 (Addgene, Cambridge, MA, USA) as a template.

Techniques: Expressing, Immunostaining, Flow Cytometry, Real-time Polymerase Chain Reaction, Cell Culture, WST-1 Assay, Marker, Staining

Journal: International Journal of Molecular Sciences

Article Title: Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism

doi: 10.3390/ijms17060800

Figure Lengend Snippet: Sca-1+/CD31− CSCs hTERT CM protects HL-1 cardiomyocytes from CoCl 2 -induced hypoxic injury. Sca-1+/CD31− CSC hTERT lysate ( A ) and Sca-1+/CD31− CSCs hTERT CM ( B ) were subjected to a mouse cytokine antibody array detecting 21 cytokines in duplicate. 1. EGF; 2. TGF-β1; 3. HGF; 4. IGF-1; 5. IGF-2; 6. MCP-1; 7. VEGF; 8. SDF-1; 9. bFGF; 10. E-Cadherin; 11. HGF R; 12. IFN-γ; 13. IL-10; 14. TNF-α; 15. IL-6; 16. IL-1α; 17. IL-1β; 18. IL-1ra; 19. Leptin; 20. CT-1; and 21. MCP-5. Solid-lined boxes indicate dominant paracrine factors expressed in Sca-1+/CD31− CSCs hTERT lysate or Sca-1+/CD31− CSCs hTERT CM; ( C ) relative quantification of cytokine levels expressed in Sca-1+/CD31− CSCs hTERT lysate and Sca-1+/CD31− CSCs hTERT CM; and ( D ) viable cells were counted using a hemocytometer after staining with 0.2% trypan blue Sca-1+/CD31− CSCs hTERT CM in HL-1 cardiomyocytes treated with or without 150 μM CoCl 2 for 24 h. Data represent mean ± SD from three independent experiments (** p < 0.01).

Article Snippet: We generated the pLPCX- hTERT -IRES- GFP vector, a Bgl II- Sal I fragment containing hTERT cDNA was amplified by PCR using pCI-neo-hEST2 (Addgene, Cambridge, MA, USA) as a template.

Techniques: Ab Array, Quantitative Proteomics, Staining

Journal: International Journal of Molecular Sciences

Article Title: Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism

doi: 10.3390/ijms17060800

Figure Lengend Snippet: Sca-1+/CD31− CSCs hTERT CM protects HL-1 cardiomyocytes from hypoxic injury partly via MCP-1-dependent mechanism. Sca-1+/CD31− CSCs hTERT were cultured in six-well plates at a density of 5 × 10 4 cells/well and transfected with 50 nM of MCP-1 siRNA duplexes or NC siRNA duplexes using Lipofectamine RNAiMAX. After a 48 h transfection, MCP-1 mRNA expression was assessed by real-time PCR ( A ); The average value of MCP-1 mRNA was normalized to that of GAPDH for each sample. The data represent the mean ± SD from three experiments. Sca-1+/CD31− CSCs hTERT CM ( B ) after transfection of NC siRNA or MCP-1 siRNA were subjected to densitometry and are presented as fold changes for MCP-1 (indicated by number 6) and IL-6 (indicated by number 15) ( C ), taking MCP-1 and IL-6 levels in NC siRNA-transfected Sca-1+/CD31− CSCs hTERT as a one-fold value, each in triplicate, * p < 0.05; ** p < 0.01 vs. NC siRNA; ( D ) representative flow cytometric analysis showing apoptotic effects on HL-1 cardiomyocytes treated with or without 150 μM CoCl 2 for 24 h in the absence or presence of Sca-1+/CD31− CSCs hTERT CM transfected with NC siRNA or MCP-1 siRNA. Bar graph of three independent experiments showing percentages of apoptotic HL-1 cardiomyocytes ( E ) treated with or without 150 μM CoCl 2 for 24 h in the absence or presence of Sca-1+/CD31− CSCs hTERT CM transfected with NC siRNA or MCP-1 siRNA (** p < 0.01); and ( F ) representative flow cytometric analysis showing apoptotic effects on HL-1 cardiomyocytes treated with or without 200 μM H 2 O 2 for 24 h in the absence or presence of Sca-1+/CD31− CSCs hTERT CM transfected with NC siRNA or MCP-1 siRNA; Bar graph of three independent experiments showing percentages of apoptotic HL-1 cardiomyocytes ( G ) treated with or without 200 μM H 2 O 2 for 24 h in the absence or presence of Sca-1+/CD31− CSCs hTERT CM transfected with NC siRNA or MCP-1 siRNA (** p < 0.01).

Article Snippet: We generated the pLPCX- hTERT -IRES- GFP vector, a Bgl II- Sal I fragment containing hTERT cDNA was amplified by PCR using pCI-neo-hEST2 (Addgene, Cambridge, MA, USA) as a template.

Techniques: Cell Culture, Transfection, Expressing, Real-time Polymerase Chain Reaction

Journal: Communications Biology

Article Title: MacroGreen, a simple tool for detection of ADP-ribosylated proteins

doi: 10.1038/s42003-021-02439-w

Figure Lengend Snippet: Comparison between MacroGreen and the wild type Af1521-GFP fusion binding to either PARylated PARP1 ( a ) or MARylated PARP10 ( b ) coated plates. In total, 50 µl reactions in 96-well Nunc MaxiSorp TM plates; detection with 1 µM MacroGreen or Af1521-GFP at room temperature c Examples of the linear assay range using MacroGreen detection of PARP1 PARylation predominantly at acidic side chains; PARP1 PARylation predominantly at serine side chains in the presence of HPF1; PARP10 MARylation at various side chains; or C2I MARylation of actin at Arg177. d Extended linear range of MAR detection on PARP10 under the same conditions. e Detection of MAR on five histone proteins (H2B, H3.1, H3.2, H3.3 and H4 as indicated) in the presence of PARP10. PARP10 MARylation alone (gray) serves as reference. Negative control reactions contained NAD + (1 mM) but not PARP10. Statistical significance of the data was calculated using a one-way ANOVA test; error bars indicate S.D. All panels – n = 4.

Article Snippet: The MacroGreen expression plasmid is available through Addgene (ID 160665).

Techniques: Comparison, Binding Assay, Negative Control

Journal: Communications Biology

Article Title: MacroGreen, a simple tool for detection of ADP-ribosylated proteins

doi: 10.1038/s42003-021-02439-w

Figure Lengend Snippet: a Verification of PARP1 inhibition by Talazoparib and of PARP10 inhibition by PJ-34 using Western blotting. b Quantification using MacroGreen of PARP1 and PARP10 inhibition. n = 4; error bars indicate S.D.

Article Snippet: The MacroGreen expression plasmid is available through Addgene (ID 160665).

Techniques: Inhibition, Western Blot

Journal: Communications Biology

Article Title: MacroGreen, a simple tool for detection of ADP-ribosylated proteins

doi: 10.1038/s42003-021-02439-w

Figure Lengend Snippet: a MacroGreen staining of DNA damage sites in U2OS DNA double-strand break reporter cells, verified by staining for γH2AX. Control experiments contained a PARP1 inhibitor. Scale bars: Main panel, 10 μm; inset, 2.5 μm. b MacroGreen fluorescence detected after staining of HEK293T cells grown in 96-well plates and treated to induce DNA damage as indicated. n = 12; error bars indicate S.D.

Article Snippet: The MacroGreen expression plasmid is available through Addgene (ID 160665).

Techniques: Staining, Control, Fluorescence