Journal: Longevity & Healthspan

Article Title: The secreted protein S100A7 (psoriasin) is induced by telomere dysfunction in human keratinocytes independently of a DNA damage response and cell cycle regulators

doi: 10.1186/2046-2395-3-8

Figure Lengend Snippet: Colony-forming efficiency analysis of NHEK transduced with p14 ARF , p16 INK4A , p53 and TRF2 ΔBΔM . NHEKs were transduced with amphotropic retroviral particles using spinfection and, 48 h later, trypsinised and seeded at clonal density (7 × 10 3 cells per 6-well plate). Cells were cultured for 2 weeks under drug selection and finally fixed and stained with Rhodamine B to reveal keratinocyte colonies. Colony-forming efficiency, displayed as percentage and relative to the respective EV control, was calculated by dividing the total number of colonies obtained per well by the total number of cells seeded per plate (7,000). Photos show wells representative of the results obtained for each construct. Legend: GFP, empty vector control for p14 ARF , p16 INK4A and p53 ; EV, empty vector control for TRF2 ΔBΔM (DN). This is the result of a single experiment.

Article Snippet: Retroviral vectors pLPC-N MYC (12540 Addgene, Cambridge, MA.) and pLPC-NMYC TRF2 ΔBΔM (16069 Addgene) were donated by Titia de Lange (Rockefeller University, NYC, USA); pBABE-puro p14 ARF , pBABE-puro p16 INK4a and pBABE-puro p53 were donated by Gordon Peters (London Research Institute, CRUK, London, UK); and pBABE-puro GFP was donated by Cleo Bishop (Blizard Institute, QMUL, London, UK).

Techniques: Transduction, Retroviral, Cell Culture, Selection, Staining, Control, Construct, Plasmid Preparation

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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cytomegalovirus disrupts Lamin A/C to control microtubule-mediated nuclear movement and cell migration

doi: 10.1073/pnas.2507831122

Figure Lengend Snippet: The effects of Lamin A/C mutants and SUN2 expression on actin filaments in HCMV-infected cells. ( A – C ) NHDFs stably expressing eGFP-tagged Lamin A/C variants; WT, nonphosphorylatable alanine mutant (S22A), or head and tail deletion mutant (ΔHC) were infected with TB40/E-UL99-mCherry at MOI 5 for 6 d. Cells were fixed in methanol (which quenches fluorescent proteins) and stained with antibodies against actin (green), gB (red), and GFP (turquoise), while DNA was stained with Hoechst (blue). ( A ) Images are representative of multiple fields of view derived from at least three independent experiments. ( B ) Enlarged examples of merged images from A which illustrate the partial restoration of actin filaments in some infected cells expressing the Lamin A/C S22A mutant; white arrows show examples of clear filaments while red arrows show examples of weak to no filament restoration. ( C ) Quantification of actin caps. n = 150 cells from three independent replicate experiments (wt: 1/50, 2/50, 1/50; S22A: 25/50, 20/50, 22/50, ΔHC: 0/50, 0/50, 1/50). Bars represent mean ± SEM; ns = not significant, **** P < 0.00001, one-way ANOVA test. ( D ) Lamin A/C expression does not restore SUN2 expression in infected cells. Lamin A/C-expressing cells were infected as described in A – C and then fixed and stained with antibodies against SUN2 (green), gB (red), and GFP (turquoise), while DNA was stained with Hoechst (blue). Representative images are shown from three independent replicate experiments. Note that methanol fixation was required for SUN2 imaging and incomplete quenching results in residual detection of concentrated GFP signal with the Lamin A/C-ΔHC variant, but its pattern is distinct and does not interfere with SUN2 detection. ( E and F ) SUN2 expression does not restore actin caps in infected cells. Control NHDFs or NHDFs expressing flag-tagged SUN2 were infected at MOI 5 for 6d then fixed and stained with antibodies against actin (green), gB (red), and Flag (turquoise), while DNA was stained with Hoechst (blue). ( E ) Representative images of uninfected or infected cells. ( F ) Quantification of actin caps in control or SUN2-expressing NHDFs infected with HCMV from three independent replicate experiments (control NHDFs: Mock; 55/35, 50/50, 44/44, or HCMV infected; 0/36, 0/44, 0/40; SUN2-expressing NHDFs: Mock; 47/47, 55/55, 48/48, or HCMV infected; 0/52, 0/47, 0/50). Bars represent mean ± SEM; **** P < 0.0001, two-way ANOVA test.

Article Snippet: Similarly, the pQCXIP retroviral vector encoding eGFP-Lamin A/C S22A was generated by amplifying the S22A variant from the pLPC-S22A-LaminA plasmid (Addgene #69066) using the same primers.

Techniques: Expressing, Infection, Stable Transfection, Mutagenesis, Staining, Derivative Assay, Imaging, Variant Assay, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cytomegalovirus disrupts Lamin A/C to control microtubule-mediated nuclear movement and cell migration

doi: 10.1073/pnas.2507831122

Figure Lengend Snippet: Lamin A/C phosphorylation and inactivation is required for HCMV-induced cell migration. NHDFs stably expressing eGFP control or eGFP-tagged Lamin A/C variants; WT, nonphosphorylatable alanine mutant (S22A), or head and tail deletion mutant (ΔHC) were infected with TB40/E-UL99-mCherry at MOI 2. ( A – C ) Time lapse imaging was performed by acquiring 1 frame per 30 min starting at 120 h.p.i. ( A and B ) Representative stills from Movies S3 and S4 are shown, with the nucleus of infected cells outlined. ( C ) Graph plots the percentage of cells wherein the displacement of the center of the nucleus was above 30 µM. n = 291 cells total from three independent biological replicates, * P < 0.05, ** P < 0.005, *** P < 0.0005, one-way ANOVA test. ( D – F ) Migration of infected cells in trans-well assays. ( D ) Schematic of trans-well assay illustrating cells expressing eGFP-Lamin constructs that accumulate in the nucleus, which is costained with Hoechst after fixation at the end-point of the assay. Cells were infected with HCMV TB40/E UL99-mCherry (MOI = 2) and transferred at 5 d.p.i. to the top of trans-well inserts with 12 µM pores. Migrating cells must pass through these pores to reach the bottom of the inserts. Samples were fixed with 4% PFA at 7 d.p.i., inserts were removed and the top (seeded cells) and bottom (migrated cells) were imaged after staining with Hoechst. ( D ) Representative images of the top and bottom of inserts. An example of a migrated cell at the bottom (white arrow) compared with those that have not migrated from the top is shown in the WT cells. Pores are visible in images of the bottom of wells (orange arrow). In the case of Lamin A/C mutants, out of focus light is visible from cells still at the top of trans-wells (blue arrows). ( E ) The number of infected cells that migrated to the other side of the transwell membrane was counted and WT numbers were set to 100% as the basis for comparisons with effects of Lamin A/C mutants. Total number of migrated cells n = 1,155 for WT, n = 296 for S22A n = 173 for ΔHC from four independent biological replicates, ns = not significant, ** P < 0.005, **** P < 0.00001, one-way ANOVA test.

Article Snippet: Similarly, the pQCXIP retroviral vector encoding eGFP-Lamin A/C S22A was generated by amplifying the S22A variant from the pLPC-S22A-LaminA plasmid (Addgene #69066) using the same primers.

Techniques: Phospho-proteomics, Migration, Stable Transfection, Expressing, Control, Mutagenesis, Infection, Imaging, Construct, Staining, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cytomegalovirus disrupts Lamin A/C to control microtubule-mediated nuclear movement and cell migration

doi: 10.1073/pnas.2507831122

Figure Lengend Snippet: Lamin A/C phosphorylation is required for HCMV-induced formation of acetylated microtubule networks. ( A – C ) NHDFs were mock infected or infected with TB40/E-UL99-mCherry at MOI 5 and then treated with DMSO solvent control or 10 µM maribavir (MBV) for 6 d. Samples were fixed and stained with antibodies against acetylated tubulin (green) and gB (red), while DNA was stained with Hoechst (blue). ( A and B ) Representative fields of view derived from at least three independent experiments illustrate the induction of acetylated microtubules during infection along with the organization, emanating from the AC and extending through the cytoplasm, along with the inhibitory effects of MBV treatment. ( C ) Graph plots the percentage of cells with robust microtubule acetylation, bars represent mean ± SEM; n = 198 cells total from three independent experiments, **** P < 0.0001, two-tailed Student’s t test. ( D – F ) NHDFs stably expressing eGFP control or eGFP-tagged Lamin A/C variants; WT, nonphosphorylatable alanine mutant (S22A), or head and tail deletion mutant (ΔHC) were infected with TB40/E-UL99-mCherry at MOI 5 for 6 d. Samples were fixed and stained with antibodies against acetylated tubulin (green), gB (red), and GFP (turquoise), while DNA was stained with Hoechst (blue). ( D and E ) Representative fields of view derived from four independent experiments and associated zoomed Insets in E . illustrate the lack (red arrows) or severe reduction (orange arrows) in the levels of acetylated microtubule networks emanating from the AC into the cytoplasm in infected cells expressing Lamin A/C mutants. Note that methanol fixation was required for microtubule imaging and incomplete quenching results in residual detection concentrated GFP signal with the Lamin A/C-ΔHC variant. ( F ) Graph plots the percentage of cells with robust microtubule acetylation derived from four independent experiments; WT cells; 22/30, 24/30, 21/30, 20/30; S22A cells; 8/30, 6/30, 9/30, 4/30; ΔHC cells; 0/30, 1/30, 2/30, 3/30. Bars represent mean ± SEM; ** P < 0.01, **** P < 0.0001, two-tailed Student’s t test.

Article Snippet: Similarly, the pQCXIP retroviral vector encoding eGFP-Lamin A/C S22A was generated by amplifying the S22A variant from the pLPC-S22A-LaminA plasmid (Addgene #69066) using the same primers.

Techniques: Phospho-proteomics, Infection, Solvent, Control, Staining, Derivative Assay, Two Tailed Test, Stable Transfection, Expressing, Mutagenesis, Imaging, Variant Assay

Journal: Journal of cell science

Article Title: Single-cell analysis reveals the Comma-1D cell line as a unique model for mammary gland development and breast cancer.

doi: 10.1242/jcs.259329

Figure Lengend Snippet: Fig. 4. Molecular characterization of Comma-1D populations. (A) The figure shows network connections of luminal and basal transcription factors in Comma- 1D subpopulations. (B) The figure shows the strategy to generate and analyze Comma-1D populations overexpressing Trp63 and Gata3. The qRT-PCR data in the schematic representation shows real results (mean±s.d.; n=3). (C) The FACS profiles show how overexpression of Trp63 and Gata3 affect the reconstitution pattern of sorted C1D-bi cells as revealed by determining the percentage of cells belonging to each population. Results are representative of n≥3. FACS profiles were collected 3 weeks after viral transduction of C1D-bi cells with the corresponding c-DNAs.

Article Snippet: For Trp63 overexpression we subcloned ΔNp63beta (Addgene #27014) in a retroviral vector (pLPCX-GFP, Addgene #65433) using the following primers designed to add XhoI forward and BamHI reverse cut sites: forward, 5′-ATCGATCGCTCGAGATGGGCTGTGATCG-3′; reverse, 5′- ATCGATCGGGATCCTTATTTTCCATTCTTGGA-3′.

Techniques: Quantitative RT-PCR, Over Expression, Transduction