Journal: Cell Reports Medicine

Article Title: PARP-1 improves leukemia outcomes by inducing parthanatos during chemotherapy

doi: 10.1016/j.xcrm.2023.101191

Figure Lengend Snippet:

Article Snippet: Human: HS-5 stromal cells , LGC Standards , ATCC Cat# CRL-11882, RRID: CVCL_3720.

Techniques: Recombinant, Derivative Assay, Staining, Negative Control, Plasmid Preparation, Caspase-3 Assay, TUNEL Assay, Reverse Transcription, Expressing, Bicinchoninic Acid Protein Assay, Western Blot, Microarray, Software, Fluorescence

Journal: iScience

Article Title: Multi-omics approach reveals posttranscriptionally regulated genes are essential for human pluripotent stem cells

doi: 10.1016/j.isci.2022.104289

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-SF3B3 (1:1000) , Proteintech , Cat# 14577-1-AP RRID: AB_2270189.

Techniques: Recombinant, Mass Spectrometry, Sequencing, Modification, Protease Inhibitor, Multiplex sample analysis, Multiplex Assay, Fractionation, Cell Culture, Bicinchoninic Acid Protein Assay, Lysis, Cloning, Gene Expression, SYBR Green Assay, Transfection, Ligation, Microarray, TaqMan Assay, Software, Cell Analysis

Journal: Stem cell research

Article Title: Generation and characterization of human induced pluripotent stem cells (iPSCs) from three male and three female patients with CDKL5 Deficiency Disorder (CDD)

doi: 10.1016/j.scr.2021.102276

Figure Lengend Snippet:

Article Snippet: Pluripotency Marker , Rabbit anti-OCT4 , 1:100 , Abcam Cat# ab19857, RRID: AB_445175.

Techniques: Derivative Assay, Sequencing, Modification

Journal: Stem cell research

Article Title: Generation and characterization of human induced pluripotent stem cells (iPSCs) from three male and three female patients with CDKL5 Deficiency Disorder (CDD)

doi: 10.1016/j.scr.2021.102276

Figure Lengend Snippet: Characterization and validation

Article Snippet: Pluripotency Marker , Rabbit anti-OCT4 , 1:100 , Abcam Cat# ab19857, RRID: AB_445175.

Techniques: Immunocytochemistry, Staining, Expressing, Microarray, Marker, Mutagenesis, Sequencing, Southern Blot

Journal: Stem cell research

Article Title: Generation and characterization of human induced pluripotent stem cells (iPSCs) from three male and three female patients with CDKL5 Deficiency Disorder (CDD)

doi: 10.1016/j.scr.2021.102276

Figure Lengend Snippet: Reagents details

Article Snippet: Pluripotency Marker , Rabbit anti-OCT4 , 1:100 , Abcam Cat# ab19857, RRID: AB_445175.

Techniques: Immunocytochemistry, Marker, Expressing, Mutagenesis, Sequencing

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: Analytic Validation of a Clinical-Grade PTEN Immunohistochemistry Assay in Prostate Cancer by Comparison to PTEN FISH

doi: 10.1038/modpathol.2016.88

Figure Lengend Snippet: Prostate cancer cases with discordant PTEN immunohistochemistry and FISH results on initial review. Case #9 : PTEN immunohistochemistry demonstrates very weak cytoplasmic immunostaining with loss of nuclear immunostaining and thus was called negative on initial review, though in retrospect it may be better classified as ambiguous due to weak staining and absence of benign glands for comparison (left). Four-color FISH image from an adjacent section that is representative of all examined cores in this tissue microarray (right) indicates that the PTEN gene does not have a detectable deletion by FISH. The enlarged inset shows that the centromeres, WAPAL, PTEN and FAS gene probes are each present as two copies. Case #10 : PTEN immunohistochemistry image (left) shows heterogeneous PTEN loss in some tumor glands (arrow) but PTEN protein is expressed by majority of other tumor glands in this core. FISH image from an adjacent section (right) was initially read as PTEN intact, but shows a focal area with hemizygous PTEN deletion recognized on re-examination guided by immunohistochemistry. The enlarged inset shows there is only one copy of the red PTEN gene probe (one red signal) and loss of both aqua FAS gene probes. Case #11 : PTEN immunohistochemistry image (left) demonstrates heterogeneous PTEN loss in some tumors glands (arrows) but not in others (arrowheads). FISH image from an adjacent section (right) shows the small area of the section that had a homozygous PTEN deletion on re-examination. The enlarged inset shows that there are no copies of the red PTEN gene probe and one copy of the aqua FAS gene probe, but retention of the adjacent WAPAL and centromere probes.

Article Snippet: Briefly, the protocol uses the Ventana automated staining platform (Ventana Discovery Ultra, Ventana Medical Systems, Tucson, AZ) and a rabbit anti-human PTEN antibody (Clone D4.3 XP; Cell Signaling Technologies, Danvers, MA).

Techniques: Immunohistochemistry, Immunostaining, Staining, Microarray

Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

doi: 10.1371/journal.ppat.1002927

Figure Lengend Snippet: (A) Microarray analysis of cellular mRNAs upregulated in HUVECs by K15 overexpression or after KSHV infection. HUVECs were transduced with a K15-expression vector (lanes 1–2) or infected with either KSHVwt or KSHVΔK15 (lanes 3–4). Depicted are the top-ranking 50 transcripts most strongly upregulated by K15 overexpression, showing an at least two-fold induction by K15 overexpression in each of two experiments performed using cells from two healthy donors (lanes 1–2). Asterisks next to fold change values within heatmap lanes 3–4 indicate genes that were induced more than two-fold in KSHVwt-infected cells compared to cells mock infected with heat-inactivated virus. Empty cells in the heatmap correspond to undetectable mRNA levels in both of the two samples compared in each lane. Arrows indicate cellular genes, inducible upon KSHVwt infection with more than two-fold elevated expression in KSHVwt- relative to KSHVΔK15-infected cells in each of the two experiments performed. (B) Quantitative PCR analysis of K15 transcript in mRNA derived from KSHVwt- or KSHVΔK15-infected cells used for the microarray experiment. (C) Quantitative PCR analysis of RCAN1/DSCR1 and VEGFA transcripts in HUVECs transduced with a retroviral K15 expression vector or with an empty control vector. (D) HUVECs from two healthy donors (Exp1–2) were infected with heat-inactivated, KSHVwt, or KSHVΔK15 and were lysed 4 days after infection. RNA was extracted, reversely transcribed and subjected to quantitative TaqMan-based PCR analysis. mRNA fold change values of KSHVwt-infected versus mock-infected (with heat-inactivated KSHVwt = HI) cells were depicted in black, values of KSHVΔK15- infected versus mock-infected cells were depicted in red. Input mRNA samples are identical to samples used in microarray experiments (compare panel A lanes 3–4).

Article Snippet: The following TaqMan gene expression assays (Applied Biosystems: #4331182) were used: Hs01120954_m1 (RCAN1); Hs00173626_m1 (VEGFA); Hs99999905_m1 (GAPDH).

Techniques: Microarray, Over Expression, Infection, Transduction, Expressing, Plasmid Preparation, Virus, Real-time Polymerase Chain Reaction, Derivative Assay, Retroviral, Control

Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

doi: 10.1371/journal.ppat.1002927

Figure Lengend Snippet: (A) HUVECs transduced with pSF91 or pSF91K15 were plated on matrigel with medium (EBM2+2%FBS) with or without VEGF (50 ng/ml) 30 hours after transduction and were assessed 6 hours later for their ability to form capillary tubes. Arrows indicate cellular junctions counted to calculate the angiogenic index. (B) Angiogenic index (number of cellular junctions) in pSF91 and pSF91K15 transduced cells. Significance levels for the indicated comparisons are marked with ‘**’ (p<0.01) (see Material and Methods). (C) Western blot showing the increased expression of both RCAN1.1 and RCAN1.4 in K15 transduced cells in medium with and without VEGF. (D) HUVECs were resuspended in supernatant collected from cells transduced with K15 or vector control for 48 hours and then were plated onto matrigel for 6 hours to score for tube formation. Medium (EBM2+2%FBS) with VEGF (50 ng/ml) was used as a positive control.

Article Snippet: The following TaqMan gene expression assays (Applied Biosystems: #4331182) were used: Hs01120954_m1 (RCAN1); Hs00173626_m1 (VEGFA); Hs99999905_m1 (GAPDH).

Techniques: Transduction, Western Blot, Expressing, Plasmid Preparation, Control, Positive Control

Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

doi: 10.1371/journal.ppat.1002927

Figure Lengend Snippet: HUVECs were transduced with pSF91K15 or the pSF91 vector and 30 hours later siRNAs against RCAN1, RCAN1.1 and RCAN 1.4 were transfected. 36 hours after transfection, cells were plated on matrigel to score for capillary tube formation (A, B) or analysed by Western blot to verify silencing of individual RCAN1 isoforms (C). Statistical significance levels for the comparisons of indicated samples are marked with ‘*’ (p<0.05) and ‘**’ (p<0.01).

Article Snippet: The following TaqMan gene expression assays (Applied Biosystems: #4331182) were used: Hs01120954_m1 (RCAN1); Hs00173626_m1 (VEGFA); Hs99999905_m1 (GAPDH).

Techniques: Transduction, Plasmid Preparation, Transfection, Western Blot

Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

doi: 10.1371/journal.ppat.1002927

Figure Lengend Snippet: (A, B) HUVECs were treated with a Calcineurin inhibitor (Cyclosporin A; 1 µM), or a PLCγ inhibitor (U73122; 20 µM) for 6 hours and were then plated on matrigel in the presence of VEGF-A to score for capillary tube formation (A). Parallel samples were analysed on Western blots to show the phosphorylation of NFAT1 and PLCγ1 and the levels of total PLCγ1, RCAN1.1 and RCAN 1.4 (B). (C, D) HUVECs were transduced with pSF91 or pSF91K15 and after 30 hours were treated with CsA or U73122 and tube formation was analysed as in . Phosphorylation of NFAT1, PLCγ1, and expression of RCAN1 isoforms and K15 was measured by Western blot (D).

Article Snippet: The following TaqMan gene expression assays (Applied Biosystems: #4331182) were used: Hs01120954_m1 (RCAN1); Hs00173626_m1 (VEGFA); Hs99999905_m1 (GAPDH).

Techniques: Western Blot, Phospho-proteomics, Transduction, Expressing

Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

doi: 10.1371/journal.ppat.1002927

Figure Lengend Snippet: HUVECs were transduced with pSF91 or pSF91K15 and (A) changes in the angiogenic index in response to K15 expression and silencing of NFAT1 or Calcineurin were analysed. (B) NFAT1 phosphorylation and expression of NFAT1, Calcineurin, RCAN1 in response to K15 overexpression and silencing of NFAT1 and Calcineurin in the absence of VEGF were analysed by Western blot. (C) Changes in the angiogenic index in response to K15 expression and silencing of PLCγ1 and NFκB p65. (D) Expression of RCAN1, PLCγ1, p65 in response to K15 overexpression and silencing of PLCγ1 or p65 in the absence of VEGF were analysed by Western blot. Transduction with pSF91, pSF91K15 and transfection with siRNA was performed as in .

Article Snippet: The following TaqMan gene expression assays (Applied Biosystems: #4331182) were used: Hs01120954_m1 (RCAN1); Hs00173626_m1 (VEGFA); Hs99999905_m1 (GAPDH).

Techniques: Transduction, Expressing, Phospho-proteomics, Over Expression, Western Blot, Transfection

Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

doi: 10.1371/journal.ppat.1002927

Figure Lengend Snippet: (A) HUVECs were treated with 50 ng/ml VEGF in 12 well plates for the indicated time points and analysed by Western blots to investigate the effects on the phosphorylation of PLCγ1 and expression of RCAN1 isoforms. (B) HUVECs were transduced with a retroviral vector expressing K15 (pSF91K15), plated in 12 well plates for the indicated time points and the phosphorylation of PLCγ1 and expression of RCAN1 isoforms was monitored by Western blot. (C) HUVECs were transduced with pSF91K15 or control vector and the angiogenic index was measured after treating the cells with VEGF at the indicated concentrations and plating them on matrigel.

Article Snippet: The following TaqMan gene expression assays (Applied Biosystems: #4331182) were used: Hs01120954_m1 (RCAN1); Hs00173626_m1 (VEGFA); Hs99999905_m1 (GAPDH).

Techniques: Western Blot, Phospho-proteomics, Expressing, Transduction, Retroviral, Plasmid Preparation, Control

Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

doi: 10.1371/journal.ppat.1002927

Figure Lengend Snippet: (A) Schematic representation of K15 showing the SH2 and SH3 binding sites in the cytoplasmic domain along with corresponding mutants. (B) HUVECs were transduced with retroviral vectors expressing K15 and the indicated mutants of the K15 SH2 and SH3 binding sites or the control vector (pSF91). Thirty hours after transduction cells were lysed and analysed by Western blot for PLCγ1 phosphorylation and expression of K15 and RCAN1 isoforms. (C) Parallel samples from the experiment shown in (B) were plated on matrigel in the absence of VEGF and scored for capillary tube formation as in .

Article Snippet: The following TaqMan gene expression assays (Applied Biosystems: #4331182) were used: Hs01120954_m1 (RCAN1); Hs00173626_m1 (VEGFA); Hs99999905_m1 (GAPDH).

Techniques: Binding Assay, Transduction, Retroviral, Expressing, Control, Plasmid Preparation, Western Blot, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

doi: 10.1371/journal.ppat.1002927

Figure Lengend Snippet: (A–C) Capillary tube formation in HUVECs infected with rKSHV.219. Seventy-two hours after infection of HUVECs, siRNA against K15 or control siRNA was microporated. Twenty-four hours after transfection the lytic cycle was induced with sodium butyrate (Na-Bu) and 10% RTA. Forty-eight hours later, cells were plated on matrigel and scored for tube formation (A). (B) Angiogenic index for the experiment shown in (A). Significance levels for the indicated comparisons of different samples are marked with ‘*’ (p<0.05) and ‘**’ (p<0.01). (C) Western blot analysis for the experiment shown in (A) to confirm the silencing of K15 expression and to quantify the expression of K8.1 (a late lytic glycoprotein), RCAN1.1/1.4, and phosphorylated PLCγ1 and phosphorylated NFAT1. (D–F) HUVEC were infected with BAC-derived KSHVwt or KSHVΔK15 for 72 hours. Following activation of the lytic cycle as in (A–C), cells were plated on matrigel for angiogenic tube formation (D). (E) Angiogenic index for the experiment shown in (D). Significance levels for the indicated comparisons are marked with ‘*’ (p<0.05). (F) Parallel samples were analysed by Western blot for the expression of K15 and K8.1 A/B.

Article Snippet: The following TaqMan gene expression assays (Applied Biosystems: #4331182) were used: Hs01120954_m1 (RCAN1); Hs00173626_m1 (VEGFA); Hs99999905_m1 (GAPDH).

Techniques: Infection, Control, Transfection, Western Blot, Expressing, Derivative Assay, Activation Assay

Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

doi: 10.1371/journal.ppat.1002927

Figure Lengend Snippet: As shown in this report, K15 directly recruits PLCγ1 and induces its phosphorylation by an unidentified kinase. The PLCγ1 dependent production of IP3 leads to increased calcium influx, Calcineurin activity, NFAT dephosphorylation and ultimately increased expression of NFAT-dependent genes, including RCAN1.1/1.4 and angiogenesis. CsA: Cyclosorin A, U73122: PLCγ1 inhibitor.

Article Snippet: The following TaqMan gene expression assays (Applied Biosystems: #4331182) were used: Hs01120954_m1 (RCAN1); Hs00173626_m1 (VEGFA); Hs99999905_m1 (GAPDH).

Techniques: Phospho-proteomics, Activity Assay, De-Phosphorylation Assay, Expressing