Journal: Oncotarget

Article Title: In vitro studies implicate an imbalanced activation of dendritic cells in the pathogenesis of murine autoimmune pancreatitis

doi: 10.18632/oncotarget.10265

Figure Lengend Snippet: BM cells of the indicated mouse cohorts were cultured for 9 days with GM-CSF (20 ng/ml) to generate BM-derived cDCs. Afterwards, the cells were exposed to LPS (1 μg/ml; right columns) or left unstimulated (left columns). The mRNA expression of A. clec4a2 , B. trem2 , C. cytip and the housekeeping gene hprt was analyzed by real-time PCR, and relative amounts of target mRNA were calculated as described in the Methods section. Data of n ≥ 6 independent cultures from different mice were used to calculate mean ± SEM. * P < 0.05 versus cDCs of the same experimental group cultured without LPS; # P < 0.05 versus identically cultured cDCs of CAST/EiJ mice. There were no significant differences between the mRNA levels in cDCs of young and adult MRL/MpJ mice for any gene and treatment protocol.

Article Snippet: The following assays were employed: Mm00454809_m1 ( cytip ), Mm04209424_g1 ( trem2 ), Mm00488795_m1 ( clec4a2 ), and Mm01545399_m1 ( hypoxanthine guanine phosphoribosyl transferase; hprt ; house-keeping gene control).

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

Journal: Oncotarget

Article Title: C1orf64 is a novel androgen receptor target gene and coregulator that interacts with 14-3-3 protein in breast cancer

doi: 10.18632/oncotarget.17826

Figure Lengend Snippet: (A) qRT-PCR to demonstrate C1orf64 silencing efficiencies with siRNA-D1 (D1) and siRNA-D2 (D2) in T-47D and MFM-223 cell lines. C1orf64 expression is relative to non-targeting siRNA (CTL). *, p< 0.01 is for D1 or D2 vs. CTL. Error Bars: ± 2SEM. (B) qRT-PCR to show the effect of C1orf64 silencing using siRNA-D1 (D1) and siRNA-D2 (D2) on PIP expression in T-47D and MFM-223 cells. *, p< 0.01 is for D1 or D2 vs. CTL. (C) qRT-PCR to assess the effect of C1orf64 silencing on the DHT-mediated induction of PIP expression in T-47D cells. CTL: CTL-siRNA + solvent control, CTL-DHT: CTL-siRNA + DHT, D1-DHT: siRNA-D1 + DHT, D2-DHT: siRNA-D2 + DHT. *, p< 0.01 is for CTL vs. CTL-DHT and **, p< 0.03 is for CTL-DHT vs. D1-DHT or D2-DHT. (D) qRT-PCR to examine the effect of C1orf64 silencing on the DHT-mediated induction of PIP expression in MFM-223 cells as outlined in (C). (E) immunoblotting to assess C1orf64 protein levels in T-47D and MFM-233 cell lines following C1orf64 overexpression. Fold change (RR) in each band density was measured relative to control in three replicate experiments. CT: control vector, OE: overexpression. (F) qRT-PCR to assess the effect of C1orf64 overexpression on PIP expression in T-47D and MFM-223 cells. CT-VEC: Control vector, C1orf64+: C1orf64 overexpression. *, p≤ 0.01 is for CT-VEC vs. C1orf64+.

Article Snippet: The gene expression levels were assessed using qRT-PCR with the following Taqman Gene Expression Assays (Life Technologies) as instructed by the manufacturer: C1orf64 (assay ID: Hs00698851_m1), SIDT1 (assay ID: Hs00214475_m1), RHOH (assay ID: Hs01877256_s1), TFAP2B (assay ID: Hs01560931_m1), SPDEF (assay ID: Hs01026050_m1), PIP (assay ID: Hs01114172_m1), and PGR (assay ID: Hs01556702_m1).

Techniques: Quantitative RT-PCR, Expressing, Solvent, Control, Western Blot, Over Expression, Plasmid Preparation

Journal: Oncotarget

Article Title: C1orf64 is a novel androgen receptor target gene and coregulator that interacts with 14-3-3 protein in breast cancer

doi: 10.18632/oncotarget.17826

Figure Lengend Snippet: (A) Fold enrichments of ChIP for PIP promoter performed with an AR antibody (AR-Ab) following the transfections of control (CT)-siRNA or C1orf64-siRNA (Duplex 1) in T-47D cells. AR binding to each promoter region (A1-A4) was assessed. Amplification of 1% of input chromatin was used as the input control and a non-specific antibody served as negative control (dashed-line). *, p< 0.03 is for C1orf64-siRNA vs. CT-siRNA in A1 and A2. Error Bars: ± 2SEM. (B) Co-immunprecipitation assays (Co-IP) to investigate the interaction between endogenous C1orf64 and AR in T-47D and MFM-223 cell lines. AR-IP was performed using an AR antibody and control experiments were conducted with a non-specific rabbit IgG. Immunoblot analysis were carried out on IP supernatants using C1orf64 and AR antibodies. Input was assessed with AR immunoblotting on 5% of lysates collected before each IP. All co-IP experiments were performed in three replicates.

Article Snippet: The gene expression levels were assessed using qRT-PCR with the following Taqman Gene Expression Assays (Life Technologies) as instructed by the manufacturer: C1orf64 (assay ID: Hs00698851_m1), SIDT1 (assay ID: Hs00214475_m1), RHOH (assay ID: Hs01877256_s1), TFAP2B (assay ID: Hs01560931_m1), SPDEF (assay ID: Hs01026050_m1), PIP (assay ID: Hs01114172_m1), and PGR (assay ID: Hs01556702_m1).

Techniques: Transfection, Control, Binding Assay, Amplification, Negative Control, Co-Immunoprecipitation Assay, Western Blot

Journal: Oncotarget

Article Title: C1orf64 is a novel androgen receptor target gene and coregulator that interacts with 14-3-3 protein in breast cancer

doi: 10.18632/oncotarget.17826

Figure Lengend Snippet: This model shows a negative interplay between AR and C1orf64. In this process, AR activation represses C1orf64 transcription and C1orf64, in turn, acts as a corepressor of AR and negatively regulates the AR-mediated induction of PIP expression and AR reporter activity. Chaperone protein 14-3-3 interacts with C1orf64 and has a regulatory function in this model. The other aspect of this interplay involves a cross-talk between the AR and ER signaling in which, AR activation has a repressive effect on the ER-mediated induction of PGR. In addition, C1orf64 is necessary for PGR expression and its repression by AR has an inhibitory effect on the positive regulation of ER activity by C1orf64. Red arrows denote a stimulatory effect and black lines indicate a repressive function.

Article Snippet: The gene expression levels were assessed using qRT-PCR with the following Taqman Gene Expression Assays (Life Technologies) as instructed by the manufacturer: C1orf64 (assay ID: Hs00698851_m1), SIDT1 (assay ID: Hs00214475_m1), RHOH (assay ID: Hs01877256_s1), TFAP2B (assay ID: Hs01560931_m1), SPDEF (assay ID: Hs01026050_m1), PIP (assay ID: Hs01114172_m1), and PGR (assay ID: Hs01556702_m1).

Techniques: Activation Assay, Expressing, Activity Assay

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: Fold changes of top ranking molecular apocrine-signature genes in two studies.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: The regulation of molecular apocrine genes by the AR-ERK feedback loop . (A) Heat map of top ranking molecular apocrine-signature genes following the inhibition of AR-ERK signaling using qPCR data. Heat map shows fold changes for gene expression relative to control in MDA-MB-453 and HCC-1954 cell lines. Treatments were carried out by CI-1040 (CI) at 2 µM and 10 µM concentrations, flutamide (FLU) at 25 nM and 40 nM concentrations, and the combination of flutamide at 25 nM or 40 nM and CI-1040 at 2 µM concentrations. Red and green colors depict up-regulation and down-regulation, respectively. Bar indicates the range of fold changes in gene expression. (B) Box plots to demonstrate relative expression of PIP to control following AR-ERK inhibition in MDA-MB-453 and HCC-1954 cell lines using qPCR. CTL: control. (C) Western blot analysis to assess PIP protein levels following AR-ERK inhibition in MDA-MB-453 and HCC-1954 cell lines. Fold changes (RR) in band densities were measured relative to the control (CTL). AR, androgen receptor; ERK, extracellular signal-regulated kinase; qPCR, quantitative PCR; RR, relative risk.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques: Inhibition, Gene Expression, Control, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: Fold changes of molecular apocrine-signature genes following treatment with AR and MEK inhibitors.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: PIP protein expression in primary breast tumors and in vivo models . (A) Immunohistochemistry (IHC) staining for PIP in ER negative (ER-) breast tumors. AR+ group: ≥20% of cells showing positive AR staining; AR- group: <20% of cells stained for AR. Percentage of cells with positive staining are demonstrated for each group. * P <0.01 is for AR+ versus AR-. Error Bars: ± 2SEM. (B) IHC staining for PIP in AR+ and AR- breast tumors. Magnification is at 60X. (C) IHC staining for PIP in xenograft tumors generated using MDA-MB-453 cell line. Control: a control tumor; FLU: a flutamide-treated tumor; PD: a PD0325901-treated tumor. Magnification is at 60X. (D) IHC for PIP in xenograft tumors. Percentage of cells positive for PIP was assessed using IHC and compared between each treatment group and control (CTL). * P <0.01 is for FLU or PD treatment versus CTL. Error Bars: ± 2SEM. AR, androgen receptor; SEM, standard error of the mean.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques: Expressing, In Vivo, Immunohistochemistry, Staining, Generated, Control

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: Transcriptional regulation of PIP by AR and CREB1 . (A) Luciferase reporter assay. The transcriptional activation of PIP promoter by PRLR, AR, CREB1, PRLR + AR, and PRLR + CREB1 expression constructs was assessed using Dual-Luciferase assays in MCF-7 cells and relative response ratios are reported. Co-transfection with the PIP reporter vector and an empty pcDNA vector was used as a control (CTL). * P <0.01, is compared to the control group. (B) Induction of PIP expression following DHT treatment. PIP expression was assessed using qPCR following DHT treatment at 30 minute, 1 hour, 3 hour, 12 hour, 24 hour, and 48 hour time-points in MDA-MB-453 and HCC-1954 cell lines. Fold changes are measured relative to the respective control at each time point. * P <0.03, is compared to the control group (dashed line). Error Bars: ± 2SEM. (C) Putative transcription factor binding sites for CREB1 in 1.5 kb promoter region of PIP. P1 (primer set 1) and P2 (primer set 2) are regions of amplification for ChIP assays. (D) ChIP assay with CREB1 antibody. The results of qPCR amplification for ChIP assays are demonstrated with two sets of primers for PIP promoter. Percentage recovery of input chromatin is shown for each primer set. *, P <0.01 is for CREB1 Ab. versus control Ab. Error Bars: ± 2SEM. (E) Western blot analysis to show CREB1 and PIP protein levels following CREB1-knockdown using siRNA in MDA-MB-453 cell line. Fold changes (RR) in band densities were measured relative to non-targeting siRNA control (CTL). Ab, antibody; AR, androgen receptor; ChIP, chromatin immunoprecipitation; DHT, dihydrotestosterone; qPCR, quantitative PCR; RR, relative risk; SEM, standard error of the mean.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques: Luciferase, Reporter Assay, Activation Assay, Expressing, Construct, Cotransfection, Plasmid Preparation, Control, Binding Assay, Amplification, Western Blot, Knockdown, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: The effect of PIP knockdown on cell invasion and viability . (A) qPCR to demonstrate PIP-knockdown efficiencies with siRNA-duplex1 (D1) and siRNA-duplex2 (D2) in MDA-MB-453 cell line. PIP expression following knockdown was assessed relative to non-targeting siRNA control (CTL) and fold change is shown for each duplex. (B) Western blot analysis to show PIP protein level following PIP-knockdown in MDA-MB-453 cell line as described in (A). Fold changes (RR) in band densities were measured relative to the control (CTL). (C) The effect of PIP expression on cell invasion. Cell invasion assays were carried out after PIP-knockdown with PIP-D1 and PIP-D2 in MDA-MB-453 cell line. Transfection with non-targeting siRNA control (CTL) was used as a control. *, P <0.03 is for each PIP-knockdown versus CTL. Error Bars: ± 2SEM. (D) MTT assay to measure cell viability following PIP-knockdown with PIP-D1 and PIP-D2 in MDA-MB-453 cell line. CTL: non-targeting siRNA control. *, P <0.03 is for each PIP-knockdown versus CTL. Error Bars: ± 2SEM. MTT, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide; qPCR, quantitative PCR; RR, relative risk; SEM, standard error of the mean.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques: Knockdown, Expressing, Control, Western Blot, Transfection, MTT Assay, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: The effect of PIP knockdown on ERK-Akt and integrin-β1signaling . (A) Western blot analysis to measure the levels of phosphorylated (Ph)-ERK, total (T)-ERK, ph-Akt, and T-Akt following PIP-knockdown with siRNA duplex1 (PIP-D1) and duplex2 (PIP-D2) in MDA-MB-453 cell line. Fold changes of Phospho/Total ratios (Ph/T-RR) were assessed relative to non-targeting siRNA control (CTL). (B) Western blot analysis to measure the level of ph-CREB1, T-CREB1, and ILK1 following PIP-knockdown as described in (A). Ph-ATF1 is the phosphorylated form of CREB-related protein that is known to be detected by this antibody. (C) Integrin-β1 immunoprecipitation (IP). IP assays were carried out with Integrin-β1 following PIP-knockdown with PIP-D1 and PIP-D2 in MDA-MB-453 cell line. Non-targeting siRNA was used as control (CTL). Western blot analysis was carried out on IP samples to measure the integrin-β1 binding to ILK1 and ErbB2. Immunoblotting with integrin-β1 antibody was used as a loading control. Fold changes (RR) of ILK1 and ErbB2 following PIP-knockdown were measured relative to that of control-siRNA. (D) Integrin-β1 immunoprecipitation following PIP-knockdown and the addition of fibronectin fragments (Fn-fs). PIP-knockdown with PIP-D1 was carried out as described in (C). Twenty-four hours after PIP-knockdown, cells were treated with α-chymotryptic fibronectin fragment 120K at 100 µg/ml concentration. Control cells were treated with vehicle only. Fold changes (RR) of ILK1 and ErbB2 following PIP-knockdown + Fn-fs were measured relative to the control. (E) The effect of fibronectin fragments on cell invasion following PIP-knockdown. Cell invasion assays were carried out after PIP-knockdown with PIP-D1 in MDA-MB-453 cell line. Transfection with non-targeting siRNA control (CTL) was used as a control. Treatment with fibronectin fragments was carried out as described in (D). Error Bars: ± 2SEM. Δ; is the difference between CTL and PIP-D1+Fn-fs groups. ERK, extracellular signal-regulated kinase; ILK1, integrin-linked kinase 1; RR, relative risk; SEM, standard error of the mean.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques: Knockdown, Western Blot, Control, Immunoprecipitation, Binding Assay, Concentration Assay, Transfection

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: Schematic diagram of the PIP signaling pathway in ER-negative breast cancer . Red arrow denotes stimulatory effect. ER, estrogen receptor; Fn, fibronectin; Fn-f, Fibronectin fragment; ITG-β1, integrin-β1.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques:

Journal: Viruses

Article Title: Ephrin B1 and B2 Mediate Cedar Virus Entry into Egyptian Fruit Bat Cells

doi: 10.3390/v17040573

Figure Lengend Snippet: The shRNA oligos used for knockdown.

Article Snippet: The antibodies used in this study include the following: anti-β-Actin (66009-1-Ig, Proteintech, Rosemont, IL, USA); anti-DYKDDDDK tag (20543-1-AP, rabbit polyclonal, Proteintech, USA); anti-DYKDDDDK tag (66008-4-Ig, mouse IgG2b, Proteintech, USA); Alexa Fluor 488 conjugated anti-mouse-IgG (AB_2534069, Invitrogen, Carlsbad, CA, USA); BV-421 conjugated anti-mouse-IgG (Poly4053, BioLegend, San Diego, CA, USA); and anti-ephrin B2 antibody (26533-1-AP, Proteintech, USA).

Techniques: shRNA, Knockdown, Sequencing

Journal: Viruses

Article Title: Ephrin B1 and B2 Mediate Cedar Virus Entry into Egyptian Fruit Bat Cells

doi: 10.3390/v17040573

Figure Lengend Snippet: (q)PCR primers.

Article Snippet: The antibodies used in this study include the following: anti-β-Actin (66009-1-Ig, Proteintech, Rosemont, IL, USA); anti-DYKDDDDK tag (20543-1-AP, rabbit polyclonal, Proteintech, USA); anti-DYKDDDDK tag (66008-4-Ig, mouse IgG2b, Proteintech, USA); Alexa Fluor 488 conjugated anti-mouse-IgG (AB_2534069, Invitrogen, Carlsbad, CA, USA); BV-421 conjugated anti-mouse-IgG (Poly4053, BioLegend, San Diego, CA, USA); and anti-ephrin B2 antibody (26533-1-AP, Proteintech, USA).

Techniques: Sequencing, Amplification

Journal: Viruses

Article Title: Ephrin B1 and B2 Mediate Cedar Virus Entry into Egyptian Fruit Bat Cells

doi: 10.3390/v17040573

Figure Lengend Snippet: The expression of ERB ephrin B1 or B2 mediates the entry of Cedar pseudotyped particles. ( A ) Scheme of pseudovirus generation. For the generation of pseudotyped particles, HEK293T cells were transfected with the transfer plasmid (pHAGE-ZsGreen-luciferase), packaging plasmid (psPAX2) together with CedV/NiV G and F. The supernatant containing the pseudotyped particles was collected at 24 h post transfection and centrifuged, and the supernatants were then directly used for infection of CHO-K1 transiently expressing ERB ephrins. The images were created with Biorender.com. ( B , D ) Optimization of CedVpp generation by transfection of different ratios of G/F plasmids as indicated ( B ), or in different amounts ( C ) or in the presence of M and/or N plasmids ( D ). Bars show the mean ±SEM. The pseudovirus particles were added onto BHK21 cells to examine the yield under different conditions. ( E , F ) Entry of pseudotyped particles expressing NiV G/F ( E ) or CedV G/F ( F ) into CHO-K1 cells transfected with plasmids encoding for ERB ephrins. Y -axis shows the fold change in luciferase induction normalized to negative control (vector ctrl.). Bars show the mean ± SEM. Statistical analysis was performed with one-way ANOVA. p < 0.05 (*), p < 0.01 and p < 0.0001 (****).

Article Snippet: The antibodies used in this study include the following: anti-β-Actin (66009-1-Ig, Proteintech, Rosemont, IL, USA); anti-DYKDDDDK tag (20543-1-AP, rabbit polyclonal, Proteintech, USA); anti-DYKDDDDK tag (66008-4-Ig, mouse IgG2b, Proteintech, USA); Alexa Fluor 488 conjugated anti-mouse-IgG (AB_2534069, Invitrogen, Carlsbad, CA, USA); BV-421 conjugated anti-mouse-IgG (Poly4053, BioLegend, San Diego, CA, USA); and anti-ephrin B2 antibody (26533-1-AP, Proteintech, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Infection, Negative Control

Journal: Viruses

Article Title: Ephrin B1 and B2 Mediate Cedar Virus Entry into Egyptian Fruit Bat Cells

doi: 10.3390/v17040573

Figure Lengend Snippet: rCedV-nTurbo635 utilizes ERB ephrins B1, B2 and A5 for cell entry. ( A ) CHO-K1 cells expressing FLAG-tagged ephrins were stained with an Alexa488 fluorophore to monitor their expression. rCedV-nTurbo635 was visualized in the Cy3 channel. Representative pictures were taken from three independent experiments. Images were processed in ImageJ/Fiji. Scale bar: 100 μm. ( B , C ) High-content microscopy ( B ) and flow cytometry ( C ) analysis of rCedV-nTurbo635 entry. ( B ) CHO-K1 cells stably expressing ERB ephrins were grown in 96-well plates, re-transfected with plasmids encoding for A ephrins and infected with rCedV-nTurbo635 at MOI 1, 2 and 5. At 24 h post infection (p.i.), high-content imaging was employed to quantify the infection rate. Graph shows the median and range from three independent experiments in technical duplicates. Statistical test: Kruskal–Wallis without Dunn’s correction. ( C ) CHO-K1 cells stably expressing ERB ephrins were re-transfected with their respective ephrins and infected with rCedV-nTurbo635 (MOI 5) at 24 h post transfection. Graph shows the infection rate of the ephrin positive population 48 h p.i. ( D ) Quantification of FLAG-tagged ephrins by flow cytometry. Data show the mean ±SD from four independent experiments capturing 30,000 events each. ( B , C ) p < 0.05 (*) and p < 0.001 (***). Statistical test: Kruskal–Wallis without Dunn’s correction.

Article Snippet: The antibodies used in this study include the following: anti-β-Actin (66009-1-Ig, Proteintech, Rosemont, IL, USA); anti-DYKDDDDK tag (20543-1-AP, rabbit polyclonal, Proteintech, USA); anti-DYKDDDDK tag (66008-4-Ig, mouse IgG2b, Proteintech, USA); Alexa Fluor 488 conjugated anti-mouse-IgG (AB_2534069, Invitrogen, Carlsbad, CA, USA); BV-421 conjugated anti-mouse-IgG (Poly4053, BioLegend, San Diego, CA, USA); and anti-ephrin B2 antibody (26533-1-AP, Proteintech, USA).

Techniques: Expressing, Staining, Microscopy, Flow Cytometry, Stable Transfection, Transfection, Infection, Imaging

Journal: Viruses

Article Title: Ephrin B1 and B2 Mediate Cedar Virus Entry into Egyptian Fruit Bat Cells

doi: 10.3390/v17040573

Figure Lengend Snippet: Ephrin B2 knockdown significantly impairs rCedV-nTurbo635 entry into ERB cells. ( A ) qPCR analysis of the ephrin expression in RaNep cells. The expression levels were normalized to the level of housekeeping gene EEF1A1. The data represent the mean ±SEM from three independent experiments. ( B , C ) qPCR analysis of the ephrin B2 ( B ) and ephrin B1 ( C ) expression in RaNep knockdown cells. The relative expression levels were normalized to the respective EFN expression level in the scrambled shRNA controls. The data show the mean± SD from at least three independent experiments. Statistical test: Kruskal–Wallis without Dunn’s correction; p < 0.05 (*) and p < 0.001 (***). ( D ) Western blot analysis of the ephrin B2 expression in RaNep knockdown cells. Representative Images were acquired from three independent experiments. ( E ) rCedV-nTurbo635 entry into RaNep cells with the knockdown of ephrin B2 or ephrin B1/B2. The ephrin knockdown cells were infected with rCedV-nTurbo635 at MOI = 1. Cell were then fixed at 48 h p.i. for flow cytometry analysis. Statistical test: one-way ANOVA corrected for multiple comparisons (Dunnett’s correction); p < 0.01 (**) and p > 0.05 (ns). ( F ) Fluorescent microscopy analysis of the rCedV-nTurbo635 entry in the RaNep scrambled shRNA control cells and the ephrin B2 and the ephrin B1/B2 KD cells. Representative Images were taken from two independent experiments. Scale bar: 100 μm.

Article Snippet: The antibodies used in this study include the following: anti-β-Actin (66009-1-Ig, Proteintech, Rosemont, IL, USA); anti-DYKDDDDK tag (20543-1-AP, rabbit polyclonal, Proteintech, USA); anti-DYKDDDDK tag (66008-4-Ig, mouse IgG2b, Proteintech, USA); Alexa Fluor 488 conjugated anti-mouse-IgG (AB_2534069, Invitrogen, Carlsbad, CA, USA); BV-421 conjugated anti-mouse-IgG (Poly4053, BioLegend, San Diego, CA, USA); and anti-ephrin B2 antibody (26533-1-AP, Proteintech, USA).

Techniques: Knockdown, Expressing, shRNA, Western Blot, Infection, Flow Cytometry, Microscopy, Control

Journal: Frontiers in Immunology

Article Title: Phosphatase of Regenerating Liver-1 (PRL-1) Regulates Actin Dynamics During Immunological Synapse Assembly and T Cell Effector Function

doi: 10.3389/fimmu.2018.02655

Figure Lengend Snippet: Expression of PRLs in mature CD4 T cells. (A) The gene expression of PRLs and other PTPs in peripheral blood CD4 T cells from n = 3 donors was analyzed by qPCR. The mean value of the ΔCT and the standard deviation (SD) for each gene is shown. Data of PRLs were compared by a one-way ANOVA. Asterisks indicate the p -value: * P ≤ 0.05, *** P ≤ 0.001. (B) Western Blot for PRL-1 and PRL-2 detection in the CD4 T cell line Jurkat (JK), in peripheral blood CD4 T cells (CD4) and in the Hela cell line. The amount of protein loaded is indicated. Numbers under the PRL-1/PRL-2 blot indicate the normalized densitometry of PRL-1 vs. PRL-2. The molecular weight (MW) markers are indicated. One representative experiment is shown. (C) Expression of PTP4A1 and PTP4A2 mRNA in Th1 effectors upon stimulation with PMA and Ionomycin for the indicated times in minutes (min). Graphs represent the relative expression (RQ) with respect to time cero ( t = 0). The mean ± SD is shown of RQ values from n = 4 different donors. Asterisks indicate the p -value of a one-sample t -test comparing each time to t = 0. Hashes indicate the p -value of a t -test comparing PTP4A1 and PTP4A2 expression at each time. * and # P ≤ 0.05, ** and ## P ≤ 0.01. (D) Western blot for PRL-1 and PRL-2 (upper left panel) and GAPDH (lower left panel) detection. The MW markers are indicated. Right panel shows the PRL-1/PRL-2 ratio. PI indicates PMA and Ionomycin stimulation. The graph shows the mean ± SD obtained from n = 4 donors analyzed. The mean of the sample was compared by a paired t -test. The asterisk indicates the p -value: * P ≤ 0.05.

Article Snippet: Taqman probes used were Hs00743856_s1 (PTP4A1), Hs00754750_s1 (PTP4A2), Hs02341135_m1 (PTP4A3), Hs00959886_g1 ( PTPN2 ), Hs00169359_m1 ( PTPN6 ), Hs00978680_m1 ( PTPN7 ), Hs04189704_m1 ( PTPRC ), Hs00934033_m1 (CD69), and Hs00272002_m1 ( GNB2L1 , housekeeping gene).

Techniques: Expressing, Gene Expression, Standard Deviation, Western Blot, Molecular Weight

Journal: Frontiers in Immunology

Article Title: Phosphatase of Regenerating Liver-1 (PRL-1) Regulates Actin Dynamics During Immunological Synapse Assembly and T Cell Effector Function

doi: 10.3389/fimmu.2018.02655

Figure Lengend Snippet: Distribution of endogenous PRL-1 at the IS. Representative images are shown of immunofluorescence of CD4 T cells interacting for up to 20 min (see material and methods) with microspheres coated with anti-CD3ε and anti-CD28 antibodies (IS-like interactions) or IgG1 as negative control for stimulation. Images represent confocal sections in the green and red channels, as well as, the merged of channels and the transmission light (TL). Calibration bar is shown in pseudocolored images. Scale bars 5 μm. (A) Examples of conjugates showing or not accumulation of PRL-1 and CD3ζ at the IS. The right panel represent the correlation of PRL-1 and CD3ζ accumulation at the IS. Dots represent individual conjugates. The p -value of the Pearson coefficient is shown. (B) Examples of conjugates showing or not accumulation of PRL-1 and the MTOC at the IS. The right panel represents the quantification of PRL-1 accumulation to the IS in relation to MTOC polarization. Dots represent individual conjugates. Groups were compared by a one-way ANOVA. Asterisks indicate the p -value: * P ≤ 0.05, ** P ≤ 0.01. (C) Two confocal sections are shown of a cell forming an IS-like interaction and one confocal section of a non-stimulated cell. Right panels represent profiles of the fluorescence intensity in the green and the red channel along lines drawn in images. Numbers indicate the correspondence between the cell and the profile. ( A – C) Data obtained from 2 experiments done with 2 different donors.

Article Snippet: Taqman probes used were Hs00743856_s1 (PTP4A1), Hs00754750_s1 (PTP4A2), Hs02341135_m1 (PTP4A3), Hs00959886_g1 ( PTPN2 ), Hs00169359_m1 ( PTPN6 ), Hs00978680_m1 ( PTPN7 ), Hs04189704_m1 ( PTPRC ), Hs00934033_m1 (CD69), and Hs00272002_m1 ( GNB2L1 , housekeeping gene).

Techniques: Immunofluorescence, Negative Control, Transmission Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: Phosphatase of Regenerating Liver-1 (PRL-1) Regulates Actin Dynamics During Immunological Synapse Assembly and T Cell Effector Function

doi: 10.3389/fimmu.2018.02655

Figure Lengend Snippet: Subcellular distribution of PRL-1 in JK cells. (A) Representative cells of Immunofluorescence experiments of JK cells overexpressing GFP-PRL-1 and stained for the pericentriolar material-1 (PCM1) or CD71. The upper and right panel represents the profile of the fluorescence intensities in the green and the red channel along the line drawn in the PRL-1/PCM1 merged image. Lower and right panel showed the Pearson's coefficient for quantifying the PRL-1 co-localization with CD71 or PCM1. Spots represent cells analyzed from n = 3 experiments. (B) Representative immunofluorescence of cell conjugates formed by JK cells overexpressing GFP-PRL-1 or GFP alone and RAJI cells labeled with CMAC (blue) and loaded with SEE. The right panel represents the quantification of the accumulation of GFP-PRL-1 or GFP alone at the IS. Dots represent individual conjugates obtained from n = 2 experiments. Groups were compared by a t -test. Asterisks indicate the p -value: *** p ≤ 0.001 (C) Immunofluorescence of cell conjugates formed by JK cells overexpressing mCit-PRL-1 or YFP alone and conjugated with RAJI cells loaded with SEE and labeled with CMAC (blue). The IS markers are shown in red. mCit-PRL-1 and YFP are shown as a pseudocolor image with the calibration bar. Co-localization is shown in a pixel map (pm) obtained at the interaction site. White pixels indicate co-localization sites. Scatter plots of green and red channels along the stack are shown. Numbers indicate Manders coefficients (MC). The interface surface obtained from a 3D reconstruction of the IS where co-localization was analyzed is shown. Scale bars 10 μm. Lower graph: Quantification of the co-localization by Pearson coefficients (R). Dots represent individual cell conjugates obtained from n = 2 (LFA-1 vs. PRL-1), n = 7 (CD3 vs. PRL-1) or n = 5 (CD3 vs. GFP) experiments. The different samples were compared by a t -test. Asterisks represent the p -values: ** P ≤ 0.01, **** P ≤ 0.0001.

Article Snippet: Taqman probes used were Hs00743856_s1 (PTP4A1), Hs00754750_s1 (PTP4A2), Hs02341135_m1 (PTP4A3), Hs00959886_g1 ( PTPN2 ), Hs00169359_m1 ( PTPN6 ), Hs00978680_m1 ( PTPN7 ), Hs04189704_m1 ( PTPRC ), Hs00934033_m1 (CD69), and Hs00272002_m1 ( GNB2L1 , housekeeping gene).

Techniques: Immunofluorescence, Staining, Fluorescence, Labeling

Journal: Frontiers in Immunology

Article Title: Phosphatase of Regenerating Liver-1 (PRL-1) Regulates Actin Dynamics During Immunological Synapse Assembly and T Cell Effector Function

doi: 10.3389/fimmu.2018.02655

Figure Lengend Snippet: Dynamic delivery of PRL-1 to the IS. (A) Right panel: Frames of a representative time-lapse confocal microscopy experiment from 13 conjugates ( n = 5 experiments) showing accumulation of GFP-PRL-1 at the IS. It is shown the distribution of GFP-PRL-1 in the pseudocolor image and in the merged image of channels (green signal). The red signal in merged images corresponds to CD3ζ-mCherry. The Raji cell is labeled with CMAC (blue). Time in seconds (s) is indicated. White arrows indicate transient accumulation of GFP-PRL1 in scanning membranes. Yellow arrowheads indicate the pericentriolar location of GFP-PRL-1. Scale bars 5 μm. The right graph represents the quantification of the accumulation of GFP-PRL-1 in scanning membranes along the time of complete (from the beginning) interactions. It is represented the mean and the SD obtained in 6 conjugates. (B) A merged image of the red (CD3ζ-mCherry) and the green (GFP-PRL-1) channel is shown of the representative time-lapse TIRFM experiment presented in Video 3. Lower and left graph represents the profile of the fluorescence intensity of the green and the red channel on the line that would cross the numbered sites. A magnified area of the region pointed by a square is shown at different times. White arrows point a CD3ζ-mCherry-containing vesicle in which GFP-PRL-1 arrives. Scale bar 2 μm. Intensity profiles show the correlation of the green and the red intensities at sites pointed by the arrows displayed in magnified areas. The right panel shows the Pearson's coefficient for the signal correlation of CD3ζ-mCherry and GFP-PRL-1 in vesicles arrived at the interface. Spots represent areas containing or not vesicles obtained from 5 cells ( n = 3 experiments). Samples Were Compared by a T-test **** P < 0.0001. (C) Green (pseudocolor) and red channels, as well as, the merged image of a frame of the time lapse TIRFM experiment presented in Video 5. (B,C) A total of 11 cells were tracked in n = 3 experiments. Time in minutes:seconds is indicated.

Article Snippet: Taqman probes used were Hs00743856_s1 (PTP4A1), Hs00754750_s1 (PTP4A2), Hs02341135_m1 (PTP4A3), Hs00959886_g1 ( PTPN2 ), Hs00169359_m1 ( PTPN6 ), Hs00978680_m1 ( PTPN7 ), Hs04189704_m1 ( PTPRC ), Hs00934033_m1 (CD69), and Hs00272002_m1 ( GNB2L1 , housekeeping gene).

Techniques: Confocal Microscopy, Labeling, Fluorescence

Journal: Frontiers in Immunology

Article Title: Phosphatase of Regenerating Liver-1 (PRL-1) Regulates Actin Dynamics During Immunological Synapse Assembly and T Cell Effector Function

doi: 10.3389/fimmu.2018.02655

Figure Lengend Snippet: PRL-1 regulates actin dynamics at the IS. (A) Green (pseudocolor) and red channels and merged image of three frames of the time-lapse TIRFM experiment shown in Video 6. Scale bars 10 μm. Time in minutes:seconds is shown. A kymograph of the green and the red channel obtained from the line drawn in the merged image is shown. The normalized intensity profile is shown of the green and red channel in the square labeled in the pseudocolor image. The time in seconds (s) between the maximal intensity in both channels is indicated. A representative cell is shown of 5 cells tracked (B) Pictures show the mCherry-β-actin (pseudocolor) in frames and kymograph of representative time-lapse TIRFM experiments of cells also expressing GFP-PRL-1, GFP-PRL-1-ΔCAAX, or GFP alone. Kymographs were obtained in lines shown in the frame. Calibration bar is indicated. Dots in lower graphs represent individual cells adhered to the activating surface obtained from n = 6 experiments. Different samples were compared by the one-way ANOVA. Asterisks represent the p -value: ** P ≤ 0.01, **** P ≤ 0.0001 (C) Immunofluorescence of IS-like structures stained for F-actin. The green (pseudocolor) and the transmission light (TL) are shown. The PB3 treatment (25 μM) and the control (vehicle) are indicated. Scale bars 5 μm. Quantification of the F-actin accumulation at the interface is shown in the right panel. Dots in the graph represent individual cell/bead conjugates analyzed in n = 4 experiments. Samples were compared by a t -test. Asterisks represent the p -value: *** P ≤ 0.001.

Article Snippet: Taqman probes used were Hs00743856_s1 (PTP4A1), Hs00754750_s1 (PTP4A2), Hs02341135_m1 (PTP4A3), Hs00959886_g1 ( PTPN2 ), Hs00169359_m1 ( PTPN6 ), Hs00978680_m1 ( PTPN7 ), Hs04189704_m1 ( PTPRC ), Hs00934033_m1 (CD69), and Hs00272002_m1 ( GNB2L1 , housekeeping gene).

Techniques: Labeling, Expressing, Immunofluorescence, Staining, Transmission Assay, Control

Journal: Frontiers in Immunology

Article Title: Phosphatase of Regenerating Liver-1 (PRL-1) Regulates Actin Dynamics During Immunological Synapse Assembly and T Cell Effector Function

doi: 10.3389/fimmu.2018.02655

Figure Lengend Snippet: PRL-1 regulates IL-2 secretion. (A) IL-2 secretion assessed by ELISA in JK cells overexpressing either mCit-PRL-1 or YFP alone and stimulated for 16 h with Raji cells loaded with SEE. Samples in each SEE concentration were compared by a paired t -tests ( n = 3 experiments). (B) PRL-1 and PRL-2 protein expression in JK cells transfected with a pool of siRNAs targeting PRL-1 or with a pool of non-targeting siRNAs (NT) as control. A lysate of the breast cancer derived cell line MCF7 was included in the WB experiment represented. (C) IL-2 secretion assessed by ELISA in JK cells transfected with the PRL-1 siRNA pool or the NT. The mean ± the SEM of n = 3 experiments is shown. Samples at different time points were compared by a t -test. The asterisks represent the p -value: * P ≤ 0.05. (D) IL-2 secretion assessed by ELISA in peripheral blood CD4 T cells treated or not with PB3 or the vehicle and stimulated for 6 h with antiCD3ε/CD28-coated beads. Concentrations of PB3 are indicated. Results were normalized to control (Vehicle) and the mean ± SD is shown of the data obtained with n = 4 experiments. Samples in each PB3 concentration were compared with the control (Vehicle) by a one-sample t -test of one tail. Asterisks represent the p -value: * P ≤ 0.05 (E) IL-2 secretion assessed by ELISA in peripheral blood CD4 T cells treated or not with the indicated inhibitor or the vehicle and stimulated for 6 h with anti-CD3ε/anti-CD28 coated beads. Results were normalized to control (Vehicle) and the mean ± SD is shown of the data obtained with n = 4 experiments. Samples were compared with the control (vehicle) by a one-sample t -test. Asterisks represent the p -value: * P ≤ 0.05.

Article Snippet: Taqman probes used were Hs00743856_s1 (PTP4A1), Hs00754750_s1 (PTP4A2), Hs02341135_m1 (PTP4A3), Hs00959886_g1 ( PTPN2 ), Hs00169359_m1 ( PTPN6 ), Hs00978680_m1 ( PTPN7 ), Hs04189704_m1 ( PTPRC ), Hs00934033_m1 (CD69), and Hs00272002_m1 ( GNB2L1 , housekeeping gene).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Transfection, Control, Derivative Assay

Journal: PloS one

Article Title: Prolactin stimulates precursor cells in the adult mouse hippocampus.

doi: 10.1371/journal.pone.0044371

Figure Lengend Snippet: Figure 1. Effects of PRL on hippocampal precursors in vitro and in vivo. The addition of exogenous PRL led to a significant increase in hippocampal (A) derived neurospheres (n = 5 experiments). As well as increasing hippocampal neurosphere number, exposure of primary hippocampal cells to exogenous PRL resulted in an increase in small and medium sized neurospheres with diameters between 40 and 150 mm (B). Following differentiation, the large KCl-activated neurospheres contained both neurons (red) and astrocytes (green) (C) whereas; the smaller PRL- and control-cultured neurospheres gave rise to GFAP-positive astrocytes exclusively (D). Infusion of PRL directly into the adult hippocampus for 7 days resulted in a significant increase in hippocampal neurosphere numbers (E). Results are expressed as mean 6 SEM. *p,0.05, **p,0.01. Scale bars are 20 mm. doi:10.1371/journal.pone.0044371.g001

Article Snippet: Infusion of PRL into the Hippocampus Osmotic mini pumps (Alzet, #1007D; 7 day infusion at a flow rate of 0.5 ml/hour) were loaded with PRL (recombinant mouse, R & D Systems) supplemented with 0.1% BSA or vehicle solution (0.9% sterile physiological saline, containing 0.1% BSA), attached to the infusion cannula and the entire apparatus incubated at 4uC overnight.

Techniques: In Vitro, In Vivo, Derivative Assay, Control, Cell Culture