Journal: Gene Therapy

Article Title: Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

doi: 10.1038/s41434-022-00380-z

Figure Lengend Snippet: A eGFP (27 kDa) and loading control β-actin (45 kDa) were assessed in retinal lysates 4 weeks after intravitreal injection of 5 × 10 9 genome copies per eye of AAV2-CBA-eGFP excluding the Kozak sequence ( n = 4) or AAV2-CBA-eGFP including the Kozak sequences ( n = 4). Eyes that were not injected were processed identically as naïve controls ( n = 2). B Quantification of the mean eGFP intensity per transduced retina is shown, corrected for total protein loaded per sample (df = 6, t = 2.93, p < 0.05, Student’s t test for No Kozak vs. Kozak). The grey bars depict the mean ± SEM. Each individual data point represents one retina. Images were taken from the same Western blot; different exposure times were needed to visualise β-actin and eGFP proteins, respectively. * p < 0.05.

Article Snippet: AAV-PGK-eGFP (Addgene plasmid #193023) was made by removing the β-globin intron preceding eGFP from one of our plasmids (Addgene plasmid #162513) [ ].

Techniques: Control, Injection, Sequencing, Western Blot

Journal: Gene Therapy

Article Title: Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

doi: 10.1038/s41434-022-00380-z

Figure Lengend Snippet: eGFP (green) is shown in the retinal wholemount quadrants ( A ), retinal eye cups ( B ) and optic chiasms ( C ); DAPI staining (blue) was performed on the retinal eye cup and the optic chiasm sections to improve histological visualisation. Autofluorescence emission from extraocular muscle and orbital fat is visible in the sections of retinal eye cups. All overview images were taken with a tile-scanning epifluorescence microscope with identical microscope settings between the experimental groups per retinal tissue. eGFP is shown throughout without amplification using a GFP antibody.

Article Snippet: AAV-PGK-eGFP (Addgene plasmid #193023) was made by removing the β-globin intron preceding eGFP from one of our plasmids (Addgene plasmid #162513) [ ].

Techniques: Staining, Microscopy, Amplification

Journal: Gene Therapy

Article Title: Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

doi: 10.1038/s41434-022-00380-z

Figure Lengend Snippet: A Brn3A staining (red) and eGFP expression (green in upper panels and grey in lower panels) in retinal wholemounts of mice were intravitreal injected with AAV2-CBA-eGFP, AAV2-CMV-eGFP, AAV2-PGK-eGFP, AAV2-sCAG-eGFP, and AAV2-SYN-eGFP. B Quantification of the mean eGFP intensity in transduced RGCs shown as average for each retinal wholemount (i) (df = 4(24), F = 11.9, p < 0.001, ANOVA with Tukey’s multiple comparison test) and as a visualisation of the eGFP intensity for each expressing RGC (ii). C Quantification of the expression efficiency of AAV2 with promoters for RGCs in retinal wholemounts (df = 4(24), F = 23.3, p < 0.001, ANOVA with Tukey’s multiple comparison test). B i, C depict the mean ± SEM in which each individual data point represents the mean value of twelve images within one retinal wholemount. B ii shows a violin plot containing the values of all RGCs expressing the eGFP transgene in which the line represents the median and the dotted patterns the quadrants. Images were taken using a confocal microscope with identical microscope settings between the experimental groups. eGFP is quantified and shown throughout without amplification using a GFP antibody. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: AAV-PGK-eGFP (Addgene plasmid #193023) was made by removing the β-globin intron preceding eGFP from one of our plasmids (Addgene plasmid #162513) [ ].

Techniques: Staining, Expressing, Injection, Comparison, Microscopy, Amplification

Journal: Gene Therapy

Article Title: Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

doi: 10.1038/s41434-022-00380-z

Figure Lengend Snippet: eGFP expression (green) and DAPI staining (blue) together with RBPMS ( A , red), Prox1 ( B , cyan), Calretinin ( C , yellow), and Vimentin ( D , orange) staining in retinal cross-sections of mice that were intravitreal injected with AAV2-CBA-eGFP, AAV2-CMV-eGFP, AAV2-PGK-eGFP, AAV2-sCAG-eGFP, and AAV2-SYN-eGFP. E Quantification of the cellular tropism by each promoter expressed as a percentage of all transduced cells assessed. Two eyes, each from a different mouse, were analysed for each promoter. Images were taken using an epifluorescent microscope with identical microscope settings between the experimental groups per investigated cell-type. eGFP is shown throughout without amplification using a GFP antibody. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer.

Article Snippet: AAV-PGK-eGFP (Addgene plasmid #193023) was made by removing the β-globin intron preceding eGFP from one of our plasmids (Addgene plasmid #162513) [ ].

Techniques: Expressing, Staining, Injection, Microscopy, Amplification

Journal: Gene Therapy

Article Title: Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

doi: 10.1038/s41434-022-00380-z

Figure Lengend Snippet: A Schematic representation of a ventral view on the mouse brain, in which the white square illustrates the location of the optic chiasm and related structures. B Microscopic overview of the optic chiasm from a mouse that had bilateral AAV2-SYN-eGFP intravitreal injections. This image is modified from the panel containing AAV2-SYN-eGFP in Fig. . C eGFP-positive fibres were found at the optic nerve, optic chiasm and optic tract in all investigated experimental groups. All images were taken using a tile-scanning epifluorescence microscope with exposure settings selected to maximise axon visualisation for each promoter. Horizontal sections were stained for DAPI, eGFP is shown throughout without amplification using a GFP antibody. Schematic was created with BioRender.com.

Article Snippet: AAV-PGK-eGFP (Addgene plasmid #193023) was made by removing the β-globin intron preceding eGFP from one of our plasmids (Addgene plasmid #162513) [ ].

Techniques: Modification, Microscopy, Staining, Amplification

Journal: Gene Therapy

Article Title: Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

doi: 10.1038/s41434-022-00380-z

Figure Lengend Snippet: A , B Sagittal and coronal sections of the mouse brain containing the LGN and SC in one hemisphere shown as a schematic and as a microscopic overview image from a mouse that had bilateral AAV2-SYN-eGFP intravitreal injections. All investigated experimental groups had eGFP-positive fibres projecting to the LGN ( C ) and SN ( D ). The overview image was taken with a tile-scanning epifluorescence microscope, whilst magnified images were taken using a confocal microscope with identical settings between experimental groups. All brain sections were stained for DAPI and eGFP. Schematic was created with BioRender.com. LGN lateral geniculate nucleus, SC superior colliculus.

Article Snippet: AAV-PGK-eGFP (Addgene plasmid #193023) was made by removing the β-globin intron preceding eGFP from one of our plasmids (Addgene plasmid #162513) [ ].

Techniques: Microscopy, Staining

Journal: Gene Therapy

Article Title: Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

doi: 10.1038/s41434-022-00380-z

Figure Lengend Snippet: A Table highlighting human donor details. B Time course of the post-mortem human retinal explant preparation, viral transduction and expression assessment. C RBPMS staining (red) and eGFP staining (green in upper panels and grey in lower panels) in post-mortem human retinal explants that were transduced with AAV2-CBA-eGFP, AAV2-CMV-eGFP, AAV2-PGK-eGFP, AAV2-sCAG-eGFP, and AAV2-SYN-eGFP ex vivo. Images were taken using a confocal microscope with identical microscope settings between the experimental groups. Human explants were stained for eGFP. D Mouse strains used for retinal explants. E Time course of the post-mortem mouse retinal explant preparation, viral transduction and expression assessment. F RBPMS staining (red) and eGFP staining (green in upper panels and grey in lower panels) in post-mortem retinal explants of C57BL/6J and B6.BOla- Wld S mice that were transduced with AAV2-SYN-eGFP ex vivo. Mouse explants were stained for eGFP. G Comparison between the number of total RGCs and the number of transduced RGCs in post-mortem mouse retinal explants transduced with AAV2-CBA-eGFP, AAV2-CMV-eGFP, AAV2-PGK-eGFP, AAV2-sCAG-eGFP, and AAV2-SYN-eGFP ex vivo [ Y = 0.176x − 5.5, Spearman’s rho = 0.864, R 2 = 0.899, p < 0.001, nonparametric Spearman correlation]. N = 7 for C57BL/6J (WT) and N = 9 for B6.BOla- Wld S .

Article Snippet: AAV-PGK-eGFP (Addgene plasmid #193023) was made by removing the β-globin intron preceding eGFP from one of our plasmids (Addgene plasmid #162513) [ ].

Techniques: Transduction, Expressing, Staining, Ex Vivo, Microscopy, Comparison

Journal: PLoS ONE

Article Title: The Coordinated P53 and Estrogen Receptor Cis-Regulation at an FLT1 Promoter SNP Is Specific to Genotoxic Stress and Estrogenic Compound

doi: 10.1371/journal.pone.0010236

Figure Lengend Snippet: (A) MCF7 cells cultured in normal medium. (C & D). HCT116 p53 +/+ cells. Gene reporter assays results are presented as the average light units and the standard errors of three independent biological replicates. The indicated reporter plasmids were transiently transfected along with the pRL-SV40 control vector. When indicated, the HCT116 p53 +/+ cells were treated with doxorubicin (Doxo “+” ) at 0.3 µg/ml for 16 hours prior to cell lysis. Luciferase activity was measured 48 hours post-transfection and normalized for transfection efficiency. Statistically significant differences are highlighted (* = p<0.01; ∧ = p<0.05, t-test). (B) Partial sequence of the 1 kb FLT1 proximal promoter. The ERE1, p53 RE and ERE2 sequences are highlighted (uppercase: bases matching the consensus sequences) and their relative position with respect to the Transcriptional Start Site (1) is shown. Also presented is a schematic drawing of the reporter constructs based in the pGL3 promoter vector containing a 1 Kb fragment of the FLT1 promoter. The specific mutations introduced to inactivate the ERE1 and ERE2 sites are indicated (bold lowercase). (C,D) Cells were transiently co-transfected with the empty pGL3 vector or the indicated reporter constructs along with an expression vector leading to overexpression of ERα (C) or ERβ (D). (E) HCT116 p53 −/− cells were used as a control for the p53-dependence of the combined effect of doxorubicin treatment and ERα or ERβ expression on the transactivation of the FLT1 promoter constructs. (F) Representative western blot showing the increase in p53 protein levels after doxorubicin treatment; β-actin was used as loading control. For each cell line, p53 protein levels were assessed in a duplicate experiment to confirm that the transfection with different reporter plasmids did not impact p53 stabilization. (G) HCT116 p53 −/− cells were transfected with the pS2/TFF1 ER reporter along with empty or overexpression vectors for ERα or ERβ. H. MCF7 cells cultured in estrogen-depleted media supplemented with 10 nM E2.

Article Snippet: The PG13 p53 reporter plasmid (obtained from B. Vogelstein, The Johns Hopkins Kimmel Cancer Center, Baltimore, Maryland, USA) , was used as a transactivation control.

Techniques: Cell Culture, Transfection, Control, Plasmid Preparation, Lysis, Luciferase, Activity Assay, Sequencing, Construct, Expressing, Over Expression, Western Blot

Journal: PLoS ONE

Article Title: The Coordinated P53 and Estrogen Receptor Cis-Regulation at an FLT1 Promoter SNP Is Specific to Genotoxic Stress and Estrogenic Compound

doi: 10.1371/journal.pone.0010236

Figure Lengend Snippet: Cells were transiently co-transfected with the FLT1-C, FLT1-T or FLT1-T-ere1 reporter constructs along with expression vectors for p53, ERα and/or ERβ. Relative luciferase activity was determined 24 hours after transfection. Presented are the average relative light units and the standard errors of three biological repeats.

Article Snippet: The PG13 p53 reporter plasmid (obtained from B. Vogelstein, The Johns Hopkins Kimmel Cancer Center, Baltimore, Maryland, USA) , was used as a transactivation control.

Techniques: Transfection, Construct, Expressing, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: The Coordinated P53 and Estrogen Receptor Cis-Regulation at an FLT1 Promoter SNP Is Specific to Genotoxic Stress and Estrogenic Compound

doi: 10.1371/journal.pone.0010236

Figure Lengend Snippet: To avoid any contribution of the endogenous p53 protein, gene reporter assays were performed in a p53-null, osteosarcoma-derived SaOS2 cell line. (A) Cells were transiently co-transfected with the FLT1-C or FLT1-T reporter constructs along with an overexpression vector for p53, p73β or p63γ as well as the pRL-SV40 control vector, followed by doxorubicin (0.3 µg/ml) or mock treatment, according to the schedule described in . Presented are the average-fold luciferase induction relative to FLT1-C mock treated and the standard errors of three replicates. Statistically significant differences relating to the impact of p53 expression alone or in combination with doxorubicin treatment are highlighted (* = p<0.01; ∧ = p<0.05, t-test). (B) The PG13 p53 reporter plasmid was used as a control for comparing the transactivation potential of p53, p73β or p63γ and the effect of the doxorubicin treatment.

Article Snippet: The PG13 p53 reporter plasmid (obtained from B. Vogelstein, The Johns Hopkins Kimmel Cancer Center, Baltimore, Maryland, USA) , was used as a transactivation control.

Techniques: Derivative Assay, Transfection, Construct, Over Expression, Plasmid Preparation, Control, Luciferase, Expressing

Journal: PLoS ONE

Article Title: The Coordinated P53 and Estrogen Receptor Cis-Regulation at an FLT1 Promoter SNP Is Specific to Genotoxic Stress and Estrogenic Compound

doi: 10.1371/journal.pone.0010236

Figure Lengend Snippet: (A) MCF7 cells were seeded into 12-well plates and transfected either with empty, FLT1-C or FLT1-T plasmids, along with the pRLSV40 control vector. 24 hours after transfection cells were treated with doxorubicin (0.3 µg/ml) or 5FU (375 µM) or mock treated, as indicated. At 16-hour post-treatment, cells were lysed for the luciferase assays. The relative averages and standard errors of at least three independent replicates are presented. (B) Western blot analysis of the p53 (DO-1 antibody), p21 (C-19 antibody) and ERα (H-184 antibody) proteins after treatment with doxorubicin and 5FU. 30 ug of total proteins were loaded in each lane. Actin (C-11 antibody) was used as loading control. All antibodies were obtained from Santa Cruz. The bands above actin are due to a residual p53 signal caused by incomplete removal of the primary Ab against p53 while re-probing for actin. (C) MCF7 cells cultured in estrogen-depleted medium for 72 hrs, were seeded into 24-well plates and transfected with empty or pS2/TFF1 reporter vector. 24 hours after transfection cells were treated with doxorubicin (0.3 µg/ml) or 5FU (375 µM) and/or with the addition of 100 nM E2. At 16-hour post-treatment, cells were lysed for the luciferase assays. The relative averages and standard errors of at least three independent replicates are presented. E2 led to a significant induction (* = p<0.01, t-test) of the pS2/TFF1 reporter that was not impacted by doxo or 5FU treatment.

Article Snippet: The PG13 p53 reporter plasmid (obtained from B. Vogelstein, The Johns Hopkins Kimmel Cancer Center, Baltimore, Maryland, USA) , was used as a transactivation control.

Techniques: Transfection, Control, Plasmid Preparation, Luciferase, Western Blot, Cell Culture

Journal: PLoS ONE

Article Title: The Coordinated P53 and Estrogen Receptor Cis-Regulation at an FLT1 Promoter SNP Is Specific to Genotoxic Stress and Estrogenic Compound

doi: 10.1371/journal.pone.0010236

Figure Lengend Snippet: (A) Schematic drawing of the annealing positions of the PCR primers utilized in the ChIP experiments to probe the individual response elements. (B, top panels) representative PCR results that evaluate the occupancy of p53 or ERα after mock, doxorubicin and 5FU treatments obtained with primers specific for the p53 RE, ERE1 and ERE1 region. (B, lower panels) same as in top panel except that the indicated FLT1-T mutant constructs were transfected. ChIP PCR reactions for p53RE, ERE1 and ERE2 were performed with the single mutant ere1 or the double mutant ere1,2 within the pGL3 based constructs after ChIP with p53 and ERα antibodies, respectively. PCR results obtained with input DNA and with IP material obtained from a negative control IgG antibody are also shown. (C) Fold change in site occupancy measured using real time PCR. Data are presented following the same order as in (A). The antibody used for the ChIP experiment was targeted at p53 for the reported analysis at the p53RE site and at ERα for the analysis at the ERE1 or ERE2 sites. Bars represent the average fold change in occupancy and standard deviations of three technical replicates.

Article Snippet: The PG13 p53 reporter plasmid (obtained from B. Vogelstein, The Johns Hopkins Kimmel Cancer Center, Baltimore, Maryland, USA) , was used as a transactivation control.

Techniques: Mutagenesis, Construct, Transfection, Negative Control, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: The Coordinated P53 and Estrogen Receptor Cis-Regulation at an FLT1 Promoter SNP Is Specific to Genotoxic Stress and Estrogenic Compound

doi: 10.1371/journal.pone.0010236

Figure Lengend Snippet: The relative expressions of FLT1 (gray bars) and p53 target gene p21 (black bars) in mock, doxorubicin or 5FU treated cells were compared using an RT-PCR approach in real time. GIMEN (neuroblastoma-derived) and HCT116 (colon adenocarcinoma) are heterozygous (CT) for the promoter SNP at the FLT1 promoter while MCF7 cells are homozygous for the p53 nonresponsive SNP allele (CC). The HCT116 p53 −/− derivative cell line was used as control for p53-independent effects. GAPDH or B2M expression levels served as normalization controls. Histograms described the average fold-induction relative to the housekeeping gene, calculated using the ΔC t method. Error bars correspond to standard errors of at least three replicates. (A) Comparison between GIMEN, HCT116 and MCF7 cells. (B) Differential impact of doxorubicin and 5FU on FLT1 expression in GIMEN cells. Since the basal levels of FLT1 expression were very low (C t ≥30), the fold of induction may appear somewhat exaggerated. C) Impact of doxorubicin and/or E2 on FLT1 and p21 mRNA expression in GIMEN cells culture in estrogen-depleted medium. The increase in FLT1 mRNA expression by combined doxorubicin and E2 treatment appeared to be statistically significant ( ∧ = p<0.05, t-test).

Article Snippet: The PG13 p53 reporter plasmid (obtained from B. Vogelstein, The Johns Hopkins Kimmel Cancer Center, Baltimore, Maryland, USA) , was used as a transactivation control.

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Control, Expressing, Comparison

Journal: Nature Communications

Article Title: Desumoylase SENP6 maintains osteochondroprogenitor homeostasis by suppressing the p53 pathway

doi: 10.1038/s41467-017-02413-3

Figure Lengend Snippet: Senp6 loss activated p53 signaling pathway and SASP. a Heatmap of RNA-seq gene expression profiles comparing Dermo1-Cre;Senp6 f/f and control primary rib chondrocytes ( n = 2). b Up-regulated pathways based on DAVID GO analyses ( p -values: Fisher Exact/EASE Score). c Enriched p53 pathway and TNFα-signaling-via-NFκB pathway in Dermo1-Cre;Senp6 f/f by GESA analysis (NES normalized enrichment score; p : nominal p -value; q : false discovery rate q -value). d Dermo1-Cre;Senp6 f/f rib cartilage (E18.5) showing higher total SUMO2/3-modified proteins, total and phosphorylated (S15) p53, and γH2AX relative to controls ( n = 3). e Chondrocyte differentiation and proliferation markers, p53 downstream targets that promote apoptosis and senescence, and SASP markers were augmented in the rib cartilage of E18.5 Dermo1-Cre;Senp6 f/f mice ( n = 3; error bars = standard deviation, * p < 0.05, relative to controls, Student’s t -test)

Article Snippet: HEK293 cells (ATCC) were transfected with PG13-Luc (with p53 binding cis-element, 0.5 μg) and Myc-TRIM28, TRIM28 K554R , TRIM28 K779R , TRIM28 K804R , TRIM28 3KR (0.5 μg) with HA-SUMO3 (0.5 μg) or Flag-SENP6 (1.5 μg) present or absent based on the experimental strategy.

Techniques: RNA Sequencing, Gene Expression, Control, Modification, Standard Deviation

Journal: Nature Communications

Article Title: Desumoylase SENP6 maintains osteochondroprogenitor homeostasis by suppressing the p53 pathway

doi: 10.1038/s41467-017-02413-3

Figure Lengend Snippet: SENP6 desumoylated and stabilized TRIM28 and suppressed p53. a Senp6 loss decreased the total TRIM28 protein in E18.5 Dermo1-Cre;Senp6 f/f rib cartilage but increased sumoylated TRIM28 (upper bands denoted by the arrow). “L” or “S” indicates long or short exposure ( n = 3). b SUMO3 modifications on TRIM28 can be removed by overexpressed SENP6 ( n = 2). c TRIM28 can be immunoprecipitated with either wild-type SENP6 or enzymatic-activity-null SENP6 (C1030A). d , e . Sumoylation-resistant TRIM28 mutations impaired TRIM28 sumoylation and p53 activation. Western blots showed that the K554R, K804R, K779R, and 3KR mutations of TRIM28 reduced TRIM28 SUMO3 modifications, and SENP6 effectively removed SUMO3 modification from all these sites ( d ). p53 reporter luciferase assays showed that the sumoylated TRIM28 enhanced p53 signaling, which was abolished by the addition of SENP6; these TRIM28 mutants induced significantly lower p53 activity than WT TRIM28 did ( n = 3; error bars = standard deviation, * p < 0.05, Student’s t -test) ( e )

Article Snippet: HEK293 cells (ATCC) were transfected with PG13-Luc (with p53 binding cis-element, 0.5 μg) and Myc-TRIM28, TRIM28 K554R , TRIM28 K779R , TRIM28 K804R , TRIM28 3KR (0.5 μg) with HA-SUMO3 (0.5 μg) or Flag-SENP6 (1.5 μg) present or absent based on the experimental strategy.

Techniques: Immunoprecipitation, Activity Assay, Activation Assay, Western Blot, Modification, Luciferase, Standard Deviation

Journal: Nature Communications

Article Title: Desumoylase SENP6 maintains osteochondroprogenitor homeostasis by suppressing the p53 pathway

doi: 10.1038/s41467-017-02413-3

Figure Lengend Snippet: Trp53 knockout partially rescued skeletal defects caused by Senp6 loss. a Prx1-Cre;Senp6 f/f ;Trp53 f/f (DKO) P0 mice had a partially rescued skeletal phenotype relative to Prx1-Cre;Senp6 f/f ;Trp53 f/+ (SKO) mice, as suggested by the smaller indentation in the scapula growth plate (red arrows), the longer clavicles (blue arrows), increased width and length of the femur (black arrows), tibia, humerus, ulna, and radius, and the moderately increased mineralization in the cranial bones (yellow arrows). Scale bars = 1 cm. b P0 DKO femoral sections show corrected growth plate diameter, perichondrium thickness (red arrows), and chondrocyte size (yellow arrows) relative to those of SKO mice. Scale bars = 100 μm for the upper panels and 50 μm for the lower panels. c p53 loss partially corrected the low bone mass and the delayed SOC formation of SKO mice at P21 as revealed by microCT imaging (SOC, red arrows; metaphasis bone, yellow arrows; proximal femur head, blue arrows; scale bars = 1 cm). H&E staining of the corresponding femurs is shown in the lower panels; scale bar = 100 μm

Article Snippet: HEK293 cells (ATCC) were transfected with PG13-Luc (with p53 binding cis-element, 0.5 μg) and Myc-TRIM28, TRIM28 K554R , TRIM28 K779R , TRIM28 K804R , TRIM28 3KR (0.5 μg) with HA-SUMO3 (0.5 μg) or Flag-SENP6 (1.5 μg) present or absent based on the experimental strategy.

Techniques: Knock-Out, Imaging, Staining

Journal: Nature Communications

Article Title: Desumoylase SENP6 maintains osteochondroprogenitor homeostasis by suppressing the p53 pathway

doi: 10.1038/s41467-017-02413-3

Figure Lengend Snippet: p53 loss rescued the cellular phenotypes of SKO growth plates. a Growth plate of DKO mice shows decreased cell apoptosis, as indicated by TUNEL staining; the representative images are on the left and quantification on the right ( n = 3; error bars = standard deviation, * p < 0.05; NS not significant, Student’s t -test; scale bar = 100 μm). b γH2AX expression was not rescued in the growth plate of DKO mice. The upper panel shows the representative IHC on the RZ and PZ of growth plate with quantification on the right; the lower panel shows western blots and quantification on the right ( n = 6, error bars = standard deviation, * p < 0.05, NS not significant, Student’s t -test; scale bar = 50 μm). c Corrected cell proliferation by Ki67 staining in the growth plate of DKO mice relative to SKO mice (quantification shown in the low panel; n = 3, error bars = standard deviation, * p < 0.05, NS not significant, Student’s t -test; scale bar = 50 μm). d Corrected cell senescence in Ad-Cre-mediated-DKO vs SKO primary rib chondrocytes, as indicated by SA-β-gal staining; representative staining images are on the left and quantifications on the right ( n = 3, error bars = standard deviation, * p < 0.05, NS not significant, Student’s t -test; scale bar = 50 μm). e Femur growth plate samples from DKO mice show the rescue of elevated p53 downstream targets as well as SASP markers ( n = 3, * p < 0.05, relative to Ctrl; and # p < 0.05, relative to SKO; error bars = standard deviation, Student’s t -test)

Article Snippet: HEK293 cells (ATCC) were transfected with PG13-Luc (with p53 binding cis-element, 0.5 μg) and Myc-TRIM28, TRIM28 K554R , TRIM28 K779R , TRIM28 K804R , TRIM28 3KR (0.5 μg) with HA-SUMO3 (0.5 μg) or Flag-SENP6 (1.5 μg) present or absent based on the experimental strategy.

Techniques: TUNEL Assay, Staining, Standard Deviation, Expressing, Western Blot

Journal: Cell Research

Article Title: KRAB-type zinc-finger proteins PITA and PISA specifically regulate p53-dependent glycolysis and mitochondrial respiration

doi: 10.1038/s41422-018-0008-8

Figure Lengend Snippet: Identification of PITA and PISA as selective regulators of p53. a The KZNF members were each transfected into HCT116 cells and the expression level of metabolism-related p53 target genes were analyzed by immunoblotting with the indicated antibodies. b ShRNA-mediated knockdown of endogenous PITA or PISA augments p53 activity. p53 activity in HCT116 p53 +/+ cells was measured by pG13L luciferase reporter gene assay Representative results of three independent reporter assay experiments are shown. c The mRNA levels of TIGAR or SCO2 in knock-down PITA or PISA HCT116 p53 +/+ cells were measured by real-time PCR. d PITA or PISA selectively regulates expression of TIGAR or SCO2 dependent on p53. Total RNA from transfected HCT116 p53 +/+ or HCT116 p53 −/− cells was subjected to real-time quantitative PCR (qPCR) analysis. e The expression levels of TIGAR or SCO2 in knockdown PITA or PISA HCT116 p53 +/+ cells were measured byimmunoblotting with the indicated antibodies. f Coimmunoprecipitation of endogenous PITA or PISA and endogenous p53 from HCT116 cells. Western blot analysis of whole-cell lysate (WCL) and immunoprecipitation (IP) with preimmune serum, PITA or PISA-specific antibody, p53 antibody or control mouse IgG are shown. g Direct interaction between PITA and p53, PISA and p53 revealed by GST pull-down assays. Both input and pull-down samples were subjected to immunoblotting (IB) with anti-His antibody. Input represents 10% of that used for pull-down. The bottom part shows the gel stained by Coomassie brilliant blue. h Mapping the region of p53 that interacts with PITA or with PISA. Myc-tagged p53 mutants were transfected into HCT116 p53 −/− cells. Endogenous PITA or PISA proteins were immunoprecipitated with anti-PITA or PISA antibody. IgG HC, heavy chain of IgG; TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain; BD, basic domain. i Interaction region mapping on PITA or PISA. Full-length or truncations of PITA or PISA and Flag-p53 were transfected into HCT116 p53 +/+ cells. WCL was immunoprecipitated with anti-Flag antibody. Both the WCL and IP samples were analyzed. j Both KRAB domain and zinc-fingers are required for inhibition of p53 activity by PITA or PISA. The PITA or PISA deletion mutants were used in the reporter gene assays. Detection of the proteins’ expression is also shown. Error bars represent mean ± SD for three independent experiments. ( b–d , j ; mean and s.e.m., n = 3. * P < 0.05: two-tailed unpaired t -test). Unprocessed original scans of blots are shown in , Figure

Article Snippet: Luciferase reporter plasmids pG13-Luc (containing 13 tandem p53-binding elements) and pRL-CMV (internal control; from Promega) were co-transfected as indicated in figure legends.

Techniques: Transfection, Expressing, Western Blot, shRNA, Knockdown, Activity Assay, Luciferase, Reporter Gene Assay, Reporter Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Control, Staining, Binding Assay, Zinc-Fingers, Inhibition, Two Tailed Test

Journal: Cell Research

Article Title: KRAB-type zinc-finger proteins PITA and PISA specifically regulate p53-dependent glycolysis and mitochondrial respiration

doi: 10.1038/s41422-018-0008-8

Figure Lengend Snippet: PITA and PISA preferentially inhibit the binding of p53 to the target genes TIGAR and SCO2, respectively. a ChIP assays in PITA-knocking down and PISA-knocking down or control p53 +/+ HCT116 cells. IP immunoprecipitation. b Multiple ChIP analysis on the consensus binding sequences of p53 targets. HCT116p53 −/− cells were transfected with a mock vector, PITA, p53, or cotransfected with p53 and PITA as indicated (top), HCT116p53 −/− cells were transfected with a mock vector, PISA, p53, or cotransfected with p53 and PISA as indicated (bottom). After 24 h, ChIP was carried out using a control mouse IgG or an anti-p53 antibody, and RT-PCR was performed for the indicated promoters. c The p53-binding sequence in the promoter or intron of SCO2 is shown. For intron 1, the PISA-binding core sequence is underlined. PISA binds to the PISA/p53-BS of SCO2 in vitro . d Supershift EMSA using nuclear extracts derived from p53 −/− HCT116 cells harboring Myc-PISA. The PISA antibody and an unlabeled competitor probe were added as indicated. EMSA was performed with 100 ng of His-PISA and biotin-labeled PISA/p53-BS oligonucleotides. e , f EMSA of the p53 protein with PISA/p53-BS and mutants. The mutant PISA/p53-BS probes (for sequences, see c ) were used in the EMSA with PISA protein. The mutant probes were used in the EMSA with the p53 protein and p53 antibody. EMSA was performed with 100 ng His-PISA and biotin-labeled mutant PISA/p53-BS. g ChIP assays in p53 +/+ HCT116 cells. The promoter or intron of the indicated p53 target genes were amplified from immunoprecipitates of anti-PITA, anti-PISA, or IgG control. GAPDH was used as an internal control. h p53 −/− and p53 +/+ HCT116 cells were co-transfected with TIGAR/p53-BS-Luc and PITA or SCO2/p53-BS-Luc and PISA, and the luciferase activity was measured. Error bars represent mean ± SD for three independent experiments. ( a , b , g , h ; mean and s.e.m., n = 3. * p < 0.05: two-tailed unpaired t -test). Unprocessed original scans of blots are shown in , Figure

Article Snippet: Luciferase reporter plasmids pG13-Luc (containing 13 tandem p53-binding elements) and pRL-CMV (internal control; from Promega) were co-transfected as indicated in figure legends.

Techniques: Binding Assay, Control, Immunoprecipitation, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Sequencing, In Vitro, Derivative Assay, Labeling, Mutagenesis, Amplification, Luciferase, Activity Assay, Two Tailed Test

Journal: Cell Research

Article Title: KRAB-type zinc-finger proteins PITA and PISA specifically regulate p53-dependent glycolysis and mitochondrial respiration

doi: 10.1038/s41422-018-0008-8

Figure Lengend Snippet: PITA and PISA regulate aerobic glycolysis and mitochondrial respiration. a Analysis of glucose consumption and lactate levels, PFK1 activity, F2,6BP activity, PK activity, NADPH levels and GSG/GSSH ratio in PITA-depleted or reintroduction of PITA HCT116 p53 +/+ cells. b Analysis of PFK1 activity, glucose consumption and lactate levels in ectopic PITA-overexpressing HCT116 p53 +/+ and HCT116 p53 −/− stable transfectants. c PK activity in HCT116 p53 +/+ cells stably transfected with PITA. d , e NADPH levels, GSG/GSSH (ratio) levels, NADH/NAD (ratio) and ROS levels in overexpression of PITA HCT116 p53 +/+ stable cell lines. f Analysis of glucose consumption and lactate levels, cytochrome c oxidase (COX) activity, in PISA-depleted or reintroduction of PISA HCT116 p53 +/+ cells. Oxygen consumption (mean TSD, nmol min −1 mg −1 protein) was measured by a Clark type oxygen microelectrode in PISA-depleted HCT116 p53 +/+ cell lines mitochondria preparations. g Cytochrome c oxidase (COX) activity, glucose consumption and lactate levels, in overexpression of PISA HCT116 p53 +/+ or HCT116 p53 −/− cell lines. h Analysis of total and single mitochondrial volume and mitochondrial numbers as described in the Experimental Procedures section in overexpression of PITA HCT116 p53 +/+ stable cell lines. i Oxygen consumption (mean TSD, nmol min −1 mg −1 protein) was measured by a Clark type oxygen microelectrode in overexpression of PISA HCT116 p53 +/+ cell lines mitochondria preparations. j After knockdown of TIGAR, PFK1 activity, glucose consumption and lactate levels in PITA-depletion HCT116 p53 +/+ and HCT116 p53 −/− cells. k After knockdown of SCO2, COX activity, glucose consumption and lactate levels in PISA-depletion HCT116 p53 +/+ and HCT116 p53 −/− cells. Error bars represent mean ± SD for three independent experiments. ( a – k ; mean and s.e.m., n = 3. * P < 0.05: two-tailed unpaired t -test)

Article Snippet: Luciferase reporter plasmids pG13-Luc (containing 13 tandem p53-binding elements) and pRL-CMV (internal control; from Promega) were co-transfected as indicated in figure legends.

Techniques: Activity Assay, Stable Transfection, Transfection, Over Expression, Knockdown, Two Tailed Test

Journal: Cell Research

Article Title: KRAB-type zinc-finger proteins PITA and PISA specifically regulate p53-dependent glycolysis and mitochondrial respiration

doi: 10.1038/s41422-018-0008-8

Figure Lengend Snippet: PITA regulates PFK1 activity and promotes aerobic glycolysis in vivo. a Schematic representation of the transgenic construct used to generate PITA transgenic (TG) mouse lines. PITA protein levels in WT and PITA TG mice at 8 weeks were assessed with both anti-Myc and anti-GAPDH antibodies. b , c Analysis of PFK1, F2,6BP and PK activity in muscle tissues from WT ( n = 8) and PITA TG mice ( n = 6) maintained on a normal diet. d NADPH levels in muscle of WT ( n = 8) and PITA TG mice ( n = 6) maintained on a normal diet. e GSG/GSSH (ratio) levels in plasma from WT ( n = 8) and PITA TG mice ( n = 6) maintained on a normal diet. f Following an overnight fast, WT ( n = 8) and PITA TG mice ( n = 6) were subjected to a hypereuglycemic clamp. Basal glucose production, whole-body and muscle glucose uptake rate during hypereuglycemic clamp, muscle (soleus and gastrocnemius) glucose uptake during the hypereuglycemic clamp. g , h Serum lactate levels and plasma glucose in WT ( n = 8) and PITA TG mice ( n = 8) mice in the fasted overnight states. i Glucose tolerance test on WT (opened circle, n = 7) and PITA TG mice (filled circle, n = 7) mice. j Left: Effect of walnut extract on rolling bar test time of WT ( n = 7) and PITA ( n = 7) mice. Right: Effect of walnut extract on locomotors time of WT and PITA mice. k Body weight of 3-month-old WT ( n = 7) and PITA TG ( n = 7) mice fed a normal chow. PITA TG mice have similar food intake ( n = 7) in comparison with WT mice ( n = 7). l Indirect calorimetry of WT (white triangle) and PITA TG mice (black triangle). Oxygen consumption (VO 2 ), carbon dioxide release (VCO 2 ), respiratory exchange ratio (RER; VCO 2 /VO 2 ) and energy expenditure per kg of body weight were determined in WT and PITA TG mice in metabolic chambers ( n = 8 per genotype). m Analysis of glucose consumption and lactate levels, PFK1 activity, NADPH level in CRISPR-Cas9 PITA MEF p53 +/+ and MEF p53 −/− cells. Data are means ± SD ( n =3). Error bars represent mean ± SD for three independent experiments. (mean and s.e.m., n = 3. * P < 0.05: two-tailed unpaired t -test). Unprocessed original scans of blots are shown in , Figure . Data in b–l represent mean ± s.e.m. * P < 0.05: two-tailed unpaired t -test

Article Snippet: Luciferase reporter plasmids pG13-Luc (containing 13 tandem p53-binding elements) and pRL-CMV (internal control; from Promega) were co-transfected as indicated in figure legends.

Techniques: Activity Assay, In Vivo, Transgenic Assay, Construct, Clinical Proteomics, Comparison, CRISPR, Two Tailed Test

Journal: Cell Research

Article Title: KRAB-type zinc-finger proteins PITA and PISA specifically regulate p53-dependent glycolysis and mitochondrial respiration

doi: 10.1038/s41422-018-0008-8

Figure Lengend Snippet: PISA regulates cytochrome c oxidase activity and dampens mitochondrial respiration in vivo. a Schematic representation of the transgenic construct used to generate PISA transgenic mouse lines. PISA protein levels in WT and two independent lines of PISA TG mice at 8 weeks were assessed with anti-Myc antibodies. b Cytochrome c oxidase (COX) activity in tissues from WT ( n = 8) and PISA TG mice ( n = 6) maintained on a normal diet. c Analysis of total and single mitochondrial volume and mitochondrial numbers (WT n = 8 and PISA TG mice n = 6). d Following an overnight fast, WT ( n = 8) and PISA TG mice ( n = 6) were subjected to a hypereuglycemic clamp. Whole-body glucose disposal rate during hypereuglycemic clamp. Serum lactate levels in the fasted overnight states. e Plasma glucose levels in WT ( n = 8) and PISA TG mice ( n = 6) mice in the fasted overnight states. f Glucose tolerance test on WT (opened circle, n = 7) and PISA TG mice (filled circle, n = 7) mice. g The amount of ATP (mean TSD, nmol min −1 mg −1 protein) produced by aerobic respiration (gray bars, O2) and glycolysis (light bars, Lac) was calculated by measuring oxygen consumption and lactate production in the mice muscle mitochondria preparations. h Plasma adiponectin, cholesterol, NEFA and triglyceride levels in WT and PISA TG mice in the fasted overnight states. i Left: Effect of walnut extract on rolling bar test time of WT and PISA TG mice. ( n = 7 per group); Right: effect of walnut extract on locomotors time of WT and PISA TG mice ( n = 7 per group). j Body weight of 3-month-old WT ( n = 8) and PISA TG ( n = 6) mice fed a normal chow. PISA TG mice have similar food intake in comparison with WT mice. k Indirect calorimetry of WT (white triangle) and PISA TG mice (gray triangle). Oxygen consumption (VO 2 ), carbon dioxide release (VCO 2 ), respiratory exchange ratio (RER; VCO 2 /VO 2 ) and energy expenditure per kg of body weight were determined in WT and PISA TG mice in metabolic chambers ( n = 8 per genotype). l Analysis of glucose consumption and lactate levels, COX activity, oxygen consumption in CRISPR-Cas9 PISA MEF p53 +/+ and MEF p53 −/− cells. Error bars represent mean ± SD for three independent experiments. (mean and s.e.m., n = 3. * P < 0.05: two-tailed unpaired t -test). Unprocessed original scans of blots are shown in , Figure . Data in b–k represent mean ± s.e.m. * P < 0.05: two-tailed unpaired t -test

Article Snippet: Luciferase reporter plasmids pG13-Luc (containing 13 tandem p53-binding elements) and pRL-CMV (internal control; from Promega) were co-transfected as indicated in figure legends.

Techniques: Activity Assay, In Vivo, Transgenic Assay, Construct, Clinical Proteomics, Produced, Comparison, CRISPR, Two Tailed Test

Journal: Cell Research

Article Title: KRAB-type zinc-finger proteins PITA and PISA specifically regulate p53-dependent glycolysis and mitochondrial respiration

doi: 10.1038/s41422-018-0008-8

Figure Lengend Snippet: PITA and PISA dissociation from p53 upon glucose starvation requires ATM-mediated phosphorylation. a pG13-Luc was cotransfected into HCT116 p53 +/+ cells with p53 and PITA or PISA as indicated. After 24 h, glucose starvation (low dosage, 5 mM) was carried out for the indicated times followed by luciferase assay. b Co-immunoprecipitation of exogenous PITA with p53 or co-immunoprecipitation of exogenous PISA with p53 in HCT116 p53 −/− cells were treated with glucose starvation was carried out for indicated times. Exogenous PITA–p53 and PISA-p53 interactions were analyzed. c ChIP assays were performed to amplify the TIGAR or SCO2 gene. HCT116 p53 +/+ cells were transfected with Myc-PITA or Myc-PISA and treated with glucose starvation. d Sh-NC, Sh-PITA and Sh-PISA of HCT116 p53 +/+ cells with or without glucose starvation were analyzed RNA sequencing, and differentially expressed genes are shown as a heatmap (ordered by P values in , Tables and ). e qRT-PCR analysis of selected genes associated p53 signaling in PITA or PISA knockdown of HCT116 p53 +/+ cells on glucose starvation (5 mM). f Effect of ATM knockdown on PITA or PISA activity in ATM wild-type cells. g HCT116 p53 +/+ cells were transfected with ATM siRNA or control siRNA and treated with GLU-5 mM. Endogenous PITA–p53 interactions or PISA-p53 were analyzed. h After knockdown of ATM, PFK1 activity, glucose consumption and lactate levels, NADPH levels and GSG/GSSH (ratio) levels in overexpression of PITA HCT116 p53 +/+ cells. i COX activity, glucose consumption and lactate levels in knockdown of ATM HCT116 p53 +/+ cells. Error bars represent mean ± SD for three independent experiments. ( c , e , f , h ; mean and s.e.m., n = 3. * P < 0.05: two-tailed unpaired t -test). Unprocessed original scans of blots are shown in , Figure

Article Snippet: Luciferase reporter plasmids pG13-Luc (containing 13 tandem p53-binding elements) and pRL-CMV (internal control; from Promega) were co-transfected as indicated in figure legends.

Techniques: Phospho-proteomics, Luciferase, Immunoprecipitation, Transfection, RNA Sequencing, Quantitative RT-PCR, Knockdown, Activity Assay, Control, Over Expression, Two Tailed Test

Journal: Cell Research

Article Title: KRAB-type zinc-finger proteins PITA and PISA specifically regulate p53-dependent glycolysis and mitochondrial respiration

doi: 10.1038/s41422-018-0008-8

Figure Lengend Snippet: PITA and PISA promote tumorigenesis. a PITA or PISA shRNA or NC (negative control) shRNA lentivirus were used to infect HCT116 p53 +/+ cells, and cell viability was measured at the indicated time points using CCK8 assays. b The human normal colonic epithelial NCM460 cells were infected with overexpression of PITA or PISA (or control) lentivirus, and the cell growth was measured by CCK8 assays. c Left, cells (2 × 10 4 ) infected with lentivirus as in a were cultured in soft agar for 2–3 weeks. The colonies were counted and the relative number of colonies in experimental group was normalized to control group. Right, soft agar assays with PITA or PISA overexpression in NCM460cells were performed as described b . d , e Depletion of PITA or PISA reduced the migration d and invasion e of colon cancer HCT116 cells. The number of migrated cells was calculated. f PITA and PISA protein levels in progression of colorectal cancer tissue ( n = 60). g Representative images of immunohistochemical staining for PITA and PISA are shown. Scale bar, 50 μm. h PITA and PISA protein levels in different type of colorectal cancer tissue ( n = 80). i Representative images of immunohistochemical staining for PITA and PISA are shown. Scale bar, 50 µm. Error bars represent mean ± SD for three independent experiments. ( a – d ; mean and s.e.m., n = 3. * P < 0.05: two-tailed unpaired t -test)

Article Snippet: Luciferase reporter plasmids pG13-Luc (containing 13 tandem p53-binding elements) and pRL-CMV (internal control; from Promega) were co-transfected as indicated in figure legends.

Techniques: shRNA, Negative Control, Infection, Over Expression, Control, Cell Culture, Migration, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Cell Research

Article Title: KRAB-type zinc-finger proteins PITA and PISA specifically regulate p53-dependent glycolysis and mitochondrial respiration

doi: 10.1038/s41422-018-0008-8

Figure Lengend Snippet: Proposed model for PITA and PISA function and regulation in unstressed and glucose starved cells. In unstressed cells, the KRAB-type zinc-finger protein PITA or PISA interacts with p53 and selectively inhibits p53-mediated transcription of metabolic gene TIGAR and SCO2 , respectively. The expression of TIGAR and SCO2 is kept at relatively low levels. In response to glucose starvation, the ATM kinase is activated by autophosphorylation and promotes PITA and PISA phosphorylation, resulting in the dissociation of PITA or PISA from p53. Low stress stimulated the transcription of TIGAR and TIGAR protein in turn inhibits the PFK1 activity, decreases the lactate level, increases NADPH level and modulates the glycolytic rate. At the later stage upon glucose starvation or the cells are subjected with prolonged high stress, the upregulation of SCO2 occurred and the SCO2 protein promotes COX activity and modulates oxidative phosphorylation. PITA and PISA coordinate with ATM to tightly control the p53 downstream effects upon metabolic stress

Article Snippet: Luciferase reporter plasmids pG13-Luc (containing 13 tandem p53-binding elements) and pRL-CMV (internal control; from Promega) were co-transfected as indicated in figure legends.

Techniques: Expressing, Phospho-proteomics, Activity Assay, Control

Journal: Marine Drugs

Article Title: Guanidine Alkaloids from the Marine Sponge Monanchora pulchra Show Cytotoxic Properties and Prevent EGF-Induced Neoplastic Transformation in Vitro

doi: 10.3390/md14070133

Figure Lengend Snippet: Alkaloids 1 – 8 and their effect on EGF-induced neoplastic transformation of JB6 P + Cl41 cells. ( A ) Structures of guanidine alkaloids 1 – 8 isolated from the marine sponge Monanchora pulchra ; ( B ) representative pictures of microscopic fields of EGF-induced colonies of JB6 P + Cl41 cells in soft agar treated with urupocidin A ( 6 ) at the indicated concentrations; ( C ) Inhibition of EGF-induced neoplastic transformation of JB6 P + Cl41 cells by compounds 1 – 4 , 7 , and 8 . INCC 50 —concentration leading to a 50% inhibition of colonies formation. Ratios of IC 50 /INCC 50 were calculated using the IC 50 values from generated by using the MTT assay. * indicates a statistically significant difference ( p < 0.05) from the control value.

Article Snippet: The JB6 P + Cl41 mouse epidermal cell line and its stable transfectants, JB6-Luc AP-1 and JB6-Luc p53 (PG-13), cells were kindly provided by Prof. Zigang Dong, Hormel Institute, University of Minnesota, MN, USA.

Techniques: Transformation Assay, Isolation, Inhibition, Concentration Assay, Generated, MTT Assay, Control

Journal: Marine Drugs

Article Title: Guanidine Alkaloids from the Marine Sponge Monanchora pulchra Show Cytotoxic Properties and Prevent EGF-Induced Neoplastic Transformation in Vitro

doi: 10.3390/md14070133

Figure Lengend Snippet: IC 50 of alkaloids 1 – 8 in human cancer HeLa cells and mouse non-malignant JB6 P + Cl41 cell lines after 48 h of treatment.

Article Snippet: The JB6 P + Cl41 mouse epidermal cell line and its stable transfectants, JB6-Luc AP-1 and JB6-Luc p53 (PG-13), cells were kindly provided by Prof. Zigang Dong, Hormel Institute, University of Minnesota, MN, USA.

Techniques: Activity Assay

Journal: Marine Drugs

Article Title: Guanidine Alkaloids from the Marine Sponge Monanchora pulchra Show Cytotoxic Properties and Prevent EGF-Induced Neoplastic Transformation in Vitro

doi: 10.3390/md14070133

Figure Lengend Snippet: Effect of alkaloids 1 – 8 on MAPK/AP-1 signaling. ( A ) Effect on basal AP-1-dependent transcriptional activity ( ○ ) and viability (□) of JB6 Cl41 cells stably expressing a luciferase reporter gene controlled by the AP-1 DNA binding sequence after 12 h of treatment. Cell viability was measured using MTT assay; ( B ) effect on the activation of JNK1/2 and ERK1/2. JB6 P + Cl41 cells were treated with the compounds 1 – 8 at the indicated concentrations for 48 h, and the level of protein expression was assessed by Western blotting. The intensities of p-JNK1/2 and p-ERK1/2 signals were quantified with Quantity One 4.6 software (Bio-Rad, Hercules, CA, USA) and normalized against the signals of total JNK1/2 and ERK1/2, correspondently; and ( C ) the effect of SP600125 (specific JNK1/2 inhibitor) on the survival of JB6 P + Cl41 cells treated with compounds 1 – 8 . Cells were co-treated with different concentrations of the individual drugs or their combination for 48 h. Cell viability was measured by MTT assay and the combinational index (CI) values were calculated with CompuSyn software (v.1.0., ComboSyn Inc., Paramus, NJ, USA) using the Chou-Talalay method. The ratio of the substances and the effects are presented in the . * indicates a statistically significant difference ( p < 0.05) from the control value.

Article Snippet: The JB6 P + Cl41 mouse epidermal cell line and its stable transfectants, JB6-Luc AP-1 and JB6-Luc p53 (PG-13), cells were kindly provided by Prof. Zigang Dong, Hormel Institute, University of Minnesota, MN, USA.

Techniques: Activity Assay, Stable Transfection, Expressing, Luciferase, Binding Assay, Sequencing, MTT Assay, Activation Assay, Western Blot, Software, Control

Journal: Marine Drugs

Article Title: Guanidine Alkaloids from the Marine Sponge Monanchora pulchra Show Cytotoxic Properties and Prevent EGF-Induced Neoplastic Transformation in Vitro

doi: 10.3390/md14070133

Figure Lengend Snippet: Effect alkaloids 1 – 8 on basal p53-dependent transcriptional activity ( ○ ) and viability (□) of JB6 Cl41 cells stably expressing a luciferase reporter gene controlled by the p53 DNA binding sequence after 12 h of treatment. Cell viability was measured using MTT assay. * indicates a statistically significant difference ( p < 0.05) from the control value.

Article Snippet: The JB6 P + Cl41 mouse epidermal cell line and its stable transfectants, JB6-Luc AP-1 and JB6-Luc p53 (PG-13), cells were kindly provided by Prof. Zigang Dong, Hormel Institute, University of Minnesota, MN, USA.

Techniques: Activity Assay, Stable Transfection, Expressing, Luciferase, Binding Assay, Sequencing, MTT Assay, Control

Journal: Marine Drugs

Article Title: Guanidine Alkaloids from the Marine Sponge Monanchora pulchra Show Cytotoxic Properties and Prevent EGF-Induced Neoplastic Transformation in Vitro

doi: 10.3390/md14070133

Figure Lengend Snippet: Summary of observed effects of compounds 1 – 8 .

Article Snippet: The JB6 P + Cl41 mouse epidermal cell line and its stable transfectants, JB6-Luc AP-1 and JB6-Luc p53 (PG-13), cells were kindly provided by Prof. Zigang Dong, Hormel Institute, University of Minnesota, MN, USA.

Techniques: Activity Assay, Phospho-proteomics, Activation Assay