Journal: Nature Communications

Article Title: Ubiquitination switches EphA2 vesicular traffic from a continuous safeguard to a finite signalling mode

doi: 10.1038/ncomms9047

Figure Lengend Snippet: ( a ) LIFEA2 consists of mCherry fused to EphA2 C-terminus and mCitrine inserted in the JMS. Inset: mCitrine JMS insertion via a coiled-coil linker that on one end matches the distance between two amino acids (3.5 Å) and on the other end matches mCitrine termini (20 Å) (PDB: 1EMA). ( b ) Fluorescence lifetime ( τ ) reports FRET in LIFEA2. First row: mCitrine fluorescence, second: mCherry fluorescence, third: τ (ns; color-coding below). First column: pre-mCherry photobleaching; second column: post-mCherry photobleaching. Graph: τ -histograms pre- and post-photobleaching. ( c ) τ of LIFEA2 on ephrinA1 stimulation in Cos-7 cells at the indicated time (min). Left images, upper row: mCitrine photon counts (donor count), lower row: τ (ns; color-coding right). Middle graph: corresponding τ -histograms (color-coding right). Right graph: relative drop in τ (Δ τ , ns) after LIFEA2 stimulation with pre-clustered ephrinA1-Fc (2 μg ml −1 ) (mean±s.e.m.). Data obtained form three independent experiments, n =7 cells. ( d ) LIFEA2 reports on a change in receptor conformation. First column: fluorescence images of LIFEA2, second: fluorescence images of EphA2–bfp, third: ratio of EphA2–bfp/LIFEA2, (arbitrary units; color-coding right), fourth: τ (ns; color-coding right), before (upper row) and 40 min after (lower row) addition of pre-clustered ephrinA1-Fc (2 μg ml −1 ). Graph: 2D histogram of τ versus EphA2–bfp/LIFEA2 ratio in single cells ( n =13 cells) pre- and 40 min post-stimulation and the corresponding linear fits. ( e ) Phosphorylation of LIFEA2 measured by its interaction with dSH2-mCherry on ephrinA1 addition for the indicated time (min). Left images, upper row: mCitrine photon counts (donor count), lower: τ (ns; color-coding right) of donor-only LIFEA2 (lacks the C-terminal mCherry). Middle graph: corresponding τ -histograms (color-coding right). Right graph: relative drop in τ (Δ τ , ns) after LIFEA2 stimulation with pre-clustered ephrinA1-Fc (2 μg ml −1 ) (mean±s.e.m.). Data obtained form three independent experiments, n =8 cells. ( f ) Western blot of anti-GFP IP LIFEA2 following stimulation with pre-clustered ephrinA1-Fc (2 μg ml −1 ). Blots probed with anti-phospho-tyrosine (PY72), anti-EphA2 and anti-GAPDH antibodies. Graph: phosphorylated fraction of EphA2 (PY72/EphA2)±s.e.m. from three blots. ( g ) Downstream signalling of endogenous EphA2, EphA2-mCitrine and LIFEA2 in Cos-7 cells on ephrinA1-Fc addition for the indicated time (min). Phosphorylated fraction of Akt (pAkt/Akt) and ERK1/2 (pERK/ERK)±s.e.m. from three blots. Scale bars: 10 μm.

Article Snippet: Primary antibodies: rabbit anti-phospho-EphA2/3/4 Y588/Y596 (pJM) (ab62256, Abcam, Cambridge, UK, 1:500); mouse anti-phospho-tyrosine (PY72) (P172.1, in vivo Biotech Services GMBH, 1:720), mouse anti-HA (MMS-101P, Covance, 1:1,000); rabbit anti-EphA2 (sc-924, Santa Cruz Biotechnologies, Santa Cruz, CA, 1:200); goat anti-EphA2 (AF-3035, R and D Systems, Minneapolis, MN, 1:500); mouse anti-EphA2 (IF7) (kind gift from M. Lackmann, 1:200); rabbit anti-c-Cbl (sc-170, Santa Cruz Biotechnologies, 1:1,000); mouse monoclonal anti-α-Tubulin (Sigma Aldrich, St. Louis, MO, 1:4,000); mouse monoclonal anti-GAPDH (CB-1001, Calbiochem, Merck Biosciences, Darmstadt, Germany, 1:3,000); living colours rabbit anti-GFP (632593, Clontech, Mountain View, CA, used for immunoprecipitation, 1:200); living colours mouse anti-GFP (632681, Clontech, Mountain View, CA, used in western blots, 1:1,000); rabbit anti-tRFP (AB234, Evrogen, Moscow, 1:2,000) and mouse anti-human IgG Fc fragment specific for clustering of Fc-fusion proteins (411455, Calbiochem), rabbit anti-Rab7 (9367, Cell Signaling Technology, Danvers, MA, 1:200), rabbit anti-Rab11a (2413, Cell Signaling Technology, used in immunofluorescence, 1:100), rabbit anti-Rab11a (ab65200, Abcam, used in western blots, 1:300), mouse anti-Rab11 (610656, BD Biosciences, used in immunofluorescence, 1:100), mouse anti-LAMP1 (ab25630, Abcam, 1:200), mouse anti-Rab5 (610281, BD Biosciences, 1:200), rabbit anti-phospho ERK-1/2 Thr/Tyr 202/204 (9101, Cell Signaling Technology, Danvers, MA, 1:500), mouse anti-ERK1/2 (Ab366991, Abcam, 1:1,000); rabbit anti phosphor-Akt Ser473 (9271, Cell Signaling Technology, Danvers, MA, 1:500); mouse anti-Akt (pan) (2920, Cell Signaling Technology, Danvers, MA, 1:1,000).

Techniques: Fluorescence, Western Blot

Journal: Nature Communications

Article Title: Ubiquitination switches EphA2 vesicular traffic from a continuous safeguard to a finite signalling mode

doi: 10.1038/ncomms9047

Figure Lengend Snippet: ( a ) Time-lapse of EphA2 and Rab11a/Rab7 colocalization in Cos-7 cells on ephrinA1 stimulation. Left panels: fluorescence images of EphA2-mCitrine (left column), bfp–Rab11a (middle), mCherry-Rab7 (right), at indicated time in min. Right graphs: ratiometric analysis of EphA2-mCitrine (WT) and Y813F-EphA2-mCitrine (Y813F) localization on different membrane compartments, with and without ectopic expression of c-Cbl (Cbl). Upper: PM (PM-EphA2), middle: Rab11a-positive RE (RE-EphA2, inset: blow-up of the first 10 min), lower: Rab7-positive late endosome (LE-EphA2). Bar graphs: average amplitude between 48–53 min±s.e.m. ( n =12–18 cells from at least three independent experiments). Upper right box: color-coding. ( b ) Immunostaining of endogenous EphA2, Rab7 and LAMP1 in Cos-7 cells before (left column) and 20 min post-stimulation of Cos-7 cells with pre-clustered ephrinA1-Fc, 2 μg ml −1 (right column). EphA2 (first row), Rab7 (second row), EphA2 (green) and Rab7 (magenta) overlay (third row), LAMP1 (fourth row), EphA2 (green) and LAMP1 (magenta) overlay (fifth row). Scale bars: 10 μm. ( c ) c-Cbl induces EphA2 ubiquitination and degradation. Left: western blot of Cos-7 total cell lysate (Total, upper) and anti-GFP IP EphA2-mCitrine or Y813F-EphA2-mCitrine (IP, lower) showing time course of stimulation with pre-clustered ephrinA1-Fc (2 μg ml −1 ). Pre-clustered Fc fragment (Fc, 2 μg ml −1 ) was used as control (dashed box). c-Cbl-bfp and HA-Ubiquitin were co-expressed. Total lysates were probed with anti-EphA2, anti-c-Cbl and anti-GAPDH antibodies. IP was probed with anti-HA for ubiquitin (Ub) and anti-EphA2 antibodies. Right graphs: upper, ratiometric quantification of EphA2/GAPDH from blots of EphA2 (red) or Y813F-EphA2 (green) (mean±s.e.m. normalized to zero time point, n =3 blots). Lower: ratiometric quantification of Ub (ubiquitin)/EphA2 from 3 blots of anti-GFP IP EphA2 (red) or Y813F-EphA2 (green), right boxes: control Fc fragment (120 min).

Article Snippet: Primary antibodies: rabbit anti-phospho-EphA2/3/4 Y588/Y596 (pJM) (ab62256, Abcam, Cambridge, UK, 1:500); mouse anti-phospho-tyrosine (PY72) (P172.1, in vivo Biotech Services GMBH, 1:720), mouse anti-HA (MMS-101P, Covance, 1:1,000); rabbit anti-EphA2 (sc-924, Santa Cruz Biotechnologies, Santa Cruz, CA, 1:200); goat anti-EphA2 (AF-3035, R and D Systems, Minneapolis, MN, 1:500); mouse anti-EphA2 (IF7) (kind gift from M. Lackmann, 1:200); rabbit anti-c-Cbl (sc-170, Santa Cruz Biotechnologies, 1:1,000); mouse monoclonal anti-α-Tubulin (Sigma Aldrich, St. Louis, MO, 1:4,000); mouse monoclonal anti-GAPDH (CB-1001, Calbiochem, Merck Biosciences, Darmstadt, Germany, 1:3,000); living colours rabbit anti-GFP (632593, Clontech, Mountain View, CA, used for immunoprecipitation, 1:200); living colours mouse anti-GFP (632681, Clontech, Mountain View, CA, used in western blots, 1:1,000); rabbit anti-tRFP (AB234, Evrogen, Moscow, 1:2,000) and mouse anti-human IgG Fc fragment specific for clustering of Fc-fusion proteins (411455, Calbiochem), rabbit anti-Rab7 (9367, Cell Signaling Technology, Danvers, MA, 1:200), rabbit anti-Rab11a (2413, Cell Signaling Technology, used in immunofluorescence, 1:100), rabbit anti-Rab11a (ab65200, Abcam, used in western blots, 1:300), mouse anti-Rab11 (610656, BD Biosciences, used in immunofluorescence, 1:100), mouse anti-LAMP1 (ab25630, Abcam, 1:200), mouse anti-Rab5 (610281, BD Biosciences, 1:200), rabbit anti-phospho ERK-1/2 Thr/Tyr 202/204 (9101, Cell Signaling Technology, Danvers, MA, 1:500), mouse anti-ERK1/2 (Ab366991, Abcam, 1:1,000); rabbit anti phosphor-Akt Ser473 (9271, Cell Signaling Technology, Danvers, MA, 1:500); mouse anti-Akt (pan) (2920, Cell Signaling Technology, Danvers, MA, 1:1,000).

Techniques: Fluorescence, Expressing, Immunostaining, Western Blot

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 2. Inhibition of p38 blocks NGF-induced neurite out- growth. A and B, PC12 cells were pretreated with the indicated con- centrations of SB203580 or 30 mM PD98059 for 30 min prior to treat- ment with 100 ng/ml NGF for 60 h. Representative images under a phase-contrast microscope (A) and quantitation of the percentage of cells with neurites (B) are shown. C and D, cells were cotransfected with pEGFP-C1 together with an empty expression vector SRa (2) or an expression vector encoding kinase-negative MKK6 (KN-MKK6), wild type p38 (WT-p38), or dominant-negative p38 (AGF-p38) (15). After 12 h the cells were treated with or without 10 mM SB203580. Then, 48 h after the transfection the cells were treated with or without 100 ng/ml NGF. Representative images of the transfected cells 60 h after NGF addition identified by the fluorescence of GFP (C) and quantitation of the percentage of cells with neurites (D) are shown.

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Microscopy, Quantitation Assay, Expressing, Plasmid Preparation, Dominant Negative Mutation, Transfection, Fluorescence

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 1. NGF induces p38 activation as well as ERK/MAPK ac- tivation. A, PC12 cells were treated with 100 ng/ml NGF or 50 mM arsenite for the indicated times (left) or with the indicated concentra- tions of NGF for 10 min (right), and the cell extracts were subjected to the immune complex kinase assay for p38 using activating transcrip- tion factor 2, ATF2, as a substrate (upper). The same cell extracts were subjected to immunoblotting with anti-phospho-p38 (middle) or anti- p38 antibodies (bottom). B, cells were pretreated with or without a p38 inhibitor SB203580 at 10 mM (lower or upper, respectively) for 30 min prior to NGF treatment as indicated, and the extracts were subjected to immunoblotting with anti-ERK/MAPK antibody. The electrophoreti- cally retarded bands represent active forms, i.e. phosphorylated forms of ERK/MAPK (ERK1 and ERK2, arrowheads) against inactive forms (arrows).

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Immune Complex Kinase Assay, Western Blot

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 3. Expression of a constitutively active MAPKK/MEK (SE- SE-KK) induces p38 activation as well as ERK/MAPK, and the p38 inhibitor blocks neurite outgrowth induced by SESE-KK. A, PC12 cells were cotransfected with pEGFP-C1 and either an empty expression vector SRa (2) or a constitutively active construct of MAPKK/MEK (SESE-KK (13); equivalent to Glu-217/Glu-221 MAPKK/ MEK in Ref. 7) expression vector and were treated with or without 10 mM SB203580. B, cells were cotransfected with HA-p38 or HA-ERK/ MAPK (MAPK) together with an empty expression vector SRa (2) or an expression vector encoding wild type MAPKK/MEK (WT-KK) or SE- SE-KK and assayed for the activity of HA-p38 or HA-MAPK. The activity of HA-p38 was also measured in cells treated with 100 ng/ml NGF for 10 min (1NGF). C, cells were cotransfected with pEGFP-C1 and either an empty expression vector SRa (2) or an SESE-KK expres- sion vector and were subjected to immunostaining with anti-phospho- p38 antibody.

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Activation Assay, Plasmid Preparation, Construct, Activity Assay, Immunostaining

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 4. EGF induces transient activation of p38 and, when combined with sustained activation of p38, causes neurite out- growth in PC12 cells. A, PC12 cells were treated with either 30 nM EGF (upper), 50 mM arsenite (upper), or 100 ng/ml NGF (lower) for the indicated times and assayed for p38 activity as described in the legend to Fig. 1A. B, cells were treated with EGF, NGF, or arsenite for the indicated times and subjected to immunostaining with anti-phospho- p38 antibody (lower; phase contrast, upper). C, cells were transfected with pEGFP-C1 together with either an empty expression vector SRa (2) or both wild type MKK6 and wild type p38 expression vectors (MKK6 & p38) and treated with or without 10 mM SB203580. 48 h after the transfection the cells were treated with or without EGF. Represent- ative images of the transfected cells 72 h after EGF addition identified by the fluorescence of GFP are shown. D, cells were treated with EGF, arsenite, or both for 1 h, washed, and then incubated in fresh medium. Representative images 60 h after the treatment under a phase-contrast microscope are shown.

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Activity Assay, Immunostaining, Transfection, Expressing, Plasmid Preparation, Fluorescence, Incubation, Microscopy

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: (a) Heatmap of EZH2 ChIP-seq signal intensity in CRPC 16DCRPC and 42DENZR cell lines (left), with overlaid H3K27Ac and H3K27Me3 histone mark ChIP-seq (right). Each horizontal line represents a 6-kb locus. (b) Representative ChIP-seq tracks surrounding the WNT5A locus in 16DCRPC and 42ENZR cells. Regions of EZH2 co-occupancy with the active H3K27Ac histone mark are highlighted. (c) Relative expression of genes bound by EZH2 alone (EZH2-none) or co-operatively with H3K27Me3 (EZH2-me) and H3K27Ac (EZH2-ac) histone marks in 42DENZR and 42FENZR cell lines. Box plot shows mean and interquartile range. (d) Heatmap of H3K27Me3 and K3K27Ac ChIP-seq signal intensity surrounding AR:EZH2 co-occupied regions in 42DENZR cells. (e) Heatmap indicating AR and EZH2 ChIP-seq signal intensity at AR:EZH2 co-occupied sites (n = 2155) in 42DENZR cells, and EZH2 signal intensity at the corresponding sites in AR-negative cell lines: NCI-H660, DU145 (GEO: GSE135623), and PC-3 (GEO: GSE123204). The shade of green (AR) or blue (EZH2) reflects binding intensity. Each horizontal line represents a 6-kb locus.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: ChIP-sequencing, Expressing, Binding Assay

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: (a) EZH2 was immunoprecipitated in 42ENZR cells, trypsin digested, and analyzed by mass spectrometry. Peptides covering 36% of EZH2 were recovered and analyzed for post-translational modifications. (n = 4 independent replicates). (b) Expression of total and phosphorylated (T350, S21, and T311 residues) EZH2 in the indicated cell lines. Protein abundance was assessed by densitometry and is reported relative to total EZH2. (c) IHC staining of pEZH2-S21 and pEZH2-T350 in serial sections from representative CRPC (n = 39) and NEPC (n = 26) patient tumours (Scale bar, 100 μm). Staining area and intensity was quantified and reported (mean ± SD; two-tailed unpaired t-test). (d) Expression of genes positively regulated by EZH2 when phosphorylated at S21 [defined by Xu et al.] in the indicated cell lines and patient tumours from the Beltran 2016 cohort. Statistical analysis was performed using a two-tailed unpaired t-test. Box plots show mean and interquartile range. ns, not significant. (e) qRT-PCR of NE lineage markers in CRPCcrEZH2 cells expressing myc-tagged EZH2S21A or EZH2S21D mutants, reported relative to empty vector transfected cells. (mean ± SD; two-tailed unpaired t-test, n = 3). Immunoblotting confirmed transgene expression. (f) Proliferation of parental 16DCRPC (control) and CRPCcrEZH2 cells stably expressing EZH2T350A and EZH2T350D phospho-mutants assessed by IncuCyte (mean ± SD, n = 3 replicates). Immunoblotting confirmed transgene expression. (g) qRT-PCR of plasticity and NE markers in VCaP and C4–2 cell lines co-transfected with EZH2 siRNA and siRNA-resistant myc-tagged EZH2WT, EZH2T350A, or EZH2T350D plasmid following treatment with ENZ (10 μM) for 7 days (mean ± SD; two-tailed unpaired t-test, n = 3).

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Immunoprecipitation, Mass Spectrometry, Expressing, Immunohistochemistry, Staining, Two Tailed Test, Quantitative RT-PCR, Plasmid Preparation, Transfection, Western Blot, Stable Transfection

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, Abundance of AR, FOXA1, SUZ12 and EED peptides detected using RIME with AR antibodies as bait. Each dot represents an independent replicate, with a solid line denoting the mean. b, SUZ12 immunoprecipitation (IP) followed by immunoblotting for AR and PRC2 subunits. The relative abundance of AR was normalized to SUZ12 pulldown. c, AR–EZH2 PLA and quantification of nuclear PLA signals (red dots) from a single plane (mean ±s.d.; P < 0.0001, two-tailed unpaired t-test; n = 3). Each dot represents the number of PLA signals in a single nucleus. Scale bar, 10 μm. d, Frequency of AR-bound genes with EZH2, SUZ12 and/or EED co-occupancy based on ChIP-seq peak annotation (±50 kb from the nearest TSS) in 42DENZR cells. e, Overlap of genomic regions co-occupied by AR and EZH2 ChIP-seq peaks (AR–EZH2 complex) with ChIP-seq peaks for the H3K27Me3 and H3K27Ac in 42DENZR cells. f, Heat map of AR and EZH2 ChIP-seq signal intensity in 16DCRPC and 42DENZR cells, with corresponding ATAC-seq peak intensity. g, Overlap of AR and EZH2 ChIP-seq peaks in 16DCRPC and 42DENZR cell lines. h, Overlap of AR and EZH2 ChIP-seq peaks in the Ptenf/f;Rb1f/f (DKO) GEMM. i, Enriched reactome pathways with genes co-occupied by AR–EZH2 in 42DENZR cells and the Ptenf/f/Rb1f/f GEMM. The size of each circular data point reflects the degree to which genes in the pathway are enriched based on RNA-seq from 42DENZR compared with 16DCRPC cells. NS, not significant. j, Expression of AR–EZH2 co-bound genes in matched prostate tumours (P1–P3) pre- and post-ENZ therapy (n = 3) from the DARANA trial. Box plot shows mean and interquartile range. Statistical analysis was performed using a paired t-test. k, Venn diagram of overlap in genes downregulated (log2FC < 1) in 42DENZR cells following depletion of AR using CRISPR (crAR) or EZH2 inhibition (10 μm GSK126; 96 h). The heat map depicts relative expression of select AR–EZH2 co-bound genes, reported relative to parental cells. l, Sequential ChIP (Re-ChIP) for selected binding sites in 42DENZR cells treated with vehicle or EZH2 inhibitor (10 μm GSK126, 96 h). Cells were first analysed by chromatin immunoprecipitation with AR antibody and then immunoprecipitated again with an AR or EZH2 antibody, as indicated. Results are reported relative to IgG control (mean ± s.d., n = 2).

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Immunoprecipitation, Western Blot, Two Tailed Test, ChIP-sequencing, RNA Sequencing Assay, Expressing, CRISPR, Inhibition, Binding Assay, Chromatin Immunoprecipitation

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, Expression of plasticity and neuroendocrine markers by real-time PCR (rtPCR) and Western blot in 16DCRPC cells with CRISPR-mediated EZH2 knockout (16DCRPC crEZH2) following 7 d ENZ treatment. Cells transfected with a non-silencing scrambled guide RNA (crSCR) served as a control. Data are reported relative to non-transfected cells (mean ± s.d., n = 3). Two-tailed unpaired t-test. b, Tumour growth velocity of CRPC cells with CRISPR-mediated EZH2 knockout transplanted subcutaneously into nude mice, followed by treatment with vehicle (veh) or ENZ (n = 5 mice per group). Box plots show mean and interquartile range. Mann–Whitney test. c, Gene expression analysis (by rtPCR) in 16DCRPC control and crEZH2 xenograft tumours at the experimental end point. Data are reported relative to vehicle-treated mice (mean ± s.d.; *P = 0.05, two-tailed unpaired t-test; n = 3 mice per treatment group). d, Strategy used to establish the 16Dreporter cell line carrying GFP and mCherry fluorescent reporters in the endogenous OCT4 and ASCL1 loci, respectively. Fluorescence-activated cell sorting (FACS) plot shows gating used to isolate the individual cell populations. HL, left homology arm; HR, right homology arm. e, Immunofluorescence images for OCT4-GFP (green) and ASCL1-mCherry (red) in CRPCreporter cells at the indicated time points after ENZ treatment. Single cells were tracked and are denoted with arrows. Scale bar, 100 μm. f, Fold change in transcript abundance of genes unique and common to the OCT4+, ASCL1+ and hybrid (OCT4+ASCL1+) FACS-isolated CRPCreporter cell populations relative to the negative population (log2FC cut-off of 1.5), by RNA-seq. g, MSigDB pathways enriched for common genes (n = 468) upregulated (defined as log2FC > 1.5) across OCT4+, ASCL1+ and hybrid (OCT4+ASCL1+) CRPCreporter populations relative to the negative population. Statistical analysis was performed using a hypergeometric test. h, EZH2 activity score, calculated on the basis of z-score-transformed expression of genes in the ‘Kondo EZH2 targets’ signature from MSigDB, in negative, OCT4+, ASCL1+ and hybrid (OCT4+ASCL1+) CRPCreporter FACS-isolated cell populations. i, Quantification of GFP+ and ASCL1+ fluorescent CRPCreporter cells following treatment with ENZ (10 μM) alone or in combination with EZH2 inhibitor (10 μM GSK126) using the IncuCyte fluorescent object counting algorithm (mean ± s.d., n = 2). Representative images at 8 d after treatment are shown. Scale bar, 50 μm.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, CRISPR, Knock-Out, Transfection, Two Tailed Test, MANN-WHITNEY, Fluorescence, FACS, Immunofluorescence, Isolation, RNA Sequencing Assay, Activity Assay, Transformation Assay

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, Heat map of AR, EZH2 and pEZH2-T350 ChIP-seq binding intensity in 42DENZR cells. Each horizontal line represents a 6-kb locus. b, Frequency of AR ChIP-seq peaks overlapping with EZH2 and pEZH2-T350 ChIP-seq peaks in 42DENZR cells. c, Distribution of AR–EZH2 and AR–pEZH2 co-bound peaks in relation to the TSS. Peaks were mapped into 5-kb bins. d, PLA analysis of the interaction between AR and pEZH2-T350, and quantification of nuclear PLA signals (red dots) from a single plane (mean ± s.d.; P = 3.8 × 10−10, two-tailed unpaired t-test; n = 3). Each dot represents the number of PLA signals in a single nucleus. Scale bar, 10 μm. e, Overlap of genes co-bound to AR–EZH2 occupied by SUZ12- and/or EED, based on ChIP-seq peak annotation in 42DENZR cells. Gene annotation was restricted to ±50 kb from TSS. f, Expression of genes with promoter-bound (defined as ±3 kb from TSS) AR alone or co-occupancy with EZH2 or pEZH2-T350 in 42DENZR and 42FENZR cell lines. Data are mean expression ± s.d., with significance assessed using a two-tailed unpaired t-test. g, Expression of AR–pEZH2 co-bound genes in matched individual patient tumours pre- and post-ENZ therapy from the DARANA trial (n = 3). Box plots show mean and interquartile range. Statistical analysis was performed using a paired t-test. h, Gene ontology signatures from MSigDB enriched for AR–EZH2 and AR–pEZH2 co-bound genes in 42DENZR cells. Statistical analysis was performed using a hypergeometric test. i, Immunohistochemical staining for AR, pEZH2-T350 and SYP (neuroendocrine marker) in serial sections from non-treated (naive) and neoadjuvant ADT/TAX-treated (4.5 months) prostate tumours from the CALGB 90203 clinical trial. Treated tumours were binned on the basis of pEZH2-T350 staining intensity, and matched NanoString-based sequencing was used to assess the expression of plasticity factors in pEZH2-low (n = 8) and pEZH2-high (n = 4) tumours. Box plots show mean and interquartile range of z-score-transformed expression values with significance assessed using a two-tailed unpaired t-test. Scale bar, 100 μm.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: ChIP-sequencing, Binding Assay, Two Tailed Test, Expressing, Immunohistochemical staining, Staining, Marker, Sequencing, Transformation Assay

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, Immunoblot of total and phosphorylated EZH2 and CDK1 in the indicated prostate cancer cell lines. HPCS, high-plasticity cell state. b, Immunoblot of EZH2 and pEZH2-T350 following CDK1 inhibition (5 μM RO-3306, 6 h). c, Immunohistochemical staining for pEZH2-T350 and pCDK1-T161 in serial sections from treatment-naive (N, n = 30), CRPC (CR, n = 40) and NEPC (NE, n = 26) clinical samples. Scale bar, 100 μm. Staining intensity was quantified (mean ± s.d.; two-tailed unpaired t-test). d, SUZ12 and EED peptides detected by RIME using EZH2 and pEZH2-T350 antibodies as bait in 42ENZR cells. Each dot represents an independent replicate, with a solid line denoting mean. Significance was defined as ≥4 peptides. e, Myc-tagged wild-type EZH2 (EZH2WT) and T350 phospho-mimicking (EZH2T350D) and phospho-dead (EZH2T350A) mutants were transiently transfected into 16DCRPC cells with endogenous EZH2 deletion for 72 h. Immunoprecipitation was performed using a Myc tag antibody. f, Distribution of pEZH2-T350, SUZ12 and EED ChIP-seq peaks in relation to the nearest TSS. The density of polycomb subunits and H3K27Ac are shown surrounding the WNT5A locus. g, Proportion of EZH2 and pEZH2-T350 ChIP-seq peaks overlapping with H3K27Me3 and H3K27Ac ChIP-seq peaks in 42DENZR cells. The distribution of H3K27Ac alone and co-occupied with pEZH2-T350 (pEZH2-ac) in relation to the TSS is shown. h, Single-sample GSEA (ssGSEA) score of MSigDB pathways in CRPCcrEZH2 cells expressing EZH2T350A or EZH2T350D mutant, and adenocarcinoma (CRPC-Adeno) and NEPC (CRPC-NE) patient specimens from the Beltran 2016 cohort4. The ASC score is shown below each cell line or individual patient. i, rtPCR and immunoblot in 42DENZR cells with EZH2 knockdown, stably expressing siRNA-resistant Myc-tagged EZH2WT or EZH2T350A mutant for 72 h. Data are reported relative to cells transfected with empty vector (EV) (mean ± s.d.; two-tailed unpaired t-test, n = 2). j, Immunohistochemical staining for EZH2 and SYP in serial sections from CRPCcrEZH2 EZH2T350A and EZH2T350D mutant xenografts treated with vehicle or ENZ. Scale bar, 100 μm. SYP staining intensity was quantified; box plots show mean and interquartile range. k, Flow cytometry plots of CD44 and NCAM1 cell surface expression in dissociated tumour cells from EZH2T350A and EZH2T350D mutant xenografts.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Western Blot, Inhibition, Immunohistochemical staining, Staining, Two Tailed Test, Transfection, Immunoprecipitation, ChIP-sequencing, Expressing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Plasmid Preparation, Flow Cytometry

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, PCA of global transcriptome in the indicated cell lines. 42DENZR cells with AR knockout (AR KO) and inhibited EZH2 activity (10 μM GSK126, 96 h) are shown. b, ASC and NEPC scores in patient tumours from ref. 5 (adenocarcinoma cluster 5, n = 28; AR+NE+, n = 10; AR−NE+, n = 3) and the indicated cell lines. c, GSEA signatures enriched (Fisher’s exact test, P < 0.05) in 42DENZR cells following AR knockout or EZH2 inhibition (10 μM GSK126, 96 h). d, Volcano plot of peptides detected by RIME using EZH2 antibodies as bait in 42DENZR cells treated with DMSO or EZH2 inhibitor (10 μM GSK126, 96 h). Statistical analysis was performed using a two-tailed unpaired t-test (n = 3). e, Immunoprecipitation of EZH2 in 42DENZR cells treated with 10 μM GSK126 for 96 h followed by immunoblotting. f, Immunoblot of SUZ12 in nuclear soluble and chromatin-bound fractions in 42DENZR cells treated with 10 μM GSK126 for 96 h. g, PLA analysis of AR–EZH2 in 42DENZR cells following EZH2 inhibition (10 μM GSK126, 96 h). Nuclear PLA signals from a single plane were quantified (mean ± s.d.; P = 3.1 × 10−16, two-tailed unpaired t-test; n = 3). Scale bar, 10 μm. h, Chromatin immunoprecipitation–PCR (ChIP–PCR) for AR at the AREs within the KLK3 enhancer in 42DENZR cells following treatment with EZH2 inhibitor (10 μM GSK126, 96 h). Results reported relative to IgG control (mean ± s.d.; P = 0.018, two-tailed unpaired t-test; n = 4). F, forward; R, reverse. i, rtPCR in 42DENZR cells treated with EZH2 inhibitor (10 μM GSK126 or GSK343, 96 h) or EED inhibitor (1 μM A-395, 96 h). Data reported relative to vehicle-treated cells (mean ± s.d., two-tailed unpaired t-test; n = 3). Western blot confirmed PRC2 inhibition. j, Confluency measured using IncuCyte (mean ± s.d., n = 2). At 48 h after seeding, cells were treated with EZH2 inhibitor (10 mM GSK126). k, Proliferation of 42DENZR cells treated with ENZ (10 μM) and EZH2 inhibitor (2 μM GSK126) alone or in combination, measured using IncuCyte. EZH2 inhibitor was removed (washout) at 96 h. Data plotted are mean ± s.d. (n = 3), with significance evaluated using a two-tailed unpaired t-test at the end point.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Knock-Out, Activity Assay, Inhibition, Two Tailed Test, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: (a) Immunoblot AR, EZH2, and H3K27Me3 (a surrogate marker of EZH2 activity) in 42DENZR cells following CRISPR-mediated AR deletion (crAR) or EZH2 inhibition (10 μM GSK126, 96 hrs). (b) Relative expression (qRT-PCR) of neuroendocrine lineage markers in 16DCRPC and C4–2 cell lines following siRNA-mediated AR silencing for 96 hours. Data are reported relative to cells transfected with a non-silencing scrambled control (mean ± SD, n = 3). A fold change >2 is considered significant. Immunoblotting confirmed AR knockdown.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Western Blot, Marker, Activity Assay, CRISPR, Inhibition, Expressing, Quantitative RT-PCR, Transfection

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: (a-b) qRT-PCR in 42DENZR (a) and 42FENZR (b) cells following siRNA-mediated EZH2 silencing (siEZH2) for the indicated time, reported relative to non-transfected control cells at day 0 (mean ± SD; two-tailed unpaired t-test, n = 3). NTC, non-targeting control. (c-d) Spheroid formation and ALDH activity in 42DENZR (c) and 42FENZR (d) cells following siRNA-mediated EZH2 silencing (siEZH2; left) or treatment with increasing dose of EZH2 inhibitor (GSK126; right) for 8 days (mean ± SD; two-tailed unpaired t-test, n = 2). (e) qRT-PCR in 42DENZR cells treated with EZH2 inhibitor (10 μM GSK126) for 7 days, followed by removal (washout) for 14 days. Expression is reported relative to cells at day 0 (mean ± SD; two-tailed unpaired t-test, n = 3). Immunoblotting confirmed on-target effect.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Transfection, Two Tailed Test, Western Blot

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Antiadipogenic effect of G. cambogia extract and the related protein expression in 3T3-L1 preadipocytes during differentiation. (A) Effect of G. cambogia extract (Ga, 300 μg/ml), FMK (3 μM) and stattic (5 μM) on RPS6KA1 and STAT3 phosphorylation in MDI-induced 3T3-L1 preadipocytes (differentiation started cells) for the indicated times (n = 4 per group). Con: MDI-untreated cells, MDI: MDI-treated cells. **p < 0.01 vs. Con, ##p < 0.01 vs. MDI. (B) Kinase activity of MAPK3/ERK1 and JAK2 in response to G. cambogia extract (n = 4 per group). The active MAPK3/ERK1 and JAK2 enzymes were used to assess kinase activity in the presence or absence of G. cambogia extract at the indicated concentrations in vitro. *p < 0.05 and **p < 0.01 vs. each control. (C) Effect of G. cambogia extract (300 μg/ml), FMK (3 μM) and stattic (5 μM) on CEBPA and PPARG expression in mature 3T3-L1 adipocytes (fully differentiated adipocytes) (n = 4 per group). Con: undifferentiated cells, Diff: mature 3T3-L1 adipocytes. **p < 0.01 vs. Con, ##p < 0.01 vs. Diff. (D) Effect of G. cambogia extract (300 μg/ml) on lipid accumulation in mature 3T3-L1 adipocytes at the indicated time points. The time table (upper) and representative images of Oil red O staining (below) are presented. EGCG (50 μM) was used as a positive control (n = 15 per group). Scale bar: 50 μm. (E) Effect of G. cambogia extract (300 μg/ml) on CEBPA and PPARG expression in mature 3T3-L1 adipocytes at the indicated time points (n = 4 per group). *p < 0.05 and **p < 0.01 vs. each group. The data are the mean ± S.D.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Expressing, Activity Assay, In Vitro, Staining, Positive Control

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Effect of CEBPB, RPS6KA1 and STAT3 regulation on autophagy-related protein expression in 3T3-L1 preadipocytes during differentiation. (A) Effect of Cebpb knockdown on CEBPB (LAP* and LAP), CEBPA, PPARG, BECN1, ATG7, ATG3, LC3, SQSTM1, ATG4B and ATG12–ATG5 expression in 3T3-L1 differentiated cells for 72 h. (B) Effect of Rps6ka1 and Stat3 knockdown on CEBPA, PPARG, BECN1, ATG7, ATG3, LC3, SQSTM1, ATG4B and ATG12–ATG5 expression in 3T3-L1 differentiated cells for 72 h (n = 4 per group). **p < 0.01 vs. each control. n.s.: not significant. (C) Effect of FMK (3 μM) and stattic (5 μM) on BECN1, ATG7, ATG3, LC3, SQSTM1, ATG4B and ATG12–ATG5 expression in 3T3-L1 differentiated cells for the indicated times (n = 4 per group). *p < 0.05 and **p < 0.01 vs. each control. The data are the mean ± S.D. (D) Schematic illustrating the interaction of CEBPB and autophagy with RPS6KA1 and STAT3. The dotted line means undisclosed facts.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Expressing

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Effect of G. cambogia extract on KLF3 expression in 3T3-L1 preadipocytes during differentiation. (A) Effect of G. cambogia extract (300 μg/ml) on KLF3, CEBPA and PPARG in 3T3-L1 differentiated cells for the indicated times (n = 4 per group). **p < 0.01 vs. each control. (B) Effect of G. cambogia extract (0–300 μg/ml) on CEBPB, KLF3, CEBPA and PPARG expression in 3T3-L1 differentiated cells after 72 h (n = 4 per group). *p < 0.05 and **p < 0.01 vs. Con. (C) Effect of FMK (3 μM) and stattic (5 μM) on CEBPB, KLF3, CEBPA and PPARG expression in 3T3-L1 differentiated cells for the indicated times (n = 4 per group). *p < 0.05 and **p < 0.01 vs. each control. (D) Effect of Cebpb, Rps6ka1 and Stat3 knockdown on KLF3 expression in 3T3-L1 differentiated cells after 72 h (n = 4 per group). **p < 0.01 vs. siCon. n.s.: not significant. (E) Effect of G. cambogia extract (300 μg/ml) on Klf3 transcript levels in 3T3-L1 differentiated cells for the indicated times (n = 9 per group). (F) Effect of G. cambogia extract (300 μg/ml) on the KLF3 protein half-life in 3T3-L1 differentiated cells after 72 h. After treatment of G. cambogia extract in cells, cycloheximide (CHX, 1.5 μg/ml) was coincubated with the indicated times (n = 4 per group). **p < 0.01 vs. each control. (G) Effect of G. cambogia extract (Ga, 300 μg/ml) for 72 h and MG132 (10 μM) and 3-MA (0.5 mM) for 24 h on SQSTM1 and KLF3 expression in 3T3-L1 differentiated cells (n = 4 per group). **p < 0.01 vs. Con. The data are the mean ± S.D.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Expressing

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Effect of G. cambogia extract on SQSTM1-mediated selective autophagic degradation of KLF3 in 3T3-L1 preadipocytes during differentiation. (A) Interaction of SQSTM1 and KLF3 in 3T3-L1 differentiated cells treated with G. cambogia extract (Ga, 300 μg/ml), FMK (3 μM) and stattic (5 μM) for 72 h. Coimmunoprecipitation (IP) was used to analyze the level of SQSTM1 that physically interacted with KLF3. The lysates were immunoprecipitated with anti-KLF3 and anti-IgG antibodies, and the precipitates were analyzed by western blotting using antibodies against SQSTM1 and KLF3 (n = 4 per group). (B) Effect of Sqstm1 knockdown on the interaction of SQSTM1 and KLF3 in 3T3-L1 differentiated cells treated with G. cambogia extract (300 μg/ml). Sqstm1 knockdown cells were treated with G. cambogia extract (300 μg/ml) for 72 h (n = 4 per group). (C) Effect of Sqstm1 knockdown on LC3, SQSTM1, KLF3, CEBPA and PPARG expression in 3T3-L1 differentiated cells treated with G. cambogia extract (300 μg/ml) for 72 h in the presence or absence of rapamycin (Ra, 10 nM) for 24 h (n = 4 per group). (D) Fluorescence photographs (left) and quantification data (below) of LC3 and KLF3 in Sqstm1 knockdown cells treated with G. cambogia extract (300 μg/ml) for 72 h in the presence or absence of rapamycin (10 nM) for 24 h. The fluorescence intensity of FITC (i.e., LC3) and TRITC (i.e., KLF3) was quantified using ImageJ software and colocalization (yellow dots indicated by white arrows) of FITC and TRITC per cell was determined and analyzed (n = 5 per group). Scale bars: 5 μm (E) Effect of Sqstm1 knockdown on G. cambogia extract- and rapamycin-mediated lipid accumulation in mature 3T3-L1 adipocytes (n = 12 per group). During full differentiation (day 0–8), cells were treated with G. cambogia extract (300 μg/ml), and rapamycin (10 nM) was added at day 3–8. Con: undifferentiated cells, Diff: mature 3T3-L1 adipocytes. *p < 0.05 and **p < 0.01 vs. each group. n.s.: not significant. The data are the mean ± S.D.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Immunoprecipitation, Western Blot, Expressing, Fluorescence, Software

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Effect of G. cambogia extract on KLF3 and CTBP2 interaction to regulate adipogenic factors in 3T3-L1 preadipocytes during differentiation. (A) Effect of G. cambogia extract (0–300 μg/ml) on CTBP2 expression in 3T3-L1 differentiated cells for 72 h (n = 4 per group). n.s.: not significant. (B) Effect of FMK (3 μM) and stattic (5 μM) on CTBP2 expression in 3T3-L1 differentiated cells for the indicated times (n = 4 per group). (C) Physical interaction of KLF3 and CTBP2 in 3T3-L1 differentiated cells treated with G. cambogia extract (Ga, 300 μg/ml), FMK (3 μM) and stattic (5 μM) for 72 h. Coimmunoprecipitation (IP) was used to analyze the interaction of KLF3 and CTBP2. The lysates were immunoprecipitated with anti-KLF3 and anti-IgG antibodies, and the precipitates were analyzed by western blotting using antibody against CTBP2 (n = 4 per group). (D) Fluorescence photographs (left) and quantification data (right) of KLF3 and CTBP2 in 3T3-L1 differentiated cells treated with G. cambogia extract (300 μg/ml), FMK (3 μM) and stattic (5 μM) for 72 h. The fluorescence intensity and colocalization of TRITC (i.e., KLF3) and FITC (i.e., CTBP2) were quantified using ImageJ software. Colocalization was analyzed using the Pearson correlation coefficient. Scale bars: 5 μm. (n = 6 per group). **p < 0.01 vs. Con, n.s.: not significant. (E) Effect of G. cambogia extract (300 μg/ml) on the transcript levels of Cebpa and Pparg in 3T3-L1 differentiated cells for the indicated times (n = 9 per group). **p < 0.01 vs. each control. The data are the mean ± S.D.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Expressing, Immunoprecipitation, Western Blot, Fluorescence, Software

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Effect of G. cambogia extract on the identified targets in HFD-induced adipose tissues. (A and B) Effect of G. cambogia extract on phospho- and total-RPS6KA1, phospho- and total-STAT3, LC3, SQSTM1, KLF3, CEBPA and PPARG expression of eWAT and iWAT in ND-fed, HFD-fed and HFD-fed mice administered a high dose of G. cambogia extract (400 mg/kg) (n = 6 per group). (C) Correlations between phospho-RPS6KA1, phospho-STAT3 and KLF3 protein expression in eWAT and iWAT (n = 6 per group). Each point represents one sample. (D and E) Immunofluorescence analysis of phospho-RPS6KA1, phospho-STAT3 and KLF3 expression in eWAT and iWAT (n = 4 per group). Nuclei were stained with DAPI (blue), and white arrows indicate phospho-RPS6KA1, phospho-STAT3 and KLF3 colocalization with DAPI. Scale bars, 50 μm. **p < 0.01 vs. ND-fed mice, #p < 0.05 and ##p < 0.01 vs. HFD-fed mice. The data are the mean ± S.D.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Analysis of the adipose tissues in the animal model. (A) Correlations between LC3-I, LC3-II, SQSTM1 and KLF3 protein expression in eWAT and iWAT (n = 6 per group). Each point represents one sample. (B) Lc3, Sqstm1, Klf3, Cebpa and Pparg transcript levels of eWAT and iWAT in ND-fed, HFD-fed and HFD-fed mice administered a high dose of G. cambogia extract (400 mg/kg) (n = 6 per group). (C) Correlations between Klf3, Lc3 and Sqstm1 transcript levels in eWAT and iWAT (n = 6 per group). Each point represents one sample. (D) Representative transmission electron micrographs of eWAT and iWAT in ND-fed, HFD-fed and HFD-fed mice administered a high dose of G. cambogia extract (400 mg/kg). The lower images are the enlarged representations of the boxed regions of the upper images. Scale bars: upper, 0.5 μm; lower, 200 nm. Autophagic vesicles are highlighted by white arrows and were quantified by counting the number of vesicles per 2 μm2 microscopic field in 4 randomly selected fields (n = 4 per group). Blue arrow: lipid droplet; green arrow: mitochondria. (E) Proposed mechanism for the antiobesity effect of G. cambogia extract in adipose tissue. **p < 0.01 vs. ND-fed mice, #p < 0.05 and ##p < 0.01 vs. HFD-fed mice. n.s.: not significant. The data are the mean ± S.D.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Animal Model, Expressing, Transmission Assay

Journal: JID Innovations

Article Title: A Series of BRAF- and NRAS-Driven Murine Melanoma Cell Lines with Inducible Gene Modulation Capabilities

doi: 10.1016/j.xjidi.2021.100076

Figure Lengend Snippet: Inducible gene modulation in murine melanoma cell lines. ( a ) Schematic depicting the generation of stable cell lines expressing inducible Cas9 and sgRNA targeting GFP or Mafg . Mouse melanoma cell lines were infected with a lentiviral construct expressing Dox-inducible Cas9 and selected with blasticidin. Subsequently, TRE-Cas9‒harboring cells were infected with GFP-U6-sgGFP or GFP-U6-sgMafg1 and selected with hygromycin, and polyclonal populations were used for experiments. ( b ) TRE-Cas9‒harboring mouse melanoma cell lines were infected with a GFP-U6-sgGFP reporter construct. Western blot showed that Dox treatment resulted in Cas9 (Flag) expression and a decrease in GFP levels. ( c ) TRE-Cas9‒harboring M10M3 and M167M1 cells were infected with GFP-U6-sgGFP or GFP-U6-sgMafg1. Cells were then treated with Dox, and Flag-Cas9, GFP, and MAFG expressions were analyzed by western blot. Cas9 was expressed on Dox treatment, GFP was decreased in sgGFP-expressing cells, and MAFG was decreased in sgMafg1-expressing cells. ( d ) M10M3 and ( e ) M167M1 cells harboring TRE-Cas9 and sgGFP or sgMafg1 were plated at a low density, and colony-forming ability was examined, which is shown as percent surface area covered by colonies. ( f ) M10M3 and ( g ) M167M1 cells harboring TRE-Cas9 and sgGFP or sgMafg1 were plated in soft agar, and anchorage-independent growth was examined. Dox, doxycycline; sgGFP, sgRNA targeting GFP; sgMafg1, sgRNA targeting Mafg .

Article Snippet: Antibodies against FLAG (1:1,000, catalog number 14793S; Cell Signaling Technology, Danvers, MA), GFP (1:2,000, catalog number 2956S; Cell Signaling Technology), MAFG (1:1,000, catalog number ab154318; Abcam, Cambridge, United Kingdom), p16INK4a (1:2,000, catalog number ab211542; Abcam), p19ARF (1:1,000, catalog number ab80; Abcam), p53 (1:1,000, catalog number 3036-100; BioVision, Milpitas, CA), phosphorylated AKT S473 (1:2,000, catalog number 9188S; Cell Signaling Technology), phosphorylated AKT T308 (1:500, catalog number 13038T; Cell Signaling Technology), AKT (1:5,000, catalog number 4691T; Cell Signaling Technology), phosphorylated ERK T202/Y204 (1:1,000, catalog number 9101S; Cell Signaling Technology), ERK (1:1,000, catalog number 4695S; Cell Signaling Technology), S100B (1:1,000, catalog number ab52942; Abcam), MART1 (1:1,000, catalog number SAB4500949-100UG; Sigma-Aldrich), Cre (1:1,000, catalog number ab190177; Abcam), MITF (1:1,000, catalog number 12590S; Cell Signaling Technology), and β-actin (1:10,000, catalog number AM4302; Thermo Fisher Scientific, Waltham, MA) were used.

Techniques: Stable Transfection, Expressing, Infection, Construct, Western Blot

Journal: Molecules

Article Title: Curcumol Synergizes with Cisplatin in Osteosarcoma by Inhibiting M2-like Polarization of Tumor-Associated Macrophages

doi: 10.3390/molecules27144345

Figure Lengend Snippet: Curcumol enhanced CDDP-induced cell apoptosis in K7M2 WT osteosarcoma cells. ( A ) Cell morphologies were shown in bright-field images after treatment with curcumol, CDDP or a combination of both for 48 h. ( B ) Curcumol, CDDP, or both were employed to treat K7M2 WT cells for 48 h. The cells were then labeled with DAPI, and fluorescence microscopy was used to analyze the nuclear alterations. ( C ) Cells were harvested after being exposed to drugs as described in ( B ), and a PI (propidium iodide) staining experiment was performed and evaluated by flow cytometry. ( D ) Statistical analysis of apoptosis cells in ( C ). ** p < 0.01; Student’s t test. ( E ) Western blotting for cleaved caspase-3 and cleaved PARP in treated K7M2 WT cells.

Article Snippet: Cleaved PARP and cleaved caspase-3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Labeling, Fluorescence, Microscopy, Staining, Flow Cytometry, Western Blot

Journal: Stem cells (Dayton, Ohio)

Article Title: Somatic stem cell marker prominin-1/CD133 is expressed in embryonic stem cell-derived progenitors.

doi: 10.1634/stemcells.2004-0232

Figure Lengend Snippet: Figure 2. Double immunofluorescence and confocal imaging analysis of prominin-1 (cell-surface labeling) and various intracellular marker proteins in R1 and P19 cells cultured for 6 hours (R1) and 48 hours (R1, P19). (A): Six-hour R1 cells showed no prominin-1–positive cells, whereas nestin-positive cells (green) were detected in approximately 28% of cells. (B): R1 (48-hour) cells showed almost no coexpression of prominin-1 (red) and SSEA-1 (green). SSEA-1–positive cells were localized in the center of ES cell aggregates, whereas prominin-1–positive cells were restricted to peripheral regions of the ES cell colonies. Prominin-1 was localized to the cell membrane (inset). (C–H): R1 (48-hour) cells showed partial coexpression of (C) prominin-1 (red) and nestin (green), (D) CK18 (green), and (E) desmin (green) and areas colabeled by (F) prominin-1 (green) and fibulin-1 (red), (G) VCP (red), and (H) FRP-2 (red), respectively, at the periphery of R1 cell aggregates. Hoechst 33342 (blue) was used to visualize cell nuclei. Insets show intracellular localization of proteins at higher magnification. As a control for the cell- surface labeling of (B–H) prominin-1, R1 (48-hour) cells after fixation with paraformaldehyde were saponin-permeabilized and incubated with prominin-1 antibody. Prominin-1 labeling was again detected at (I) the peripheral region of cell aggregates (red). (K–L): To control unspecific binding, R1 (48-hour) cells were labeled only with the secondary antibodies (Cy3 goat anti-rat antibody [for rat monoclonal 13A4 antibody] [K], fluorescein isothiocyanate mouse anti-rabbit [for rabbit polyclonal antibody] [L]). (M–S): P19 cells cultured for 48 hours showed prominin-1 (red) expression in almost all cells and a partial coexpression with (M) SSEA-1 (green) and (N) nestin (green) but no colabeling with (O) desmin (green). P19 cells were colabeled by (P) prominin-1 (green) and fibulin-1 (red), (R) VCP (red), and (S) FRP-2 (red), respectively. Bars = 30 μm. Abbreviations: ES, embryonic stem; FRP, frizzled-related protein; SSEA, stage-specific embryonic antigen; VCP, valosin containing protein.

Article Snippet: After three washes with Ca/Mg-PBS, cells were incubated with fluorochrome-conjugated secondary antibodies (Cy3 anti-mouse immunoglobulin M [IgM], rat immunoglobulin G [IgG], or goat IgG [1:600] and/or fluorescein isothiocyanate [FITC] anti-mouse IgG or IgM, rabbit IgG, and rat IgG [1:100]; Jackson ImmunoResearch, Dianova, Hamburg, Germany, http://www.jacksonimmuno.com).

Techniques: Immunofluorescence, Imaging, Labeling, Marker, Cell Culture, Membrane, Control, Incubation, Binding Assay, Expressing

Journal: Stem cells (Dayton, Ohio)

Article Title: Somatic stem cell marker prominin-1/CD133 is expressed in embryonic stem cell-derived progenitors.

doi: 10.1634/stemcells.2004-0232

Figure Lengend Snippet: Figure 3. Semiquantitative immunofluorescence, FACS, and Western blot analysis of prominin-1 in P19 (48-hour) and R1 cells. (A): Semi- quantitative immunofluorescence analysis for prominin-1–positive Hoechst 33342–labeled P19 cells (48 hours); R1 cells cultured in the presence of LIF (6-hour, 48-hour, and 7-day colonies); and R1 cells dif- ferentiated for 2–28 days in the absence of LIF. For each value, 10 areas (~1,000 cells) of each coverslip (n = 3) of three independent experiments were analyzed. Values represent mean ± SD. (B–F): FACS analysis of prominin-1 immunoreactivity in (B) P19 cells (48 hours), (C) R1 6-hour cells, (D) R1 48-hour cells, (E) R1 7-day colonies, and (F) R1 cells after differentiation for 14 days (7 + 14 days) in the absence of LIF. The his- tograms represent the fluorescence of the prominin-1–immunolabeled cells (black) compared with corresponding control cells (gray; labeled with only secondary antibody) after gating the cells in a forward/side- scatter dot plot. (G): Western blotting analysis of prominin-1 (top panel, arrow) in undifferentiated R1 cells (48-hour and 7-day colonies) and R1 cells after further differentiation for 21 days (7 + 21 days). Adult kid- ney membrane and P19 (48-hour) cells were used as positive control. In each sample, the total amount of protein was visualized by Coomassie- blue staining (C.B.) (bottom panel). (H): Quantification of prominin- 1 immunoreactivity during the differentiation of R1 cells. Prominin-1 immunoreactivity detected in (G) was quantified by densitometric scan- ning, normalized to the amount of protein detected by C.B. (reference band indicated by asterisks) (G), and plotted as percentage of prominin- 1 immunoreactivity detected in undifferentiated R1 cells (7-day colo- nies). Each value represents the mean of two independent experiments. Abbreviations: FACS, fluorescence-activated cell sorter; FITC, fluo- rescein isothiocyanate; LIF, leukemia inhibitory factor.

Article Snippet: After three washes with Ca/Mg-PBS, cells were incubated with fluorochrome-conjugated secondary antibodies (Cy3 anti-mouse immunoglobulin M [IgM], rat immunoglobulin G [IgG], or goat IgG [1:600] and/or fluorescein isothiocyanate [FITC] anti-mouse IgG or IgM, rabbit IgG, and rat IgG [1:100]; Jackson ImmunoResearch, Dianova, Hamburg, Germany, http://www.jacksonimmuno.com).

Techniques: Immunofluorescence, Western Blot, Labeling, Cell Culture, Fluorescence, Immunolabeling, Control, Membrane, Positive Control, Staining

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β accumulates at sites of DNA damage in an ATM/H2AX/MDC1-dependent manner. ( A ) U2OS cells were microirradiated, fixed 5 min later, and immunostained for WRAP53β and γH2AX, a marker for DNA DSBs. Nuclei were stained with DAPI in all immunofluorescence experiments. ( B ) U2OS cells were treated with siControl or two different WRAP53β targeting oligonucleotides (siWRAP53#1 and siWRAP53#2) for 48 h, irradiated (6 Gy, 1-h recovery) or left untreated, fixed after pre-extraction with cytoskeleton (CSK) buffer, and immunostained for WRAP53β and γH2AX. ( C ) U2OS cells were irradiated (6 Gy), fixed, and immunostained for WRAP53β at the indicated time points. Quantification is given as the percentage of the 100 cells counted in each experiment whose nuclei contained WRAP53β IRIF. ( D ) U2OS cells were treated with the inhibitors or siRNAs, as indicated, for 6 h or 48 h, respectively; irradiated (6 Gy, 1-h recovery); fixed after pre-extraction with CSK buffer; and immunostained for WRAP53β and γH2AX. ( E ) Quantification of the results in D , as the percentage of the 100 cells counted in each experiment whose nuclei contained WRAP53β IRIF. The error bars depict the SEM; n = 3; (***) P < 0.001 as determined by Student’s t -test. ( F ) ChIP assay showing the recruitment of WRAP53β to the I-PpoI-induced DSB at chromosome 1 in MCF7 cells stably expressing ddI-PpoI. The time indicated is hours after the addition of 4-OHT. The I-PpoI cleavage site on chromosome 1 is located at distance 0. Cells were cultivated in medium containing 0.1% FBS for 24 h before DSB induction. Data are shown as the mean of two independent experiments. The Y -axis displays the fold change in relative occupancy normalized to the control.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Marker, Staining, Immunofluorescence, Irradiation, Extraction, Stable Transfection, Expressing, Control

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β promotes recruitment of repair proteins to DSBs. ( A ) U2OS cells were transfected with siControl or siWRAP53#2 oligonucleotides for 24 h, exposed to IR (6 Gy) or left untreated, and, 1 h later, immunostained for γH2AX, MDC1, BRCA1, 53BP1, and RAD51. ( B ) U2OS cells treated as in A and then immunostained for RNF168 and conjugated ubiquitin (with the FK2 antibody). In the case of GFP-RNF8 staining, following treatment with oligonucleotides for 24 h, the cells were transiently transfected with the GFP-RNF8 plasmid for 8 h, exposed to IR (6 Gy), allowed to recover for 1 h, and then fixed and analyzed. ( C ) Quantification of the results in A and B as the percentage of 200 cells counted in each experiment whose nuclei contained IRIF. In the case of GFP-RNF8, only successfully transfected cells were counted. ( D ) U2OS cells were treated with the siRNAs indicated for 24 h, irradiated (6 Gy), allowed to recover for 1 h, and then subjected to Western blotting for WRAP53β, H2AX, and β-actin. The error bars depict the SEM. n = 3; (**) P < 0.01; (***) P < 0.001, as determined by Student’s t -test.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Transfection, Ubiquitin Proteomics, Staining, Plasmid Preparation, Irradiation, Western Blot

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β binds MDC1 and RNF8 via their FHA domains. ( A ) U2OS cells were either left untreated or irradiated with 6 Gy of IR, and, 30 min, later immunoprecipitation of WRAP53β was performed, followed by immunoblotting of WRAP53β, MDC1, GFP-RNF8, and β-actin. ( B ) U2OS cells were transfected with the indicated HA-MDC1 constructs for 16 h and irradiated with 2 Gy, and, 30 min later, immunoprecipitation of WRAP53β was performed, followed by immunoblotting of WRAP53β and HA-MDC1. ( C ) Schematic illustration of RNF8 deletion constructs. ( D ) U2OS cells were transiently transfected with EGFP-RNF8 plasmids, HA-MDC1, and Flag-WRAP53β for 16 h; irradiated; and subjected to immunoprecipitation of GFP followed by immunoblotting for GFP-RNF8, Flag-WRAP53β, and HA-MDC1. (HC) Heavy chain of the antibody. U2OS ( E ) and H1299 ( F ) cells were transiently transfected with Flag-RNF8 plasmids, HA-MDC1, and EGFP-WRAP53β for 16 h; irradiated; and subjected to Flag immunoprecipitation followed by immunoblotting for the indicated proteins. ( G ) Schematic illustration of the domain architecture of MDC1 and RNF8, where black lines mark WRAP53β- and MDC1-binding sites. Numbers indicate amino acids.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Irradiation, Immunoprecipitation, Western Blot, Transfection, Construct, Binding Assay

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β facilitates MDC1–RNF8 interaction through its WD40 domain. ( A ) U2OS cells were treated with the siRNAs indicated for 48 h and with GFP-RNF8 for 24 h (all samples), irradiated with 6 Gy, and, 30-min later, subjected to immunoprecipitation of WRAP53β followed by immunoblotting of WRAP53β, MDC1, RNF8, γH2AX, and β-actin. ( B ) Immunoprecipitation of MDC1 in irradiated (6 Gy, 15-min recovery) U2OS cells treated with the siRNA indicated for 48 h or ATM inhibitor (ATMi) for 24 h. All samples were transfected with GFP-RNF8 for 16 h. ( C ) U2OS cells were treated with the siRNAs indicated for 48 h or ATM inhibitor for 16 h, irradiated with 6 Gy, allowed to recover for 15 min, and then subjected to Western blotting of MDC1, WRAP53β, γH2AX, and β-actin. ( D ) Schematic illustration of EGFP-tagged deletion constructs of WRAP53β. ( E ) U2OS cells were transiently transfected with the indicated EGFP-WRAP53β plasmids and Flag-RNF8 for 16 h, irradiated, and subjected to GFP immunoprecipitation followed by immunoblotting for MDC1, Flag-RNF8, and GFP-WRAP53β. (HC) Heavy chain of the antibody. ( F ) U2OS cells were transfected with siControl or siWRAP53#2 oligonucleotides for 8 h followed by transfection of EGFP-Empty or EGFP-WRAP53β WD40 (1–7) for 16 h, exposed to IR (6 Gy), and, after 1 h, immunostained for 53BP1 followed by quantification of the results. The graph in A shows the percentage of 100 GFP transfected cells in each experiment whose nuclei were 53BP1-positive. The error bars depict the SEM. n = 3; (*) P < 0.05, as determined by Student’s t -test. ( G ) U2OS cells were transiently transfected with the indicated EGFP-WRAP53β plasmids, HA-MDC1, and Flag-RNF8 for 16 h; irradiated; and subjected to immunoprecipitation of GFP followed by immunoblotting for HA-MDC1, Flag-RNF8, and GFP-WRAP53β. ( H ) Schematic illustration of how WRAP53β scaffolds the MDC1–RNF8 complex. Upon DNA damage, WRAP53β binds MDC1 and RNF8 simultaneously via its WD40 domain and facilitates their interaction.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Irradiation, Immunoprecipitation, Western Blot, Transfection, Construct

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β promotes HR and NHEJ. ( A ) U2OS cells were treated with the siRNAs indicated for 24 h, exposed to 6 Gy of IR, fixed 1 h or 24 h later, and immunostained for γH2AX. ( B ) Quantification of the results in A showing the percentage of nuclei containing >10 γH2AX foci ( n = 200). ( C , D ) HR ( C ) and NHEJ ( D ) efficiency following treatment of the cells with the siRNA indicated for 48 h. DR-GFP (HR) and EJ5-GFP (NHEJ) reporter systems were used in the FACS analysis, with siRAD51 and siArtemis as positive controls. ( E ) Cells were transfected with siRNA for 24 h, exposed to IR (3 Gy), harvested at the time points indicated, and subjected to flow cytometry. Nonirradiated cells were treated with siRNA alone for 60 h. The error bars depict the SEM. n = 3; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, as determined by Student’s t -test.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Transfection, Flow Cytometry

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β protects cells against accumulation of spontaneous DNA damage. ( A ) U2OS cells were treated with siControl or two different siWRAP53 oligonucleotides (siWRAP53#1 and siWRAP53#2) for 24 h, fixed, and immunostained for WRAP53β and γH2AX. ( B ) The percentage of nuclei in A containing >10 γH2AX foci was quantified in the 200 cells counted for each experiment. ( C ) After treating U2OS with siWRAP53#2 or siControl for 24 h or 48 h, DNA damage was assessed by the alkaline comet assay. The error bars depict the SEM. n = 3; (**) P < 0.01; (***) P < 0.001, as determined by Student’s t -test.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Alkaline Single Cell Gel Electrophoresis

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: Schematic model of WRAP53β function in the DDR pathway. (Step 1) In response to IR, γH2AX and MDC1 accumulate at DSBs independently of WRAP53β. ATM-mediated phosphorylation of MDC1 makes MDC1 competent to bind RNF8. However, RNF8 is not yet localized at DSBs. (Step 2) WRAP53β is recruited to sites of DNA damage in an ATM-, H2AX-, and MDC1-dependent manner. Simultaneous binding of MDC1 and RNF8 to WRAP53β facilitates their direct interaction and retention of RNF8 at DSBs. (Step 3) Once assembled at DSBs, RNF8 catalyzes ubiquitylation of H2AX. Ubiquitylation at DSBs enables recruitment and accumulation of 53BP1, BRCA1, and RAD51 and subsequent DNA repair.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Phospho-proteomics, Binding Assay

Journal: Nanotoxicology

Article Title: Reactive oxygen species-mediated p38 MAPK regulates carbon nanotube-induced fibrogenic and angiogenic responses

doi: 10.3109/17435390.2011.647929

Figure Lengend Snippet: Single-walled carbon nanotube (SWCNT)-induced fibrogenic effects are mediated through p38 mitogen-activated protein kinase (MAPK) pathway. A. CRL-1490 cells were pretreated for 1 h with LY294002 (10 μM), SP200165 (10 μM), SB203580 (10 μM) and PD98059 (10 μM) and then treated with Survanta (SRVT) (1.5 μg/ml) or SWCNTs (10 μg/ml). Cell supernatants were analysed for soluble collagen content by Sircol® collagen assay after 24 h. B. CRL-1490 cells were pretreated for 1 h with LY294002 (10 μM), SP200165 (10 μM), SB203580 (10 μM) or PD98059 (10 μM) and then treated with SRVT (150 μg/ml) or SWCNTs (10 μg/ml) for 24 h and analysed for cell growth. C. CRL-1490 cells were treated with various concentrations of SRVT (0–3.75 μg/ml) and SWCNTs (0–25 μg/ml) for 6 h after which they were washed with PBS and extracted with SDS sample buffer. The cell extracts were separated on 10% polyacrylamide-SDS gels, transferred and probed with antibodies against phospho-p38 MAPK and total p38 MAPK. The immunoblot signals were quantified by densitometry. D. Cells were either left untreated or pretreated with NAC (10 mM) or catalase (CAT) (1000 U/ml) for 1 h, followed by SRVT (1.5 μg/ml) or SWCNTs (10 μg/ml) for 6 h. Cell lysates were prepared and analysed for phospho-p38 MAPK and total p38 MAPK. The immunoblot signals were quantified by densitometry. E. CRL-1490 cells were pretreated for 1 h with SB203580 (10 μM) and then treated with SRVT (1.5 μg/ml) or SWCNTs (10 μg/ml) for 3 h. Samples were analysed for reactive oxygen species (ROS) production by measuring DCF fluorescence intensity. Plots are mean ± SD (n = 3). *, p < 0.05 versus nontreated control. **, p < 0.05 versus nontreated control and corresponding SRVT treatment. #, p < 0.05 versus SWCNT control and corresponding SRVT treatment.

Article Snippet: Antibodies against phospho-p38 MAPK and total p38 MAPK were obtained from Cell Signaling Technology, Inc. (Beverly, MA).

Techniques: Sircol Collagen Assay, Western Blot, Fluorescence

Journal: Nanotoxicology

Article Title: Reactive oxygen species-mediated p38 MAPK regulates carbon nanotube-induced fibrogenic and angiogenic responses

doi: 10.3109/17435390.2011.647929

Figure Lengend Snippet: Single-walled carbon nanotube (SWCNT)-induced TGF-β1 activation through reactive oxygen species (ROS)-mediated p38 mitogen-activated protein kinase (MAPK) pathway. A. CRL-1490 cells were treated with various concentrations of Survanta (SRVT) (0–3.75 μg/ml) and SWCNTs (0–25 μg/ml) for 24 h and analysed for TGF-β1 by ELISA. B. Cells were pretreated for 1 h with NAC (10 mM), catalase (CAT) (1000 U/ml), SB203580 (10 μM), TGF-β1 (100 ng/ml) or TGF-β1 inhibitor (LY364947) (10 μM) and then treated with SRVT (1.5 μg/ml) and SWCNTs (10 μg/ml) for 24 h and analysed for TGF-β1 by ELISA. C. CRL-1490 cells were pretreated for 0.5 h with TGF-β1 (100 ng/ml) or TGF-β1 inhibitor (10 μM) and then treated with SRVT (1.5 μg/ml) and SWCNTs (10 μg/ml) for 3 h. Samples were analysed for ROS production by measuring DCF fluorescence intensity. Plots show relative fluorescence intensity over nontreated control at the peak response time of 3 h after the treatment. D. Cells were either left untreated or pretreated with TGF-β1 (100 ng/ml) or TGF-β1inhibitor (LY364947) (10 μM) for 1h, followed by SWCNTs (10μg/ml) for 6h. Cell lysates were prepared and analysed for phospho-p38 MAPK and total p38 MAPK. The immunoblot signals were quantified by densitometry. E. CRL-1490 cells were pretreated for 1 h with TGF-β1 (100 ng/ml) or TGF-β1 inhibitor (LY364947) (10 μM) and then treated with SRVT (1.5 μg/ml) or SWCNTs (10 μg/ml) for 24 h and analysed for cell growth. F. CRL-1490 cells were pretreated for 1 h with TGF-β1 (100 ng/ml) or TGF-β1 inhibitor (10 μM) and then treated with SRVT (1.5 μg/ml) and SWCNTs (10 μg/ml). Cell supernatants were analysed for soluble collagen content by Sircol® collagen assay after 24 h. G. Cells were pretreated for 1 h with TGF-β1 (100 ng/ml) or TGF-β1 inhibitor (10 μM) and then treated with SRVT (1.5 μg/ml) and SWCNTs (10 μg/ml) for 24h and analysed for collagen I by western blotting. Blots were reprobed with β-actin antibody to confirm equal loading of the samples. The immunoblot signals were quantified by densitometry. Plots are mean ± SD (n = 4). *, p < 0.05 versus nontreated control. **, p < 0.05 versus nontreated control and corresponding SRVT treatment. #, p < 0.05 versus SWCNT control and corresponding SRVT treatment.

Article Snippet: Antibodies against phospho-p38 MAPK and total p38 MAPK were obtained from Cell Signaling Technology, Inc. (Beverly, MA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Western Blot, Sircol Collagen Assay

Journal: Nanotoxicology

Article Title: Reactive oxygen species-mediated p38 MAPK regulates carbon nanotube-induced fibrogenic and angiogenic responses

doi: 10.3109/17435390.2011.647929

Figure Lengend Snippet: Single-walled carbon nanotube (SWCNT)-induced VEGF plays a key role in fibrogenesis. A. CRL-1490 cells were treated with various concentrations of Survanta (SRVT) (0–3.75 μg/ml) and SWCNTs (0–25 μg/ml) for 24 h and analysed for VEGF by ELISA. B. CRL-1490 cells were either left untreated or pretreated with NAC (10 mM), catalase (CAT) (1000 U/ml) or SB203580 (10 μM) for 1 h and then treated with SRVT (1.5 μg/ml) or SWCNTs (10 μg/ml) for 24 h. Samples were analysed for VEGF by ELISA. C. CRL-1490 cells were pretreated for 0.5 h with VEGF (100 nm) or VEGF inhibitor (CBO-P11) (10 μm) and then treated with SRVT (1.5 μg/ml) or SWCNTs (10 μg/ml) for 3 h. Samples were analysed for reactive oxygen species (ROS) production by measuring DCF fluorescence intensity. Plots show relative fluorescence intensity over nontreated control at the peak response time of 3 h after the treatment. D. Cells were either left untreated or pretreated with VEGF (100 nm) or VEGF inhibitor (CBO-P11) (10 μm) for 1 h, followed by SWCNTs (10 μg/ml) for 6 h. Cell lysates were prepared and analysed for phospho-p38 mitogen-activated protein kinase (MAPK) and total p38 MAPK. The immunoblot signals were quantified by densitometry. E. CRL-1490 cells were pretreated for 1 h with VEGF (100 nm) or VEGF inhibitor (CBO-P11) (10 μm) and then treated with SRVT (1.5 μg/ml) or SWCNTs (10 μg/ml) for 24 h and analysed for cell growth. F. CRL-1490 cells were pretreated for 1 h with VEGF (100 nm) or VEGF inhibitor (10 μm) and then treated with SRVT (1.5 μg/ml) or SWCNTs (10 μg/ml) for 24 h. Cell supernatants were analysed for soluble collagen content by Sircol® collagen assay. G. Cells were either left untreated or were pretreated with TGF-β1 (100 ng/ml) and TGF-β1 inhibitor (LY364947) (10 μM) followed by SRVT (1.5 μg/ml) or SWCNTs (10 μg/ml) for 24 h. Samples were analysed for VEGF by ELISA. H. CRL-1490 cells were pretreated for 1 h with VEGF (100 nm) or VEGF inhibitor (10 μm) and then treated with SRVT (1.5 μg/ml) or SWCNTs (10 μg/ml) for 24 h and analysed for TGF-β1 by ELISA. Plots are mean ± SD (n = 3). *, p < 0.05 versus nontreated control, **, p < 0.05 versus nontreated control and corresponding SRVT treatment. #, p < 0.05 versus SWCNT control and corresponding SRVT treatment.

Article Snippet: Antibodies against phospho-p38 MAPK and total p38 MAPK were obtained from Cell Signaling Technology, Inc. (Beverly, MA).

Techniques: Enzyme-linked Immunosorbent Assay, Fluorescence, Western Blot, Sircol Collagen Assay