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

Article Title: Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

doi: 10.1073/pnas.1601053113

Figure Lengend Snippet: (A) Schematic representation of a 3-plex SILAC approach for profiling phosphorylation dynamics in resting and BCR-stimulated DG75 cells. DG75 cells were cultured in SILAC medium as indicated and were left untreated, or were BCR-stimulated, for 2, 5, 10, or 20 min. Daudi cells were stimulated for 2 and 10 min. Lysates were mixed in a 1:1:1 ratio and digested with trypsin. Resulting phosphopeptides were enriched by either SCX/TiO2 chromatography (global phosphoproteome analysis; GPome) or phosphotyrosine immunoprecipitation (pY-IP; pYome analysis), and analyzed by LC-MS/MS. For analysis of protein expression levels, proteins were separated by 1D-PAGE, digested with trypsin, and analyzed by LC-MS/MS (see SI Materials and Methods for details). (B) Schematic representation of a 2-plex SILAC approach for profiling phosphorylation changes upon inducible CD79a knockdown or upon SYK inhibition. DG75 cells were cultured in SILAC medium and treated as indicated. Lysates were processed as described in A. (C) DG75 and Daudi cells were loaded with the ratiometric Ca2+-chelator INDO-1-AM and subjected to BCR-induced Ca2+ flux analysis by flow cytometry.

Article Snippet: Antibodies against the following proteins were used: SLP65, pSLP65, PLCγ2, pPLCγ2, ERK, pERK, SYK, pSYK, BTK, pBTK, CD79a, pCD79a, CBL, pCBL, ACTN4, actin (all from Cell Signaling Technology), pTyr (4G10; Millipore) and ARFGEF2 (Abcam).

Techniques: Cell Culture, Chromatography, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Expressing, Inhibition, Flow Cytometry

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

Article Title: Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

doi: 10.1073/pnas.1601053113

Figure Lengend Snippet: Tonic BCR signaling. (A) CD79a shRNAs are toxic for BL cell lines. The figure shows the fraction of GFP-positive, shRNA-expressing cells relative to the GFP-negative, shRNA-negative fraction at the times indicated (normalized to day 0). Data are representative of three experiments. (B) CD79a and actin immunoblots of lysates derived from DG75 cells that were treated with doxycycline for 18 h to express either unspecific shRNAs (Control) or shRNAs targeting CD79a. (C) BCR cell surface expression in DG75 control cells or CD79a knockdown cells was monitored by flow cytometry 18 h after shRNA induction. (D and E) Unsupervised clustering analysis of all p-sites that were regulated upon BCR stimulation/CD79a knockdown/SYK inhibition. Values for each p-site (row) in all conditions (columns) are colored based on the z-score of the log2-transformed SILAC ratios.

Article Snippet: Antibodies against the following proteins were used: SLP65, pSLP65, PLCγ2, pPLCγ2, ERK, pERK, SYK, pSYK, BTK, pBTK, CD79a, pCD79a, CBL, pCBL, ACTN4, actin (all from Cell Signaling Technology), pTyr (4G10; Millipore) and ARFGEF2 (Abcam).

Techniques: shRNA, Expressing, Western Blot, Derivative Assay, Flow Cytometry, Inhibition, Transformation Assay

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

Article Title: Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

doi: 10.1073/pnas.1601053113

Figure Lengend Snippet: (A) BCR cell surface expression was monitored by flow cytometry (red line, DG75; blue line, Daudi). (B) Scatter plots showing the fold-change of p-sites on mainly serines/threonines (GPome, Left) and mainly tyrosines (pYome, Right) as determined by quantitative MS upon BCR stimulation versus CD79a knockdown and SYK inhibition. Selected phosphorylated proteins and p-sites are highlighted.

Article Snippet: Antibodies against the following proteins were used: SLP65, pSLP65, PLCγ2, pPLCγ2, ERK, pERK, SYK, pSYK, BTK, pBTK, CD79a, pCD79a, CBL, pCBL, ACTN4, actin (all from Cell Signaling Technology), pTyr (4G10; Millipore) and ARFGEF2 (Abcam).

Techniques: Expressing, Flow Cytometry, Inhibition

Journal: Scientific Reports

Article Title: Patient derived mutation W257G of PPP2R1A enhances cancer cell migration through SRC-JNK-c-Jun pathway

doi: 10.1038/srep27391

Figure Lengend Snippet: ( a–d ) SKOV3 and HEC-251 cells stably expressing PPP2R1A were serum starved for 12 h. These cells were lysed. The phosphorylation levels of AKT, p44/42, SRC, FAK, SAPK/JNK, and c-Jun and the expression levels of SRC, p44/p42, p21, PR65, b-actin, and c-Jun were analyzed by western blotting. ( e ) After cells were wounded by scratching the surface with a pipette tip, cells were treated with PP2 or SP600125 at indicated concentrations. The images of wound-scratch assays were taken at 0 and 15 h post scratching. The relative closed-wound distance was calculated after measuring the width of at least four wounds. *p < 0.05, **p < 0.01; one-tailed Student’s t test.

Article Snippet: The following antibodies were used: PR65 (sc-15355, Santa Cruz, CA, USA), PP2Ac (05-421, Millipore), FLAG M2 (F1804, Sigma Aldrich), HA (sc-7392, Santa Cruz), phospho-AKT (Thr308) (9275, Cell Signaling), phospho-AKT (Ser473) (9271, Cell Signaling), phospho-SRC (Tyr416) (6943, Cell Signaling), phospho-FAK (Tyr397) (3283, Cell Signaling), phospho-PP2Ac (Tyr307) (ab32104, Abcam), SRC (2108, Cell Signaling), p21 (2946, Cell Signaling), β-actin (sc-4778, Santa Cruz), phospho-SAPK/JNK (Thr183, Tyr185) (9251, Cell Signaling), phospho-c-Jun (Ser73) (9164, Cell Signaling), phospho-c-Jun (Ser63) (9261, Cell Signaling), and c-Jun (sc-1694, Santa Cruz).

Techniques: Stable Transfection, Expressing, Western Blot, Transferring, One-tailed Test

Journal: PLoS Pathogens

Article Title: SopF, a phosphoinositide binding effector, promotes the stability of the nascent Salmonella -containing vacuole

doi: 10.1371/journal.ppat.1007959

Figure Lengend Snippet: (A) HeLa cells were transfected with a plasmid encoding for EGFP-SopF for 18 h and then subject to confocal fluorescence microscopy or sequential detergent fractionation. Left panel shows a representative confocal microscopy image. EGFP-SopF (greyscale), DNA (blue). Scale bar is 10 μm. Right panel shows immunoblotting analysis. Cells were collected and subject to sequential detergent fractionation. Equal volumes of saponin-soluble, TX-100-soluble, and SDS-soluble fractions were separated by SDS-PAGE and subject to immunoblotting with antibodies against GFP, Hsp27 (cytosol), calnexin (membranes) and lamin A/C (nucleus). Molecular mass markers are indicated on the left. Results are representative of two independent experiments. (B) As for (A) except HeLa cells were transfected with a plasmid encoding for FLAG-SopF. FLAG-SopF was detected by immunostaining (left panel) or immunoblotting (right panel) with anti-FLAG antibodies. (C) SopF partially colocalizes with actin-binding proteins found at cell adhesion sites. HeLa cells were transfected with pFLAG-SopF for 18 h, then fixed and immunostained with anti-FLAG, anti-moesin, anti-lamellipodin and anti-vasodilator-stimulated phosphoprotein (VASP) antibodies. Representative confocal microscopy images show FLAG-SopF in green and moesin, lamellipodin or VASP in red. Scale bars are 10 μm. Insets show enlargements of boxed areas.

Article Snippet: Membranes were blocked at room temperature for 1 h with Tris-buffered saline (TBS) containing 5% (w/v) skim milk powder and 0.1% (v/v) Tween-20 (TBST-milk), then incubated with the following primary antibodies overnight at 4˚C: mouse anti-FLAG M2 affinity isolated (1:2,000 dilution; Sigma), mouse anti-HA.11 ascites (1:2,000; BioLegend), rabbit polyclonal anti-GFP (1:40,000; Thermo), mouse anti-β-lactamase (clone 8A5.A10, 1:2,000 dilution; Thermo), mouse anti-Hsp27 (clone G31, 1:20,000; Cell Signaling), rabbit polyclonal anti-calnexin (1:40,000; Enzo), rabbit polyclonal anti-lamin A/C (1:5,000; Cell Signaling) or mouse anti-LAMP-1 (clone H4A3, 1:1,000 dilution; Developmental Studies Hybridoma Bank).

Techniques: Transfection, Plasmid Preparation, Fluorescence, Microscopy, Fractionation, Confocal Microscopy, Western Blot, SDS Page, Immunostaining, Binding Assay

Journal: PLoS Pathogens

Article Title: SopF, a phosphoinositide binding effector, promotes the stability of the nascent Salmonella -containing vacuole

doi: 10.1371/journal.ppat.1007959

Figure Lengend Snippet: (A) HeLa epithelial cells were infected with S . Typhimurium wild type (WT), Δ sopF , Δ sopF pSopF (comp), Δ sopF pSopF(1–345), Δ sopF pSopF(1–367) or Δ sopF pACYC177 (empty vector) bacteria. The proportion of cytosolic bacteria was determined by CHQ resistance assay at 90 min p.i. (upper panel) or GAL8 recruitment at 1 h p.i. (lower panel, all bacteria are constitutively expressing mCherry for fluorescence detection). Upper panel: data represent the mean ± SD (n≥3 independent experiments). Lower panel: Data represent the mean ± SD (total of >600 bacteria per strain from n≥3 independent experiments). Asterisks indicate data significantly different from WT infection (one-way ANOVA with Dunnett’s post-hoc test). (B) HeLa cells were transfected with plasmids encoding for FLAG-SopF(1–367) or FLAG-SopF(1–345) for 18 h. Cells were fixed and immunostained with anti-FLAG antibodies. DNA was stained with Hoechst 33342. Representative confocal microscopy images show FLAG-SopF in greyscale and DNA in blue. Scale bars are 10 μm. (C) Subcellular fractionation of transfected cells. HeLa cells were transfected with plasmids encoding for FLAG-SopF, FLAG-SopF(1–367) or FLAG-SopF(1–345) for 18 h, then collected and subjected to sequential detergent fractionation. Equal volumes of saponin-soluble, TX-100-soluble, and SDS-soluble fractions were separated by SDS-PAGE and subject to immunoblotting with antibodies against the FLAG epitope, Hsp27 (cytosol), calnexin (membranes) and lamin A/C (nucleus). Molecular mass markers are indicated on the left. Results are representative of two independent experiments. (D) C-terminal truncations of SopF lose plasma membrane association in the S . cerevisiae mss4 tet-off strain. Wild type (WT) and mss4 tet-off yeast strains were transformed with plasmids encoding for yEGFP-SopF, yEGFP-SopF C370S, yEGFP-SopF(1–367) or yEGFP-SopF(1–345) and the subcellular localization of SopF in live cells was visualized by widefield fluorescence microscopy. Representative fluorescence images are shown. Scale bars are 2 μm. The role of a potential lipidation site in SopF localization was assessed by site-directed mutagenesis of the Cys370 residue (C370S). The grey box depicts a domain of unknown function (DUF), DUF3626, spanning amino acid residues 178–338 of SopF. (E) Quantification of SopF localization in WT and mss4 tet-off yeast strains that were transformed and visualized as described in (D). Subcellular localization was categorized as cytosol, internal membrane sites (IMS), plasma membrane (PM), or IMS and PM. Results are expressed as the mean percentage of total yeast transformants (n = 300 cells from three independent transformations).

Article Snippet: Membranes were blocked at room temperature for 1 h with Tris-buffered saline (TBS) containing 5% (w/v) skim milk powder and 0.1% (v/v) Tween-20 (TBST-milk), then incubated with the following primary antibodies overnight at 4˚C: mouse anti-FLAG M2 affinity isolated (1:2,000 dilution; Sigma), mouse anti-HA.11 ascites (1:2,000; BioLegend), rabbit polyclonal anti-GFP (1:40,000; Thermo), mouse anti-β-lactamase (clone 8A5.A10, 1:2,000 dilution; Thermo), mouse anti-Hsp27 (clone G31, 1:20,000; Cell Signaling), rabbit polyclonal anti-calnexin (1:40,000; Enzo), rabbit polyclonal anti-lamin A/C (1:5,000; Cell Signaling) or mouse anti-LAMP-1 (clone H4A3, 1:1,000 dilution; Developmental Studies Hybridoma Bank).

Techniques: Infection, Plasmid Preparation, Expressing, Fluorescence, Transfection, Staining, Confocal Microscopy, Fractionation, SDS Page, Western Blot, FLAG-tag, Transformation Assay, Microscopy, Mutagenesis

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: (A) HEK293T cells were transfected with p53, Isg15-modifying enzymes and difference oncogenes (Src, Ras, Myc). In some cases, proteosome inhibitor MG132 (25 μM) was added. After Ni-beads pulldown, Isg15-modified p53 was analyzed by Western blotting with 1801 antibody. (B) Different oncogenes increase the interaction between p53 and Herc5. HEK293T cells were transfected with HA-p53, Flag-Herc5 and difference oncogenes (Src, Ras, Myc). Flag-Herc5 was immunoprecipitated using anti-Flag M2-conjugated beads, and p53 in the complexes was detected by Western blotting. (C) Isg15 siRNA knockdown enhances Src-mediated p53 stabilization. HEK293T cells were transfected with HA-p53, Src, and control or Isg15 siRNAs. Cell lysates were analysed by Western blotting using indicated antibodies. (D) Overexpression of Ras or Myc increases Src phosphorylation as a read-out of its activity. HEK293T cells were transfected with Ras or Myc. The Src phosphorylation was analyzed by Western blotting using phospho-Src Tyr416 antibody.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: Transfection, Modification, Western Blot, Immunoprecipitation, Over Expression, Activity Assay

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: (A) Isg15 knockdown by siRNA enhances Src activity-mediated p53 stabilization. The Src mutant (8A7F, R388A/Y527F) was stably transfected in HCT116 cells with Flag-tagged endogenous p53. The cells were transfected with control or Isg15 siRNA. 48h after transfection, the cells were treated with imidazole (5 mM) to activate Src activity. Cytosolic (C) and Nuclear (N) fractions were then separated, and the level of p53 was analyzed by Western blotting. Parp and RhoGDI were used as nuclear and cytosolic markers, respectively. (B) The increase of Src activity by chemical rescue promotes p53 tyrosine phosphorylation. The Src mutant (8A7F, R388A/Y527F) was stably transfected in HCT116 cells with Flag-tagged endogenous p53. After treatment of MG132 (25 μM) for 1.5h, the cells were treated with imidazole (5 mM) to activate Src activity, and collected at the indicated times. Cytosolic (C) and Nuclear (N) fractions were then separated. p53 was immunoprecipitated using anti-flag M2 beads and analyzed with phospho-Tyrosine antibody. Parp and RhoGDI were used as nuclear and cytosolic markers, respectively.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: Activity Assay, Mutagenesis, Stable Transfection, Transfection, Western Blot, Immunoprecipitation

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: (A) Src phosphorylates p53 in vitro . In vitro kinase assay was performed by incubating purified His-tagged p53 with Src. The products were analysed by Western blotting using anti-phospho-Tyrosine antibody. (B) Src increases Tysosine phosphorylation of p53 in HEK293T cells. His-p53 was co-transfected Src and analyzed by Western blotting with 1801 antibody after Ni-beads pulldown. (C) Src phosphorylates p53 at Tyr126 and Tyr220. HEK293T cells were transfected with Src and WT, Y126F, Y220F, or 2F (Y126F+Y220F) His-53. p53 was precipitated with Ni-beads and analyzed with phospho-Tyrosine antibody. The results were quantified by densitometry and analyzed by GelPro software (lower panel). (D) Phospho-mimicking mutations ofTyr126 and Tyr220 increases p53 ISGyaltion. HEK293T cells transfected with WT, Y126D, Y220D, or 2D (Y126D+Y220D) mutants of p53 were analyzed for p53 ISGylation after Ni-beads pull-down. (E) Phospho-mimicking mutations of Tyr126 and Tyr220 increase p53 interaction with Herc5. HEK293T cells were transfected with WT, Y126D, Y220D, or 2D (Y126D+Y220D) p53 mutants and Flag-Herc5. Flag-Herc5 was immunoprecipitated and p53 was analyzed by Western blotting and results were quantified by densitometry (lower panel). (F) Y220C mutation increases p53 ISGylation. HEK293T cells transfected with WT, Y220C, or Y220D p53 mutants together with Isg15-modifying enzymes were analyzed for p53 ISGylation after Ni-beads pull-down.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: In Vitro, Kinase Assay, Purification, Western Blot, Transfection, Software, Immunoprecipitation, Mutagenesis

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: (A) Isg15 knockout increases unfolding and folding form of p53 in the transformed cells. Lysates from V-Src transformed mouse embryo fibroblasts (MEFs) (WT or Isg15 knockout) were immunoprecipitated with p53 antibodies Ab1620 or Ab240. The immunoprecipitated p53 was analysed by Western blotting. (B) Knockout of Isg15 increases the expression of p21 gene in transformed cells. RT-PCR was performed to analyse the p21 expression of V-Src transformed WT or Isg15 knockout MEFs cells. (C&D) Isg15 knockout enhances p53-mediated inhibition of transformation. (C) V-Src transformed WT, Isg15 knockout, or Isg15/p53 double knockout MEFs were grown in soft agar. Colonies were stained with MTT and counted 3 weeks later. (D) Transformed MEFs were injected into NSG nude mice. Tumors were collected and analyzed 21d after injection.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: Knock-Out, Transformation Assay, Immunoprecipitation, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Inhibition, Double Knockout, Staining, Injection

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: (A) Isg15 knockout impairs K-ras-induced lung tumors. 10-week-old Kras mice (control mice, n=10 (including 5 WT and 5 Isg15 Het mice); Isg15 knockout mice, n=8) were sacrificed, and lung lesions were counted (shown with arrows). (B) Isg15 knockout decrease the proliferation of K-ras-induced lung cancer. The lung collected from Isg15 Het or Isg15 knockout K-ras mice was sectioned and stained with Ki67 antibody. The positive cells of Ki67 in tumor region were showed and quantified. (C) Knockout of Isg15 increases the expression of p21 gene in K-ras-induced lung cancer. The K-ras-induced tumors were dissected from lungs containing cancer lesions. RT-PCR was performed to analyse the p21 mRNA expression. (D) Knockdown of Isg15 enhances the Nutlin-mediated inhibition of cell proliferation. MTT assay were performed with MCF7 cells transfected with either control, Isg15 siRNA, p53 siRNA or a combination of siRNAs in the absence or presence of Nutlin (3 μM). The difference of proliferating activity between 24h to 72h after seeding was shown in graphs.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: Knock-Out, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Inhibition, MTT Assay, Transfection, Activity Assay

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: (A) Knockdown of Isg15 increases DNA damage-induced p53 response. HCT116 cells were transfected with control or Isg15 siRNAs, or together with p53 siRNA. Cells were irradiated with 8 Gy of IR (upper panel) or treated with UV (30 J/m 2 ) (lower panel) and collected at the time points indicated. Cell lysates were analysed by Western blotting using indicated antibodies. (B) Knockdown of Isg15 increases DNA damage-induced inhibition of survival. HCT116 cells that transfected as described in (A) were processed in colony formation assay after treatment with 1Gy of IR. 3000 cells were plated for each condition. (C) Isg15 knockout increases unfolding and folding form of p53 in the Isg15 knockdown cells. HCT116 cells were transfected with control or Isg15 siRNAs, then were irradiated with 8 Gy of IR for 6h. The cell lysates were collected and immunoprecipitated with p53 antibodies Ab1620 or Ab240. For testing the specificity of the conformation-specific antibody, the lysates were incubated at 37°C for 8 min to denature folded p53.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: Transfection, Irradiation, Western Blot, Inhibition, Colony Assay, Knock-Out, Immunoprecipitation, Incubation

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: In normal cells, ISGylation primarily targets misfolded dominant-negative form of p53 thus promoting a total p53 activity (1). In cancer cells, the level of Isg15 is increased and the presence of different oncogenes promotes the interaction between p53 and Herc5 to increase the p53 ISGlyation. As a result, native p53 is targeted by Isg15-dependent degradation reducing the overall p53 activity.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: Dominant Negative Mutation, Activity Assay

Journal: bioRxiv

Article Title: Coupling of Polo kinase activation to nuclear localization by a bifunctional NLS is required during mitotic entry

doi: 10.1101/202440

Figure Lengend Snippet: a. Mutation of NLS residues or T182D substitution abrogates the KD-PBD interaction. GST-PBD or GST-bound sepharose beads were incubated with lysates of cells transfected with different forms of Flag-KD (with the IDL) as indicated. Pull-down products were analyzed by Western blots. b. Mutation of NLS residues or T182D substitution abrogates the Polo-Map205 interaction. Cells were transfected as indicated and PrA-Map205 was purified. Samples were analyzed by Western blotting. c. Bioluminescence Resonance Energy Transfer (BRET) reveals the impact of T182D and NLS7A mutations on the KD-PBD interaction in live cells. HEK293T cells were transfected with a fixed amount of Luc-KD (with the IDL) expression vector and increasing amounts of PBD-GFP expression vector. A third plasmid expressing a Map205 fragment (Map) which stabilizes the PBD-KD complex was co-transfected. When the KD and PBD interact, the luciferase (Luc) moiety, upon reaction with coelenterazine, transfers energy to GFP, which then fluorescences (BRET). Differences in BRET 50 (the GFP/Luc ratio at which BRET is half-maximal) reflect differences in affinity. AU: arbitrary units. Error bars: standard deviation of triplicate values from a representative experiment. d. Mutation of NLS residues increases Polo kinase activity. Immunoprecipitated Polo-GFP (WT and mutants) were used in kinase reactions using casein as a substrate. For Polo inhibition, BI2536 was added at 300 nM. Reactions were analyzed by autoradiography, Western blots, and amido black (total protein). e. Mutation of NLS-interacting PBD residues prevents the KD-PBD interaction. Experiment as in (a). f. Mutation of NLS-interacting PBD residues increases Polo kinase activity. Experiment as in (d). g. Model for coupling of Polo activation and nuclear localization. See text for details. The crystal structure of a complex between the KD (green) and the PBD (cyan) of zebrafish Polo and the inhibitory peptide from Drosophila Map205 (red) (Protein Data Bank accession no. 4J7B ) was used for structure rendering with PyMOL 1.4.

Article Snippet: Primary antibodies used in Western blotting and immunofluorescence were anti-Flag M2 produced in mouse (#F1804, Sigma), anti-GST from rabbit (#2622, Cell Signaling Technology), anti-GFP from rabbit (#A6455, Invitrogen), anti-GFP from mouse (#1218, abcam), peroxidase-conjugated ChromPure rabbit IgG (#011-030-003, for PrA detection, Jackson ImmunoResearch), anti-α-tubulin DM1A from mouse (#T6199, Sigma), anti-Myc 9E10 from mouse (#sc-40, Santa Cruz Biotechnology, Inc.), mouse monoclonal antiPolo MA294 (a gift of D. Glover, University of Cambridge, Cambridge, UK), anti-RFP from rabbit (#62341, abcam), anti-Lamin Dm0 (DSHB Hybridoma Product ADL84.12, ADL84.12 was deposited to the DSHB by Fisher, P.

Techniques: Mutagenesis, Incubation, Transfection, Western Blot, Purification, Bioluminescence Resonance Energy Transfer, Expressing, Plasmid Preparation, Luciferase, Standard Deviation, Activity Assay, Immunoprecipitation, Inhibition, Autoradiography, Activation Assay

Journal: bioRxiv

Article Title: Mesoderm-specific transcript reduces ciliary sphingomyelin levels to promote tendon stem/progenitor cells osteochondrogenesis in traumatic heterotopic ossification

doi: 10.1101/2025.03.01.640937

Figure Lengend Snippet: (a) Fibroblast-like TSPCs extracted from mice for subsequent experiments (scale bar 100 um). (b) Flow cytometry analysis of the expression of Pdgfra and Prrx1 (stem/progenitor cell surface markers) on TSPCs. (c) Immunofluorescence images of ARL13B (red) and α-tubulin (green) mark the cilia in shNC, shIFT88, and shARL3 TSPCs. DAPI staining indicates nuclei (blue), scale bar 10 um (upper) and 30 um (lower). (d) The percentage of ciliated TSPCs marked by ARL13B show the cilia counts in the control group and ciliary gene knockdown groups. (e) Cilia length in TSPCs with or without shIFT88 or shARL3 treatment (n= 25/group). (f) Representative images of alizarin red staining (upper) and alcian blue staining (lower) in TSPCs with or without shIFT88 or shARL3 treatment after 3 weeks of in vitro osteogenic or chondrogenic differentiation. (g) qPCR showed relative osteogenesis-related genes ( Alpl, Runx2, Bglap, Sp7 ) and chondrogenesis-related genes ( Col2a1, Sox9, Acan ) mRNA expression in TSPCs with or without shIFT88 or shARL3 treatment after 3 weeks of in vitro osteogenic or chondrogenic differentiation. Data are presented as means ± SD of three independent assays. Statistical analyses were performed by one-way ANOVA analyses with Tukey’s post hoc test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. ns, P > 0.05.

Article Snippet: Primary antibodies used are as follows: ARL13B (1:500, #17711-1-AP, Proteintech Group Inc.); Acetyl-α-Tubulin (Lys40) (1:800, #12152, Cell Signaling Technology) ; PDGFRα (1:100, #sc-338, Santa Cruz); CD31 (1:500, #11265-1-AP, Proteintech Group Inc.); F4/80 (1:500, #28463-1-AP, Proteintech Group Inc.); TNMD(1:100, #PA5-112767, Invitrogen); α-SMA(1:500, #14395-1-AP, Proteintech Group Inc.); MEST (1:250, #11118-1-AP, Proteintech Group Inc.).

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Staining, Control, Knockdown, In Vitro

Journal: bioRxiv

Article Title: Mesoderm-specific transcript reduces ciliary sphingomyelin levels to promote tendon stem/progenitor cells osteochondrogenesis in traumatic heterotopic ossification

doi: 10.1101/2025.03.01.640937

Figure Lengend Snippet: (a) The volcano plot visualizes the differential expressed genes in injury site of the tHO mice 3 weeks after burn/tenotomy compared to the control mice. (b) RNA-seq performed between shIft88 and shNC TSPCs after 3-7 days of in vitro osteogenic differentiation and differential expressed genes are visualized. (c) Venn diagram shows the common differential expressed genes among three genesets and identified MEST, PTN, COL2A1 as the key genes. (d) The expression density of Mest in each cluster are shown by violin plot and UMAP plot. (e) qPCR shows relative Mest mRNA expression in TSPCs with or without shIFT88 or shARL3 treatment during in vitro osteogenic (left) or chondrogenic (right) differentiation. (f) Western blot shows MEST protein expression levels in TSPCs with or without shIFT88 or shARL3 treatment during in vitro osteogenic (left) or chondrogenic (right) differentiation. (g) UMAP plot shows the expression of Mest at different time points (Day0 ,3, 7, 21) during tHO development. (h) Immunohistochemistry images demonstrates MEST in tHO tendon tissues at different time post burn/tenotomy injury (scale bar 100 um). (i) Immunofluorescence images of α-tubulin (green) and MEST (red) in mouse primary TSPCs. DAPI staining indicates nuclei (blue), scale bar 10 um. Data are presented as means ± SD of three independent assays. Statistical analyses were performed by one-way ANOVA analyses with Tukey’s post hoc test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.

Article Snippet: Primary antibodies used are as follows: ARL13B (1:500, #17711-1-AP, Proteintech Group Inc.); Acetyl-α-Tubulin (Lys40) (1:800, #12152, Cell Signaling Technology) ; PDGFRα (1:100, #sc-338, Santa Cruz); CD31 (1:500, #11265-1-AP, Proteintech Group Inc.); F4/80 (1:500, #28463-1-AP, Proteintech Group Inc.); TNMD(1:100, #PA5-112767, Invitrogen); α-SMA(1:500, #14395-1-AP, Proteintech Group Inc.); MEST (1:250, #11118-1-AP, Proteintech Group Inc.).

Techniques: Control, RNA Sequencing, In Vitro, Expressing, Western Blot, Immunohistochemistry, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Mesoderm-specific transcript reduces ciliary sphingomyelin levels to promote tendon stem/progenitor cells osteochondrogenesis in traumatic heterotopic ossification

doi: 10.1101/2025.03.01.640937

Figure Lengend Snippet: (a) Representative images of alizarin red staining (upper) and alcian blue staining (lower) in TSPCs with or without siMest treatment after 3 weeks of in vitro osteogenic or chondrogenic differentiation. (b) qPCR shows relative Mest and osteogenic differentiation-related ( Alpl, Runx2, Bglap ) or chondrogenic differentiation-related genes ( Col2a1, Sox9, Acan ) mRNA expression in TSPCs with or without siMest treatment after 3 weeks of in vitro osteogenic or chondrogenic differentiation. (c) tSNE map shows Mest-high and Mest-low subclusters divided from Mesenchymal cluster. The expression level of Mest, Runx2, Sox9 , and Acan in Mest-high and Mest-low subclusters were visualize by violin plots. (d) Immunofluorescence images of α-tubulin (green) mark the cilia of TSPCs. DAPI staining indicates nuclei (blue), scale bar 10 um. Ciliated TSPCs proportion (cilia counts) marked by α-tubulin and cilia length (n= 25/group) in the siNC and siMest group. (e) Micro-CT images show tHO of lower limbs in shNC and shMest groups (n= 4/group). Quantification of tHO degree by bone volume (BV, mm^3) and bone mineral dendity (BMD, g/cc). Data are presented as means ± SD of three independent assays. Statistical analyses were performed by Student’s t-test for two-group comparison and one-way ANOVA analyses with Tukey’s post hoc test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. ns, P > 0.05.

Article Snippet: Primary antibodies used are as follows: ARL13B (1:500, #17711-1-AP, Proteintech Group Inc.); Acetyl-α-Tubulin (Lys40) (1:800, #12152, Cell Signaling Technology) ; PDGFRα (1:100, #sc-338, Santa Cruz); CD31 (1:500, #11265-1-AP, Proteintech Group Inc.); F4/80 (1:500, #28463-1-AP, Proteintech Group Inc.); TNMD(1:100, #PA5-112767, Invitrogen); α-SMA(1:500, #14395-1-AP, Proteintech Group Inc.); MEST (1:250, #11118-1-AP, Proteintech Group Inc.).

Techniques: Staining, In Vitro, Expressing, Immunofluorescence, Micro-CT, Comparison

Journal: bioRxiv

Article Title: Mesoderm-specific transcript reduces ciliary sphingomyelin levels to promote tendon stem/progenitor cells osteochondrogenesis in traumatic heterotopic ossification

doi: 10.1101/2025.03.01.640937

Figure Lengend Snippet: (a) Immunofluorescence images of α-tubulin (green) marked cilia in ciliated and non-ciliated mouse primary TSPCs after cilia isolation. DAPI staining indicates nuclei (blue). (b) OPLS-DA plot shows the differences between NC (blue, n= 7) and SIM (green, n= 6) groups. (c) Circular diagram of the classification statistics of differential lipids. (d) Bubble chart of lipid classification. The horizontal axis represents the quantity range of lipids, and the vertical axis represents the logarithmic transformation of fold change. Each circle represents a lipid substance. The larger the circle, the higher the variable importance. Different colors represent the primary classification, and lipids of the same classification are arranged together. (e) The volcano plot visualizes the differential expressed lipids in SIM group compared to the NC group. (f) Relative abundance of sphingomyelin and triglyceride are significantly different in SIM and NC group. (g) KEGG enrichment analysis of differential expressed lipids between SIM and NC group (C: cellular processes; E: environmental information processing; H: human diseases; M: metabolism; O:organismal systems). (h) The expression density of Sgms2 in each cluster (left) and in Mesenchymal subcluster (right) are shown by UMAP plot. (i) UMAP plot shows the expression of Sgms2 in Mesenchymal subcluster at different time points (Day0 ,3, 7, 21) during tHO development.

Article Snippet: Primary antibodies used are as follows: ARL13B (1:500, #17711-1-AP, Proteintech Group Inc.); Acetyl-α-Tubulin (Lys40) (1:800, #12152, Cell Signaling Technology) ; PDGFRα (1:100, #sc-338, Santa Cruz); CD31 (1:500, #11265-1-AP, Proteintech Group Inc.); F4/80 (1:500, #28463-1-AP, Proteintech Group Inc.); TNMD(1:100, #PA5-112767, Invitrogen); α-SMA(1:500, #14395-1-AP, Proteintech Group Inc.); MEST (1:250, #11118-1-AP, Proteintech Group Inc.).

Techniques: Immunofluorescence, Isolation, Staining, Transformation Assay, Expressing

Journal: Breast Cancer Research : BCR

Article Title: PRKCQ promotes oncogenic growth and anoikis resistance of a subset of triple-negative breast cancer cells

doi: 10.1186/s13058-016-0749-6

Figure Lengend Snippet: Protein tyrosine kinase C theta isoform ( PRKCQ ) overexpression promotes multiple oncogenic activities in breast epithelial cells. a Non-transformed, immortalized MCF-10A cells overexpressing vector control or PRKCQ were generated, and assessed for their ability to survive in suspension cultures using the Cell Death ELISA assay. b The ability of MCF-10A cells overexpressing vector control or PRKCQ in Transwell Boyden chamber assays in the presence or absence of epidermal growth factor ( EGF ) was evaluated. The number of cells that migrated 18 hours after plating in the insert was counted. Five fields of migrated cells (×10 objective) were counted and averaged. c MCF-10A cells expressing vector control or PRKCQ (1 × 10^4 cells) were plated on day 1 in growth media lacking EGF. The number of cells were counted on the indicated days and plotted. d Cell-cycle profile analysis of empty vector or PRKCQ-expressing cells was performed by fluorescence-activated cell sorting after propidium iodide staining of cells cultured in growth media +/- EGF: * p < 0.05, ** p < 0.01, *** p < 0.001. PKC protein kinase C

Article Snippet: Antibodies directed against the following proteins were obtained from the indicated suppliers: AbCam: rabbit monoclonal protein kinase C (PKC-θ) (EPR1487(2)); Cell Signaling Technologies: phospho-retinoblastoma (Rb) (S807/811) and total Rb, phospho-p44/p42 MAPK (ERK1/2) (T202/Y204) and total p44/42 MAPK (ERK1/2), phospho-Akt (S473) and total (pan-) Akt, PKC isoform antibody sampler kit (for PKCδ, PKCα, PKCμ, PKCζ) phospho-PKCθ (T538), phospho-PKCα/βII (T638/641), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin.

Techniques: Over Expression, Transformation Assay, Plasmid Preparation, Generated, Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence, FACS, Staining, Cell Culture

Journal: Breast Cancer Research : BCR

Article Title: PRKCQ promotes oncogenic growth and anoikis resistance of a subset of triple-negative breast cancer cells

doi: 10.1186/s13058-016-0749-6

Figure Lengend Snippet: Extracellular signal-related ( Erk )/mitogen-activated protein kinase (MAPK) activation is required for protein tyrosine kinase C theta isoform ( PRKCQ )-mediated epidermal growth factor ( EGF )-independent growth. a MCF-10A cells overexpressing pBabe vector control or wild-type PRKCQ were cultured in growth media lacking EGF. Lysates were probed with the indicated antibodies. b PRKCQ or vector control expressing MCF-10A cells were cultured in growth media +/- EGF. Cells grown in the absence of EGF were treated for 24 hours with dimethyl sulfoxide ( DMSO ) or mitogen-activated protein kinase kinase (MEK) inhibitor U0126 (5 μM). Cells were lysed and probed with the indicated antibodies. c MCF-10A cells expressing vector control or PRKCQ were cultured in growth media lacking EGF and treated with DMSO or U0126 (5 μM) for the indicated number of days. DMSO or U0126 was replaced every 2–3 days. Cell numbers were counted on the indicated days and plotted. Rb retinoblastoma

Article Snippet: Antibodies directed against the following proteins were obtained from the indicated suppliers: AbCam: rabbit monoclonal protein kinase C (PKC-θ) (EPR1487(2)); Cell Signaling Technologies: phospho-retinoblastoma (Rb) (S807/811) and total Rb, phospho-p44/p42 MAPK (ERK1/2) (T202/Y204) and total p44/42 MAPK (ERK1/2), phospho-Akt (S473) and total (pan-) Akt, PKC isoform antibody sampler kit (for PKCδ, PKCα, PKCμ, PKCζ) phospho-PKCθ (T538), phospho-PKCα/βII (T638/641), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin.

Techniques: Activation Assay, Plasmid Preparation, Cell Culture, Expressing

Journal: Breast Cancer Research : BCR

Article Title: PRKCQ promotes oncogenic growth and anoikis resistance of a subset of triple-negative breast cancer cells

doi: 10.1186/s13058-016-0749-6

Figure Lengend Snippet: Protein tyrosine kinase C theta isoform ( PRKCQ ) kinase activity is required for epidermal growth factor ( EGF )-independent growth, Extracellular signal-related kinase ( Erk activity) and retinoblastoma ( Rb ) phosphorylation. a MCF-10A cells expressing vector control, constitutively active (A148E) or kinase-inactive (K409R) PKCθ, were cultured in growth media lacking EGF. Cell lysates were probed with the indicated antibodies. b MCF-10A cells expressing vector control, active or kinase-dead PKCθ, were cultured in media lacking EGF for the indicated number of days and counted

Article Snippet: Antibodies directed against the following proteins were obtained from the indicated suppliers: AbCam: rabbit monoclonal protein kinase C (PKC-θ) (EPR1487(2)); Cell Signaling Technologies: phospho-retinoblastoma (Rb) (S807/811) and total Rb, phospho-p44/p42 MAPK (ERK1/2) (T202/Y204) and total p44/42 MAPK (ERK1/2), phospho-Akt (S473) and total (pan-) Akt, PKC isoform antibody sampler kit (for PKCδ, PKCα, PKCμ, PKCζ) phospho-PKCθ (T538), phospho-PKCα/βII (T638/641), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Cell Culture

Journal: Breast Cancer Research : BCR

Article Title: PRKCQ promotes oncogenic growth and anoikis resistance of a subset of triple-negative breast cancer cells

doi: 10.1186/s13058-016-0749-6

Figure Lengend Snippet: Protein tyrosine kinase C theta isoform ( PRKCQ ) is preferentially expressed in the triple-negative breast cancer ( TNBC ) subtype and breast cancer cell lines. PRKCQ transcript levels in triple-negative and hormone-receptor-positive or human epidermal growth factor (Her2)-positive patient tumors in The Cancer Genome Atlas dataset ( TCGA ) ( a ) and METABRIC dataset ( b ) were compared. c PKCθ protein expression in breast cancer cell lines was assessed by western analysis. Expression of PRKCδ, another novel PKC isoform, was also examined. GAPDH glyceraldehyde-3-phosphate dehydrogenase

Article Snippet: Antibodies directed against the following proteins were obtained from the indicated suppliers: AbCam: rabbit monoclonal protein kinase C (PKC-θ) (EPR1487(2)); Cell Signaling Technologies: phospho-retinoblastoma (Rb) (S807/811) and total Rb, phospho-p44/p42 MAPK (ERK1/2) (T202/Y204) and total p44/42 MAPK (ERK1/2), phospho-Akt (S473) and total (pan-) Akt, PKC isoform antibody sampler kit (for PKCδ, PKCα, PKCμ, PKCζ) phospho-PKCθ (T538), phospho-PKCα/βII (T638/641), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin.

Techniques: Expressing, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: PRKCQ promotes oncogenic growth and anoikis resistance of a subset of triple-negative breast cancer cells

doi: 10.1186/s13058-016-0749-6

Figure Lengend Snippet: Protein tyrosine kinase C theta isoform ( PRKCQ ) downregulation inhibits growth of triple-negative breast cancer (TNBC) cells in culture and triple-negative breast tumor xenografts. a Lentiviral PRKCQ short hairpin ( sh )RNA vector-infected TNBC cells were lysed and probed for expression of protein kinase C ( PKC ) family members. b TNBC cells expressing empty vector control ( EV ) or PRKCQ shRNA (90, 54 or 16) were grown in monolayer cultures for the indicated number of days and counted. c TNBC cells expressing vector control or PRKCQ shRNA (90, 54 or 16) were cultured in suspension for 24 hours. Cell death (anoikis) was assessed using the Cell Death ELISA. d TNBC cells expressing EV control or PRKCQ shRNA vectors were cultured in 3-D Matrigel TM cultures in chamber slides for the indicated number of days. e MDA-231-Luc-D3H2LN cells (5 × 10 5 ) expressing vector control or PRKCQ shRNA (90 or 54) were injected subcutaneously into the flank of 6-week-old, female nude mice. Tumor size was measured and recorded every 3–4 days. All figures are representative of three independent experiments except for the xenograft study, which was performed twice. Scale bars represent 200 μM. GAPDH glyceraldehyde-3-phosphate dehydrogenase

Article Snippet: Antibodies directed against the following proteins were obtained from the indicated suppliers: AbCam: rabbit monoclonal protein kinase C (PKC-θ) (EPR1487(2)); Cell Signaling Technologies: phospho-retinoblastoma (Rb) (S807/811) and total Rb, phospho-p44/p42 MAPK (ERK1/2) (T202/Y204) and total p44/42 MAPK (ERK1/2), phospho-Akt (S473) and total (pan-) Akt, PKC isoform antibody sampler kit (for PKCδ, PKCα, PKCμ, PKCζ) phospho-PKCθ (T538), phospho-PKCα/βII (T638/641), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin.

Techniques: Plasmid Preparation, Infection, Expressing, shRNA, Cell Culture, Enzyme-linked Immunosorbent Assay, Injection

Journal: Neoplasia (New York, N.Y.)

Article Title: Loss of Pin1 Suppresses Hedgehog-Driven Medulloblastoma Tumorigenesis 1

doi: 10.1016/j.neo.2017.01.002

Figure Lengend Snippet: GLI1 interacts with PIN1. (A) Detection of the GLI1/PIN1 interaction using the Y2H assay. In this Y2H experiment, PIN1 is fused to the GAL4 DNA-binding (DB) domain and GLI1 is fused to the GAL4 activation domain (AD). The six Y2H controls have been previously described . The DB-PIN1 and AD-GLI1 fusion proteins interact with each other, leading to the activation of the HIS3 reporter gene and allowing yeast cells to grow on selective medium lacking histidine. Experiment was replicated three times. (B) HA-GLI1 co-purifies with SBP-FLAG-PIN1 (top panels), and HA-PIN1 co-purifies with SBP-FLAG-GLI1 (bottom panels). Immunoprecipitation (IP) of SBP-FLAG–tagged PIN1 (top panels) or SBP-FLAG–tagged GLI1 (bottom panels) in HEK293T cells in the presence of HA-tagged GLI1 or HA-tagged PIN1, respectively. WB: Western blot; SBP: Streptavidin binding peptide tag. Experiment was replicated three times. (C) Endogenous GLI1 co-purifies with endogenous PIN1 in MED-311FH cells, a low-passage, patient-derived cell line derived from a medulloblastoma tumor. The IP of endogenous PIN1 was performed using an α-PIN1 monoclonal antibody on a protein extract derived from Shh-N ligand-treated MED-311FH cells, followed by Western blot analyses using either α-PIN1 or α-GLI1 antibody. Experiment was replicated twice. WB: Western blot; IP: immunoprecipitation. (D) PIN1 interacts with GLI1 in a phosphorylation-dependent manner. GST pulldown with glutathione beads of bacteria-purified GST-tagged PIN1 in the presence of a protein extract derived from mammalian HEK293T cells transduced with HA-tagged GLI1 and in the presence or absence of calf intestinal alkaline phosphatase (CIP). Experiment was replicated twice. WB: Western blot. (E) PIN1 leads to increased GLI1 protein abundance in vitro . Analysis of GLI1 protein levels in the presence of an increasing level of WT PIN1 and mutant PIN1 W34A in HEK293T cells. HEK293T cells were transfected with 300 ng of HA-GLI1 protein expression vector as well as 0, 50, 150, or 450 ng of the SBP-FLAG-PIN1 or the SBP-FLAG-PIN1 W34A protein expression vector. Experiment was replicated twice. WB: Western blot; SBP: Streptavidin binding peptide tag.

Article Snippet: The following primary antibodies were used: PIN1 (Santa Cruz Biotechnology, Santa Cruz, CA, sc15340 and sc46660), GLI1 (Cell Signaling, Danvers, MA, #2534, #2643; Novus, Littleton, CO, NB600-600), NeuN (Zymed, Thermo Fisher Scientific, Waltham, MA, #18-7373), Ki67 (Abcam, Cambridge, MA, ab16667), HA (Roche, Reinach, Switzerland, #12013819001), FLAG (Sigma, St. Louis, MO, A8592), and β-actin (Cell Signaling, #5125).

Techniques: Y2H Assay, Binding Assay, Activation Assay, Immunoprecipitation, Western Blot, Derivative Assay, Purification, Transduction, In Vitro, Mutagenesis, Transfection, Expressing, Plasmid Preparation

Journal: Neoplasia (New York, N.Y.)

Article Title: Loss of Pin1 Suppresses Hedgehog-Driven Medulloblastoma Tumorigenesis 1

doi: 10.1016/j.neo.2017.01.002

Figure Lengend Snippet: PIN1 promotes Hh signaling. (A) GLI-dependent luciferase reporter assay. Shh-Light2 is an NIH 3T3 cell line stably transfected with GLI-dependent Firefly luciferase and constitutive Renilla luciferase reporters, which can be used to study modulation of Hh signaling . Shh-N–mediated activation of luciferase activities was measured upon CRISPR/Cas9-mediated depletion of either GLI1 or PIN1. Control: Shh-Light2 cells stably transfected with empty CRISPR/Cas9 vector. First, for each one of the 10 conditions tested, the Firefly luciferase activity signals were measured using the Dual-Luciferase Reporter Assay System (Promega) and were normalized by the Renilla luciferase signals. Second, the 10 signal ratios were normalized by the signal ratio obtained in the [control medium/control cell line] condition. Shown are means ±S.D. of three biological replicates. We fit analysis of variance (ANOVA) models to log-transformed data and tested whether the increase with Shh-N versus no treatment was smaller under Gli1 and Pin1 KD conditions than the same increase under control conditions. The difference of fold-changes was significant for all four KD conditions tested; [***] P < 3 × 10 −5 . Inset: Western blot analyses validate the CRISPR/Cas9-mediated depletion of PIN1 and GLI1. sg: CRISPR/Cas9 single guide RNA; WB: Western blot. (B) Expression analysis by quantitative RT-PCR of Hh target genes upon treatment with SAG (0.3 μM) and/or juglone (5 μM) in MED-311FH. Shown are means ± S.D. of two biological replicates measured twice each. Statistical significances of the difference between the “ SAG ” and “ SAG + juglone” conditions were computed using one-way ANOVA models on log-transformed data for each gene; [*] P < .05, [**] P < .01.

Article Snippet: The following primary antibodies were used: PIN1 (Santa Cruz Biotechnology, Santa Cruz, CA, sc15340 and sc46660), GLI1 (Cell Signaling, Danvers, MA, #2534, #2643; Novus, Littleton, CO, NB600-600), NeuN (Zymed, Thermo Fisher Scientific, Waltham, MA, #18-7373), Ki67 (Abcam, Cambridge, MA, ab16667), HA (Roche, Reinach, Switzerland, #12013819001), FLAG (Sigma, St. Louis, MO, A8592), and β-actin (Cell Signaling, #5125).

Techniques: Luciferase, Reporter Assay, Stable Transfection, Transfection, Activation Assay, CRISPR, Plasmid Preparation, Activity Assay, Transformation Assay, Western Blot, Expressing, Quantitative RT-PCR