Journal: The Journal of Biological Chemistry

Article Title: Rho-associated Coiled-coil Kinase (ROCK) Protein Controls Microtubule Dynamics in a Novel Signaling Pathway That Regulates Cell Migration *

doi: 10.1074/jbc.M112.394965

Figure Lengend Snippet: ROCK regulates MT acetylation in U2OS cells. U2SO cells treated with 10 μm Y-27632 or vehicle were lysed, and the levels of acetyl tubulin, α-tubulin, pMLC, and GAPDH were determined by immunoblotting (A) or by immunofluorescence microscopy after staining for pMLC (red), nuclei (blue), and acetylated MTs (green) (B). Immunoblots of lysates from U2OS cells transiently expressing constitutively active F-ROCK1 (50 kDa), F-ROCK1-KD, or FLAG vector probed with anti-acetyl-tubulin, GAPDH (loading control), pMLC, and anti-FLAG Abs (C) and immunofluorescence microscopy (D). Red, FLAG; blue, nuclei; green, acetylated MTs. Scale bar = 50 μm.

Article Snippet: In vitro tubulin polymerization assays were performed using the tubulin polymerization assay kit (catalog no. BK006P, Cytoskeleton).

Techniques: Western Blot, Immunofluorescence, Microscopy, Staining, Expressing, Plasmid Preparation, Control

Journal: The Journal of Biological Chemistry

Article Title: Rho-associated Coiled-coil Kinase (ROCK) Protein Controls Microtubule Dynamics in a Novel Signaling Pathway That Regulates Cell Migration *

doi: 10.1074/jbc.M112.394965

Figure Lengend Snippet: TPPP1 phosphorylation on Ser-32, Ser-159, and Ser-107 in vivo and in vitro by ROCK does not alter its MT polymerizing activity. A, in vitro kinase assay showing that the phosphorylation of the TPPP1S32A, TPPP1S107A, and TPPP1S159A proteins by ROCK1Δ4 (ROCK1) is reduced in comparison with that of TPPP1 (top panel). The numbers below the top panel represent the ratio between the phosphorylation level of wild-type TPPP1 and that of the single TPPP1 mutants. AR, autoradiography. B, the triple S32A/S107A/S159A mutations abolished TPPP1 phosphorylation by ROCK1. C, in vitro tubulin polymerization assay in the presence of bacterially purified GST-TPPP1 and in vitro ROCK1 phosphorylated GST-TPPP1. D, overexpression of TPPP1, TPPP13Ala, or TPPP13Glu in U2OS cells increased MT levels (green) as shown by immunofluorescence microscopy (nuclei, blue). Scale bar = 50 μm.

Article Snippet: In vitro tubulin polymerization assays were performed using the tubulin polymerization assay kit (catalog no. BK006P, Cytoskeleton).

Techniques: In Vivo, In Vitro, Activity Assay, Kinase Assay, Comparison, Autoradiography, Polymerization Assay, Purification, Over Expression, Immunofluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Rho-associated Coiled-coil Kinase (ROCK) Protein Controls Microtubule Dynamics in a Novel Signaling Pathway That Regulates Cell Migration *

doi: 10.1074/jbc.M112.394965

Figure Lengend Snippet: ROCK phosphorylation of TPPP1 inhibits HDAC6 binding and tubulin acetylation. A, inhibition of ROCK activity increases the interaction between TPPP1 and HDAC6. U2OS cell extracts treated with Y-27632 (lane 4) or vehicle (lane 3) were incubated with IgG (lane 2) or rabbit anti-TPPP1 polyclonal Abs (lanes 3 and 4). The immunoprecipitated proteins (IP) and total cell extracts were analyzed by immunoblotting (IB) for TPPP1 and HDAC6. B, immunoblots of extracts from A immunoprecipitated with mouse anti-HDAC6 mAb and probed as in A. C, ROCK phosphorylation of TPPP1 inhibits its binding to HDAC6. Extracts from U2OS cells expressing F-TPPP1 and its mutants were purified with anti-FLAG M2-agarose, and total lysates and IP were immunoblotted and probed with anti-HDAC6 and anti-FLAG mAbs. D, cell extracts from C were immunoprecipitated with anti-HDAC6 or IgG Abs. Total lysates and IPs were analyzed by immunoblotting as in C. E, immunoblot of lysates from U2OS cells stably expressing F-TPPP1 and its mutants were analyzed for acetyl tubulin, α-tubulin, GAPDH (loading control), and F-TPPP1. F, the U2OS cell lines described in E were stained for nuclei (blue) and acetylated MTs (green) and analyzed by immunofluorescence microscopy. Scale bar = 50 mm. The numbers below the top panels in A, B, D, and E represent the fold changes in the indicated protein levels.

Article Snippet: In vitro tubulin polymerization assays were performed using the tubulin polymerization assay kit (catalog no. BK006P, Cytoskeleton).

Techniques: Binding Assay, Inhibition, Activity Assay, Incubation, Immunoprecipitation, Western Blot, Expressing, Purification, Stable Transfection, Control, Staining, Immunofluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Rho-associated Coiled-coil Kinase (ROCK) Protein Controls Microtubule Dynamics in a Novel Signaling Pathway That Regulates Cell Migration *

doi: 10.1074/jbc.M112.394965

Figure Lengend Snippet: TPPP1 regulates the microtubule network. TPPP1 regulates the microtubule network by promoting tubulin polymerization and by binding to HDAC6 to inhibit its activity. This leads to increased acetylation of the HDAC6 substrates α-tubulin. Increased MT acetylation results in decreased cell migration and invasion. When TPPP1 is phosphorylated by ROCK, it is unable to bind to HDAC6, resulting in increased HDAC6 activity in the cell, which leads to decreased acetylated-tubulin levels and increased cell migration and invasion.

Article Snippet: In vitro tubulin polymerization assays were performed using the tubulin polymerization assay kit (catalog no. BK006P, Cytoskeleton).

Techniques: Binding Assay, Activity Assay, Migration

Journal: REPRODUCTION

Article Title: Decreased STAT3 in human idiopathic fetal growth restriction contributes to trophoblast dysfunction

doi: 10.1530/rep-14-0622

Figure Lengend Snippet: Figure 4 (A) Relative quantitation of STAT3 mRNA normalised to 18S rRNA gene was performed in isolated trophoblasts from first-trimester placentae (nZ12) and term placentae (nZ10). (B) Immunoblot of STAT3 protein in first-trimester villous cytotrophoblasts. A representative immunoblot for total STAT3 (79 kDa) and the loading control tubulin (49 kDa)isdepicted.(C)Semi-quantitationofSTAT3proteinnormalisedto tubulin in cultured CTB from first-trimester and term placentae is shown. The asterisk (*) value denotes significantly increased levels for total STAT3 protein in in vitro differentiated ST compared with CTB (P!0.05).

Article Snippet: The membranes were stripped and reprobed with rabbit anti-human tubulin (Imgenex, San Diego, CA, USA) for cultured cell-derived protein, or with ACTB for tissue protein extracts (Imgenex) to allow relative protein levels to be determined.

Techniques: Quantitation Assay, Isolation, Western Blot, Control, Cell Culture, In Vitro

Journal: REPRODUCTION

Article Title: Decreased STAT3 in human idiopathic fetal growth restriction contributes to trophoblast dysfunction

doi: 10.1530/rep-14-0622

Figure Lengend Snippet: Figure 5 (A) Relative quantitation of STAT3 mRNA normalised to 18S rRNA gene was performed in BeWo cells treated with either 10 mM forskolin (FSK) or DMSO vehicle control. (B) STAT3 protein in FSK- induced BeWo cells. A representative immunoblot of total STAT3 (79 kDa) and the loading control tubulin (49 kDa) is depicted. (C) Semi- quantitation of STAT3 protein normalised to tubulin in BeWo cells cultured with FSK or vehicle DMSO control is shown. The asterisk (*) value denotes significantly increased levels for total STAT3 protein in BeWo cells cultured with FSK compared with DMSO control (P!0.05). Each experimental condition within an experiment was performed at least in duplicate, and all cell culture (passages 42–44) experiments were repeated at least on three independent occasions.

Article Snippet: The membranes were stripped and reprobed with rabbit anti-human tubulin (Imgenex, San Diego, CA, USA) for cultured cell-derived protein, or with ACTB for tissue protein extracts (Imgenex) to allow relative protein levels to be determined.

Techniques: Quantitation Assay, Control, Western Blot, Cell Culture

Journal: REPRODUCTION

Article Title: Decreased STAT3 in human idiopathic fetal growth restriction contributes to trophoblast dysfunction

doi: 10.1530/rep-14-0622

Figure Lengend Snippet: Figure 6 (A) Relative quantitation of STAT3 mRNA normalised to 18S rRNA gene was performed in BeWo cells treated with NC or STAT3 siRNA. Gene expression was determined using the 2KDDCT method (Livak & Schmittgen 2001). Data are expressed as meanGS.E.M. *P!0.05, Mann–Whitney U test. (B) Immunoblot of STAT3 protein in BeWo cells transfected with STAT3 siRNA. Immunoblotting of STAT3 protein in BeWo cells cultured with NC and/or STAT3 siRNA as described in ‘Materials and methods’ section. A representative immunoblot of total STAT3 (79 kDa) and the loading control tubulin (49 kDa) is depicted. (C) Semi-quantitative analysis of STAT3 protein in BeWo cells transfected with STAT3 siRNA was normalised to tubulin. The asterisk (*) value denotes significantly decreased levels of total STAT3 protein in STAT3 siRNA-treated cells compared with NC (P!0.05). Each experimental condition within an experiment was performed at least in duplicate, and all cell culture (passages 51 and 52) experiments were repeated at least on three independent occasions.

Article Snippet: The membranes were stripped and reprobed with rabbit anti-human tubulin (Imgenex, San Diego, CA, USA) for cultured cell-derived protein, or with ACTB for tissue protein extracts (Imgenex) to allow relative protein levels to be determined.

Techniques: Quantitation Assay, Gene Expression, MANN-WHITNEY, Western Blot, Transfection, Cell Culture, Control

Journal: REPRODUCTION

Article Title: Decreased STAT3 in human idiopathic fetal growth restriction contributes to trophoblast dysfunction

doi: 10.1530/rep-14-0622

Figure Lengend Snippet: Figure 7 (A) Relative quantitation of CGB mRNA normalised to 18S rRNA was performed in BeWo cells treated with NC and/or STAT3 siRNA. b-hCG production in BeWo cells transfected with STAT3 siRNA. The protein level of b-hCG secreted in media was determined by ELISA. Asterisk (*) denotes significance (P!0.005, nZ3, Mann–Whitney U test). (B) Real-time PCR analysis of TP53 in BeWo cells transfected with NC or STAT3 siRNA. Data are expressed as meanGS.E.M. *P!0.05, Mann–Whitney U test. A representative immunoblot of total TP53 (55 kDa) and the loading control tubulin (49 kDa) is depicted. Semi- quantitative analysis of TP53 protein in BeWo cells transfected with NC or STAT3 siRNA was normalised to tubulin. Data are expressed as meanGS.E.M. *P!0.05, Mann–Whitney U test. (C) Real-time PCR analysis of caspases 3 and 8 in BeWo cells transfected with NC or STAT3 siRNA. Data are expressed as meanGS.E.M. *P!0.05, Mann–Whitney U test. Caspase activity assays were performed in BeWo cells cultured with NC and/or STAT3 siRNA as described in ‘Materials and methods’ section. The asterisk (*) value denotes significantly increased levels of caspases 3 and 8 activity in STAT3 siRNA-treated cells compared with NC (P!0.05). (D) Real-time PCR analysis of SNAT2 in BeWo cells transfected with NC or STAT3 siRNA. Statistical comparisons were performed using the Mann–Whitney U test. A representative immunoblot of total SNAT2 (59 kDa) and the loading control tubulin (49 kDa) is depicted. Semi-quantitative analysis of SNAT2 protein in BeWo cells transfected with STAT3 siRNA was normalised to tubulin. The asterisk (*) value denotes significantly decreased levels of SNAT2 protein in STAT3 siRNA-treated cells compared with NC (P!0.05).

Article Snippet: The membranes were stripped and reprobed with rabbit anti-human tubulin (Imgenex, San Diego, CA, USA) for cultured cell-derived protein, or with ACTB for tissue protein extracts (Imgenex) to allow relative protein levels to be determined.

Techniques: Quantitation Assay, Transfection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Western Blot, Control, Activity Assay, Cell Culture

Journal: iScience

Article Title: Merkel cell polyomavirus T-antigens regulate DICER1 mRNA stability and translation through HSC70

doi: 10.1016/j.isci.2021.103264

Figure Lengend Snippet: AUF1 is not involved in DICER1 regulation in MCC (A) Western blot analysis of DICER1 expression upon AUF1 silencing in MCPyV+ (WaGa and MKL1) and MCPyV− (MCC26) cell lines. Two different siRNAs targeting AUF1 (siAUF1#1 and siAUF#2) were used. siCTR was applied as a negative control. (B) Quantification of DICER1 expression in Figure 6 A. DICER1 expression was normalized to GAPDH and then siCTR. Error bars, SEM. Each biological replicate is presented as a circle. (C) RT-qPCR analysis of DICER1 expression in WaGa cells transfected with siCTR or siAUF1 after actinomycin D (Act. D) treatment. This experiment was conducted in parallel to the HSC70 silencing experiments described in Figure 2 I. p value was assessed by two-way ANOVA. ns, not significant. (D) Western blot analysis of HSC70 and AUF1 in nuclear and cytoplasmic fractions of MCPyV+ cell lines. α-TUBULIN and HISTONE3 were used as cytoplasmic and nuclear markers, respectively. (E) Western blot analysis of HSC70 and AUF1 in cytosolic and nuclear compartments of MCC26 cells transfected with LT339 or pCTR. Lamin A/C was used as nuclear or nuclear membrane marker, whereas GAPDH was used as cytoplasmic marker. Red arrows indicate trLT of LT339.

Article Snippet: Mouse monoclonal anti-α-TUBULIN antibody (B-7) , Santa Cruz Biotechnology , sc-5286 RRID: AB_628411.

Techniques: Western Blot, Expressing, Negative Control, Quantitative RT-PCR, Transfection, Membrane, Marker

Journal: iScience

Article Title: Merkel cell polyomavirus T-antigens regulate DICER1 mRNA stability and translation through HSC70

doi: 10.1016/j.isci.2021.103264

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-α-TUBULIN antibody (B-7) , Santa Cruz Biotechnology , sc-5286 RRID: AB_628411.

Techniques: Virus, Recombinant, Transfection, Electroporation, Bicinchoninic Acid Protein Assay, Reverse Transcription, Gene Expression, Polymerase Chain Reaction, Luciferase, Mutagenesis, In Vitro, Construct, shRNA, Software

Journal: Nucleic Acids Research

Article Title: The proapoptotic dp5 gene is a direct target of the MLK-JNK-c-Jun pathway in sympathetic neurons

doi: 10.1093/nar/gkp175

Figure Lengend Snippet: Expression of dominant negative c-Jun reduces the induction of a dp5 reporter construct and the endogenous dp5 RNA after NGF withdrawal. ( a ) c-JunΔ169 binds to the dp5 ATF site as a heterodimer with ATF2. An oligonucleotide containing the dp5 ATF site was incubated with in vitro translated c-Jun, c-JunΔ169, Δ169m0, ATF2 or unprogrammed rabbit reticulocyte lysate (RRL) as indicated, and an EMSA experiment was performed. Only the regions of the gel that contain the protein/DNA complexes and the unbound probe are shown, to make the figure more compact. ( b ) Co-injection of an expression vector for dominant negative c-Jun (JunΔ169) reduces induction of a dp5 reporter construct after NGF withdrawal. Sympathetic neurons were microinjected with dp5 -LUC + ALL (20 ng/µl), pRL-TK (10 ng/µl) and pcDNA1 or pCDJunΔ169 (100 ng/µl). Cells were maintained +NGF or –NGF for ∼20 h and then luciferase activity was measured. Firefly luciferase levels were normalized and luciferase activity was calculated relative to the level +NGF, which was set as 1, for either pcDNA1 or pCDFLAGΔ169. The mean of nine independent experiments ±SEM is shown. In the case of empty vector the increase in reporter gene activity after NGF withdrawal was significant: * P < 0.002, Student's t -test. For pCDJunΔ169 the increase in reporter activity after NGF deprivation was not significant: # P > 0.1, Student's t -test. ( c ) Sympathetic neurons infected with AdvJunΔ169 or AdvlacZ express recombinant proteins of the predicted size. Sympathetic neurons were cultured for 5 days in vitro and then infected with recombinant adenoviruses as indicated. After overnight infection, the cells were refed with fresh SCG medium containing NGF. Forty eight hours later, protein extracts were prepared and immunoblots performed with antibodies to β-galactosidase, α-tubulin (a loading control) and the FLAG epitope. The positions of protein molecular weight markers run on the same 12% gel are shown. ( d ) RT–PCR analysis of dp5 and nfm mRNA levels in adenovirus-infected neurons expressing β-galactosidase or the JunΔ169 protein. Sympathetic neurons were cultured for 5 days in vitro and then infected with the recombinant adenoviruses indicated. After overnight infection, the cells were refed with fresh SCG medium containing NGF (+NGF) or anti-NGF antibody (-NGF). Sixteen hours later, RNA was isolated and semi-quantitative RT–PCR performed with primers specific for dp5 or nfm . Thirty-five cycles of PCR were performed and the products were run on a 2.5% agarose gel. ( e ) AdvJunΔ169 reduces the increase in dp5 RNA levels after NGF withdrawal. RT–PCR analysis of dp5 and nfm mRNA levels in adenovirus-infected neurons was carried out as described in (d). dp5 mRNA levels were normalized to nfm RNA levels to control for any differences in the amount of cDNA used for each RT–PCR reaction. The induction factor (the normalized dp5 RNA level –NGF/+NGF) was then calculated for each virus. The average of five experiments ± SEM is shown.

Article Snippet: The following primary antibodies were used: the M2 anti-FLAG epitope mouse monoclonal antibody (Sigma-Aldrich), a mouse monoclonal anti-β-galactosidase antibody (Promega), a rat monoclonal anti-α-tubulin antibody (Serotec Ltd, Kidlington, UK), a mouse monoclonal anti-c-Jun antibody (BD Transduction Laboratories), a mouse monoclonal phospho-c-Jun (serine 63) antibody (KM-1; Santa Cruz Biotechnology, Inc.), a rabbit polyclonal phospho-c-Jun (serine 73) antibody (9164; Cell Signalling Technology), a rabbit polyclonal anti-ATF2 antibody (C-19; Santa Cruz Biotechnology, Inc.), a rabbit polyclonal phospho-ATF2 (threonine 71) antibody (9221; Cell Signalling Technology), a rabbit polyclonal ERK1/2 antibody (Cell Signalling Technology).

Techniques: Expressing, Dominant Negative Mutation, Construct, Incubation, In Vitro, Injection, Plasmid Preparation, Luciferase, Activity Assay, Infection, Recombinant, Cell Culture, Western Blot, Control, FLAG-tag, Molecular Weight, Reverse Transcription Polymerase Chain Reaction, Isolation, Quantitative RT-PCR, Agarose Gel Electrophoresis, Virus

Journal: Cell Proliferation

Article Title: The 5.8S pre‐rRNA maturation factor, M‐phase phosphoprotein 6, is a female fertility factor required for oocyte quality and meiosis

doi: 10.1111/cpr.12769

Figure Lengend Snippet: MPP6 depletion impeded meiotic progression. (A) Immunofluorescence showed that MPP6 enriched within the nucleus at the GV stage, within spindles at GVBD and MI stage, at cytokinesis ring at the TI stage, and at spindle poles at MII stage. DNA in blue, tubulin in green, MPP6 in red. (B) Western blot showed that the MPP6 protein level remained constant during meiosis. (C) Western blot showed that chariot‐mediated antibody transfection significantly reduced the endogenous MPP6 level. (D) Quantification of (C). (E) Immunofluorescence showed that MPP6 intensity was significantly reduced by chariot‐mediated antibody transfection. Tubulin in green, MPP6 in red. (F) Bright‐field (BF) image showed that MPP6 depletion significantly decreased the percentage of GVBD oocytes at 3 hours of in vitro maturation (IVM). Numbers in the images indicate the number of GVBD oocytes/number of total oocytes. (G) Quantification of GVBD rate at different IVM time points (0‐3 hours). (H) Immunofluorescence showed that at 2.5 hours of IVM, when GVBD was about to occur, MPP6 depletion significantly increased lamin intensity on the nuclear envelope. DNA in blue, lamin in green. (I) Quantification of (H). (J) Bright‐field (BF) image showed that MPP6 depletion significantly decreased the percentage of MII oocytes at 14.5 hours of IVM. Numbers in the images indicate the number of MII oocytes/number of total oocytes. (K) Quantification of MII rate (1PB) at different IVM time points (8‐14.5 hours). Scale bars in A, E and H, 20 µm. Scale bar in F and J, 100 µm. * P < .05; ** P < .01; *** P < .001

Article Snippet: Primary antibodies were mouse anti‐β‐actin (Cat#: A5316‐100); mouse anti‐GAPDH (Cat#: 30201ES60; YEASEN); mouse polyclonal a anti‐α‐Tubulin (cat#:G3115); mouse polyclonal a anti‐β‐Tubulin (cat#:sc‐5274); mouse monoclonal anti‐alpha tubulin (Acetyl Lys40) (cat#:bsm‐33235M; Bioss); rabbit polyclonal anti‐mphosh6 (cat#:10695‐1‐AP; Proteintech); rabbit polyclonal anti‐BubR1 (cat#:ab28193; Abcam); human anti‐centromere CREST (Cat#: 15‐234; Antibodies Incorporated); mouse polyclonal anti‐cyclin B1 (Cat#: B8566); rabbit polyclonal anti‐Phosphorylation CDK1 (Thr14) (Cat#: OAAN02724; Aviva Systems Biology); and rabbit anti‐Phosphorylation AKT (s473) (Cat#: 9271; Cell Signaling Technology).

Techniques: Immunofluorescence, Western Blot, Transfection, In Vitro

Journal: Cell Proliferation

Article Title: The 5.8S pre‐rRNA maturation factor, M‐phase phosphoprotein 6, is a female fertility factor required for oocyte quality and meiosis

doi: 10.1111/cpr.12769

Figure Lengend Snippet: MPP6 depletion caused spindle disorganization and spindle checkpoint activation. (A) Immunofluorescence showed that MPP6 depletion significantly altered the spindle geometry, decreased spindle microtubule intensity and increased the chromosome displacement at 7.5 hour of IVM. Tubulin (green) was detected by the acetylated tubulin antibody. (B‐D) Quantification of A. MPP6 depletion significantly decreased spindle length and width and increased the ratio of chromosome displacement/spindle length. (E) Quantification showed that MPP6 depletion significantly reduced acetylated tubulin intensity. (F) The speed of microtubule disassembly by nocodazole in MPP6‐depleted oocytes was significantly faster than in control oocytes. Oocytes were stained with acetylated tubulin antibody. (G) Quantification of (F). (H) BUBR1 staining on kinetochores in MI oocytes significantly increased after MPP6 depletion. DNA in blue, BUBR1 in green, kinetochores in red. (I) Quantification of (H) the percentage of BUBR1‐positive oocytes significantly increased. Scale bar, 20 µm. * P < .05; ** P < .01; *** P < .001

Article Snippet: Primary antibodies were mouse anti‐β‐actin (Cat#: A5316‐100); mouse anti‐GAPDH (Cat#: 30201ES60; YEASEN); mouse polyclonal a anti‐α‐Tubulin (cat#:G3115); mouse polyclonal a anti‐β‐Tubulin (cat#:sc‐5274); mouse monoclonal anti‐alpha tubulin (Acetyl Lys40) (cat#:bsm‐33235M; Bioss); rabbit polyclonal anti‐mphosh6 (cat#:10695‐1‐AP; Proteintech); rabbit polyclonal anti‐BubR1 (cat#:ab28193; Abcam); human anti‐centromere CREST (Cat#: 15‐234; Antibodies Incorporated); mouse polyclonal anti‐cyclin B1 (Cat#: B8566); rabbit polyclonal anti‐Phosphorylation CDK1 (Thr14) (Cat#: OAAN02724; Aviva Systems Biology); and rabbit anti‐Phosphorylation AKT (s473) (Cat#: 9271; Cell Signaling Technology).

Techniques: Activation Assay, Immunofluorescence, Staining

Journal: Cell Proliferation

Article Title: The 5.8S pre‐rRNA maturation factor, M‐phase phosphoprotein 6, is a female fertility factor required for oocyte quality and meiosis

doi: 10.1111/cpr.12769

Figure Lengend Snippet: MPP6 depletion caused abnormality in oocyte maturation and fertilization. (A) At 14.5 hours of IVM, MPP6 depletion significantly altered the MII spindle geometry and disrupted chromosome congression. Tubulin in green, DNA in red. Arrows marked uncompressed chromosomes. (B‐D) Quantification of A. MPP6 depletion significantly increased spindle length, chromosome displacement and the ratio of chromosome displacement/spindle length. (E) Chromosome spreading of MII oocytes and kinetochore counting showed that the percentage of MII oocytes with aneuploidy significantly increased. DNA in blue, kinetochores in red. (F) Quantification of (E). (G) Percentage of fertilized oocytes with 2‐PN (2 pronuclear) significantly reduced after MPP6 depletion. DNA in blue, tubulin in green, F‐actin (stained with phalloidin) in red. Arrows marked pronucleus. (H) Quantification of (G). Scale bar, 20 µm. * P < .05; ** P < .01; *** P < .001

Article Snippet: Primary antibodies were mouse anti‐β‐actin (Cat#: A5316‐100); mouse anti‐GAPDH (Cat#: 30201ES60; YEASEN); mouse polyclonal a anti‐α‐Tubulin (cat#:G3115); mouse polyclonal a anti‐β‐Tubulin (cat#:sc‐5274); mouse monoclonal anti‐alpha tubulin (Acetyl Lys40) (cat#:bsm‐33235M; Bioss); rabbit polyclonal anti‐mphosh6 (cat#:10695‐1‐AP; Proteintech); rabbit polyclonal anti‐BubR1 (cat#:ab28193; Abcam); human anti‐centromere CREST (Cat#: 15‐234; Antibodies Incorporated); mouse polyclonal anti‐cyclin B1 (Cat#: B8566); rabbit polyclonal anti‐Phosphorylation CDK1 (Thr14) (Cat#: OAAN02724; Aviva Systems Biology); and rabbit anti‐Phosphorylation AKT (s473) (Cat#: 9271; Cell Signaling Technology).

Techniques: Staining

Journal: Cell Proliferation

Article Title: The 5.8S pre‐rRNA maturation factor, M‐phase phosphoprotein 6, is a female fertility factor required for oocyte quality and meiosis

doi: 10.1111/cpr.12769

Figure Lengend Snippet: MPP6 is important for the normal activities of key meiotic kinases. (A) MPP6 depletion significantly increased p‐cdk1 level and decreased cyclin B1 level. (B) Quantification of (A). (C) Immunofluorescence staining showed that MPP6 depletion significantly reduced p‐Akt at spindle poles. DNA in blue, tubulin in green, p‐Akt in red. (D) Western blot showed that MPP6 depletion significantly reduced p‐Akt level. (E) Quantification of (D). Scale bar, 20 µm. * P < .05

Article Snippet: Primary antibodies were mouse anti‐β‐actin (Cat#: A5316‐100); mouse anti‐GAPDH (Cat#: 30201ES60; YEASEN); mouse polyclonal a anti‐α‐Tubulin (cat#:G3115); mouse polyclonal a anti‐β‐Tubulin (cat#:sc‐5274); mouse monoclonal anti‐alpha tubulin (Acetyl Lys40) (cat#:bsm‐33235M; Bioss); rabbit polyclonal anti‐mphosh6 (cat#:10695‐1‐AP; Proteintech); rabbit polyclonal anti‐BubR1 (cat#:ab28193; Abcam); human anti‐centromere CREST (Cat#: 15‐234; Antibodies Incorporated); mouse polyclonal anti‐cyclin B1 (Cat#: B8566); rabbit polyclonal anti‐Phosphorylation CDK1 (Thr14) (Cat#: OAAN02724; Aviva Systems Biology); and rabbit anti‐Phosphorylation AKT (s473) (Cat#: 9271; Cell Signaling Technology).

Techniques: Immunofluorescence, Staining, Western Blot

Journal: Cell Proliferation

Article Title: The 5.8S pre‐rRNA maturation factor, M‐phase phosphoprotein 6, is a female fertility factor required for oocyte quality and meiosis

doi: 10.1111/cpr.12769

Figure Lengend Snippet: Ageing‐associated quality decrease of oocytes is partially correlated with reduced MPP6 level. (A) RT‐PCR showed that pre‐rRNA with 5'ETS primer pair and mature 5.8S rRNA with F0 + R0 primer pair both significantly decreased within ageing oocytes, and 5.8S pre‐RNA with F0 + R2 remained unchanged. (B) Quantification of (A). (C) RT‐PCR showed that the MPP6 mRNA level significantly decreased in ageing oocytes. (D) Quantification of (C). (E) Immunofluorescence staining showed that the MPP6 level significantly decreased within ageing oocytes. DNA in blue, tubulin in green, MPP6 in red. (F) Western blot showed that good overexpression of exogenous MPP6‐EGFP protein could be achieved by mRNA injection into oocytes. Red arrows marked endogenous MPP6 and exogenous MPP6‐EGFP. (G) RT‐PCR showed that MPP6‐EGFP overexpression significantly reduced 5.8S pre‐rRNA amplified with F0 + R2 primer pair, while did not change 5'ETS pre‐rRNA. (H) Quantification of (G). (I) ROS level in ageing oocytes was significantly higher than in young oocytes, while injection of MPP6‐EGFP mRNA into ageing oocytes significantly reduced the ROS level. (J) Quantification of (I). (K) Mitochondria aggregation in ageing oocytes was significantly higher than in young oocytes, while injection of MPP6‐EGFP mRNA into ageing oocytes significantly reduced the mitochondria aggregation. (L) Quantification of (K). Scale bar, 20 µm. * P < .05

Article Snippet: Primary antibodies were mouse anti‐β‐actin (Cat#: A5316‐100); mouse anti‐GAPDH (Cat#: 30201ES60; YEASEN); mouse polyclonal a anti‐α‐Tubulin (cat#:G3115); mouse polyclonal a anti‐β‐Tubulin (cat#:sc‐5274); mouse monoclonal anti‐alpha tubulin (Acetyl Lys40) (cat#:bsm‐33235M; Bioss); rabbit polyclonal anti‐mphosh6 (cat#:10695‐1‐AP; Proteintech); rabbit polyclonal anti‐BubR1 (cat#:ab28193; Abcam); human anti‐centromere CREST (Cat#: 15‐234; Antibodies Incorporated); mouse polyclonal anti‐cyclin B1 (Cat#: B8566); rabbit polyclonal anti‐Phosphorylation CDK1 (Thr14) (Cat#: OAAN02724; Aviva Systems Biology); and rabbit anti‐Phosphorylation AKT (s473) (Cat#: 9271; Cell Signaling Technology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Western Blot, Over Expression, Injection, Amplification

Journal: Molecular Medicine Reports

Article Title: Co-expression of podoplanin and fibroblast growth factor 1 predicts poor prognosis in patients with lung squamous cell carcinoma

doi: 10.3892/mmr.2017.6830

Figure Lengend Snippet: Podoplanin regulated FGF1 expression and angiogenesis in vitro . Confirmation of podoplanin knockdown by siRNA in NCI-H226 cells using (A) RT-qPCR. (B) Western blot analysis showed podoplanin knockdown and FGF1 expression in NCI-H226 cells. (C) RT-qPCR was used to demonstrate the effect of podoplanin knockdown on FGF1 mRNA. (D) Cell supernatants were subjected to western blotting for FGF1. Cell counts and α-tubulin of the lysates of the same batch cells was used as loading control. (E) Effect of podoplanin on the formation of tube-like structures by human umbilical vein endothelial cells. Original magnification, ×200. (F) Comparison of the numbers of branch points of endothelial tubes in podoplanin-knockdown and NC groups. Each experiment was repeated at least three times. *P<0.05 and **P<0.01. FGF1, fibroblast growth factor 1; siRNA, small interfering RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NC, negative control.

Article Snippet: The following primary antibodies were used: Monoclonal rabbit anti-human podoplanin antibody (1:1,000; cat. no. 9047; Cell Signaling Technology, Inc., Danvers, MA, USA); monoclonal mouse anti-human FGF1 antibody (1:500; cat. no. H00002246-M02; Abnova Corporation, Taipei, Taiwan); and monoclonal mouse anti-human α-tubulin antibody (1:5,000; cat. no. 66031-1-Ig; ProteinTech Group, Inc., Chicago, IL, USA) as a loading control.

Techniques: Expressing, In Vitro, Knockdown, Quantitative RT-PCR, Western Blot, Control, Comparison, Small Interfering RNA, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: Cancer letters

Article Title: Rosiglitazone reduces cell invasiveness by inducing MKP-1 in human U87MG glioma cells.

doi: 10.1016/j.canlet.2008.11.033

Figure Lengend Snippet: Fig. 1. Rosiglitazone reduces the gelatinolytic activity and the expression of matrix metalloproteinase-2 (MMP-2), and decreases cell invasiveness in U87MG cells. U87MG Cell were treated with different concentrations of RGZ (0–30 lM). (A) Cultured media were collected and their gelatinolytic activity were measured by zymography. (B) Total cell lysates were subjected to Western blot analysis probed with anti-MMP-2 or anti-a-tubulin antibodies. (C) Cell viability was tested by MTT assay after a 24 h incubation. (D) U87MG cells (1 105 cells) were incubated in serum-free DMEM medium and placed in the upper part of transwell chamber seperated by a Matrigel coated filter. After 24 h, cells on the bottom side of the filter were fixed, stained, and counted. Data represent the means ± SEM of three independent experiments. (E) U87MG cells were treated with RGZ (30 lM) in the absence or presence of bisphenol A diglycidyl ether, (BADGE; 10 lM) in serum-free medium for 24 h. Media were then collected for zymographic assay. (F) U87MG cells (1 105 cells) were incubated with RGZ (30 lM) in the absence or presence of BADGE (10 lM) in serum-free medium and placed in the upper part of transwell chamber. Cell invasiveness was assessed as described above. Data represent the means ± SD of three independent experiments. Statistically significant (*p < 0.05, as compared with the basal secretion; N.S., no significant difference) was determined by Student’s t-test.

Article Snippet: All antibodies specific for MMP-2, MKP-1, phosphop38, phospho-ERK, phospho-JNK, and a-tubulin and MKP-1 siRNA were purchased from Santa Cruz Biochemicals (Santa Cruz, CA).

Techniques: Activity Assay, Expressing, Cell Culture, Zymography, Western Blot, MTT Assay, Incubation, Staining

Journal: Cancer letters

Article Title: Rosiglitazone reduces cell invasiveness by inducing MKP-1 in human U87MG glioma cells.

doi: 10.1016/j.canlet.2008.11.033

Figure Lengend Snippet: Fig. 2. Rosiglitazone induces de novo MAP kinase phosphatase-1 expression via a ROS-dependent pathway. (A) U87MG cells were treated with RGZ (0– 30 lM) for 24 h, and cell lysates were immunoblotted with antibodies specific for phospho-p38 MAPK, phospho-ERK, phospho-JNK, or a-tubulin. In (B), U87MG cells were treated with RGZ (0–30 lM) for 24 h, and MKP-1 protein level was measured by Western blot analysis. In (C), U87MG cells were treated with RGZ (0–30 lM) for 24 h, and RNA were extracted for RT-PCR analysis. (D) U87MG cells were pretreated with triptolide (10 ng/ml) for 30 min, cells were then treated with RGZ (30 lM) for 24 h. MKP-1 expression was analyzed by Western blotting. (E) U87MG cells were pretreated with actinomycin D (Act. D) (1 lM) or cycloheximide (CHX) (10 lM) for 30 min before being treated with RGZ (30 lM) for 24 h. MKP-1 protein was detected by Western blot analysis using anti-MKP-1 antibodies. (F) U87MG cells were treated with different concentrations of an ROS scavenger N-acetyl-L-cysteine (l-NAC) (0.3– 1 mM) for 30 min prior to the addition of RGZ (30 lM) and incubated for 24 h. MKP-1 protein expression was detected by Western blot analysis. The protein levels of a-tubulin serve as equal loading control for all western blots analysis.

Article Snippet: All antibodies specific for MMP-2, MKP-1, phosphop38, phospho-ERK, phospho-JNK, and a-tubulin and MKP-1 siRNA were purchased from Santa Cruz Biochemicals (Santa Cruz, CA).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Incubation, Control

Journal: Cancer letters

Article Title: Rosiglitazone reduces cell invasiveness by inducing MKP-1 in human U87MG glioma cells.

doi: 10.1016/j.canlet.2008.11.033

Figure Lengend Snippet: Fig. 5. Ectopic expression of MKP-1 reduces MMP-2 activity and cell invasiveness in U87MG and GBM8401 cells. U87MG or GBM8401 cells were stably expressed with either control vector (pcDNA3.1) or wild-type MKP-1 (pcDNA3.1/MKP-1). (A) Media were collected for MMP-2 zymography. (B) Cell lysates were immunoblotted with antibodies specific for MMP-2, MKP-1, phospho-p38, phospho-ERK, phosphor-JNK, or a-tubulin. (C) Cell invasiveness was analyzed by an in vitro Matrigel invasion assay. Data are presented as the means ± SD of three independent experiments performed in triplicate. (*p < 0.05, as compared with the control).

Article Snippet: All antibodies specific for MMP-2, MKP-1, phosphop38, phospho-ERK, phospho-JNK, and a-tubulin and MKP-1 siRNA were purchased from Santa Cruz Biochemicals (Santa Cruz, CA).

Techniques: Expressing, Activity Assay, Stable Transfection, Control, Plasmid Preparation, Zymography, In Vitro, Invasion Assay

Journal: Pharmaceutics

Article Title: Anti-Breast Cancer Properties and In Vivo Safety Profile of a Bis-Carbazole Derivative

doi: 10.3390/pharmaceutics17040415

Figure Lengend Snippet: Docking simulations suggested that 1 binds a region proximal to the Paclitaxel-binding site of tubulin. The most important amino acid residues involved in the interactions are evidenced. Cyan ribbons: Tubulin, α-subunit, Salmon ribbon Tubulin β-subunit. Yellow sticks, 1 binding mode. Oxygen atoms belonging to evidenced aminoacids are colored in red, while nitrogen in blue. Bromide moieties are evidenced in amaranth.

Article Snippet: The mouse anti-Bid (sc-373939) and anti-β-Actin (69879) and the rabbit anti-β Tubulin (9104) were obtained from Santa Cruz Biotechnology and employed at a 1:100 dilution.

Techniques: Binding Assay

Journal: Pharmaceutics

Article Title: Anti-Breast Cancer Properties and In Vivo Safety Profile of a Bis-Carbazole Derivative

doi: 10.3390/pharmaceutics17040415

Figure Lengend Snippet: In vitro tubulin polymerization assay. The assembly of tubulin into microtubules was followed by measuring the turbidity (absorbance, A) at 350 nm for 3600 s at 37 °C. The polymerization curves, in the presence of 1 (1 μM) or not (CTRL, DMSO), are shown. Moreover, the graphic shows the curves obtained using two reference molecules, Vinblastine and Paclitaxel, (both at 10 μM concentration), used as tubulin-destabilizing and tubulin-stabilizing agents, respectively.

Article Snippet: The mouse anti-Bid (sc-373939) and anti-β-Actin (69879) and the rabbit anti-β Tubulin (9104) were obtained from Santa Cruz Biotechnology and employed at a 1:100 dilution.

Techniques: In Vitro, Polymerization Assay, Concentration Assay

Journal: Pharmaceutics

Article Title: Anti-Breast Cancer Properties and In Vivo Safety Profile of a Bis-Carbazole Derivative

doi: 10.3390/pharmaceutics17040415

Figure Lengend Snippet: Immunostaining studies on MDA-MB-231 cells treated with compound 1 (0.3 µM), Vinblastine, Paclitaxel (both at 1 µM), or vehicle (CTRL, DMSO) for 24 h. In the control experiment (CTRL), cells exhibited a regular arrangement of cytoskeleton. Vinblastine and Paclitaxel, instead, dramatically impacted the tubulin network. Vinblastine produced tubulin crystal formation (panel ( B ), V), whereas Paclitaxel induced tubulin bundles and thickened fibers (panel ( B ), P). Compound 1 exposure produced a morphology similar to Paclitaxel treatment (white arrows). Panels ( A ): DAPI, excitation/emission wavelength 350 nm/460 nm. Panels ( B ): β-tubulin (Alexa Fluor ® 488) excitation/emission wavelength 490 nm/515 nm. Panels ( C ): overlay. Images were taken at 20×, and representative fields were shown.

Article Snippet: The mouse anti-Bid (sc-373939) and anti-β-Actin (69879) and the rabbit anti-β Tubulin (9104) were obtained from Santa Cruz Biotechnology and employed at a 1:100 dilution.

Techniques: Immunostaining, Control, Produced

Journal: International Journal of Molecular Sciences

Article Title: RIG-I Promotes Tumorigenesis and Confers Radioresistance of Esophageal Squamous Cell Carcinoma by Regulating DUSP6

doi: 10.3390/ijms24065586

Figure Lengend Snippet: Effect of overexpression and silencing of downstream differential gene DUSP6 on radioresistance of ESCC cells. ( A ) Differentially expressed genes in the MAPK signaling pathway after RIG-I knockdown in KYSE510 cells were analyzed using RNA-seq. ( B ) TPM, log2, and p values of downregulated genes in the MAPK pathway were determined using RNA-seq analysis. shRNA-control was used as the control. ( C ) mRNA expression of screened DUSP6, NGFR, and DDIT3 genes in KYSE150 and KYSE510 cells was verified using RT-PCR. The mRNA values of three independent experiments were shown as dot, triangle or block. GAPDH was used as an inner control. Overexpression or knockdown of DUSP6 in KYSE450 and KYSE510 cells. ( D ) DUSP6 protein expression was quantified using Western blotting and ( E ) mRNA levels were quantified using qRT-PCR. Empty vector and shRNA-control were used as controls. The mRNA values of three independent experiments were shown as dot, triangle or block. GAPDH was used as an internal control. ( F ) KYSE450 and KYSE510 were exposed to different doses of ionizing radiations (0, 2, 4, 6, and 8 Gy, 2 Gy/min) and DUSP6 protein expression levels were quantified. GAPDH was used as an inner control. After stable DUSP6 overexpression or depletion using lentiviral vectors, ( G ) KYSE450 and KYSE510 cells were exposed to different doses (0, 2, 4, 6, and 8 Gy, 2 Gy/min) and cultured for 14 days to measure radiosensitivity using colony formation assay; cell survival curve fitted by the single-hit multi-target model. α-tubulin was used as an internal control. Empty vector and shRNA-control were used as controls. Data are shown as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The same membrane was stripped and reprobed with mouse anti-GAPDH, anti-β-actin, and anti-α-tubulin monoclonal antibodies (Proteintech Group Inc.) as internal controls.

Techniques: Over Expression, Knockdown, RNA Sequencing, shRNA, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Cell Culture, Colony Assay

Journal: International Journal of Molecular Sciences

Article Title: RIG-I Promotes Tumorigenesis and Confers Radioresistance of Esophageal Squamous Cell Carcinoma by Regulating DUSP6

doi: 10.3390/ijms24065586

Figure Lengend Snippet: RIG-I enhances the radioresistance of ESCC cells by regulating DUSP6 in vivo or in vitro. Knockdown of DUSP6 in RIG-I overexpressing cells was successfully established. ( A ) DUSP6 protein levels were validated using Western blotting, and ( B ) DUSP6 mRNA levels were validated using qRT-PCR in KYSE450 cells. α-tubulin and GAPDH were used as internal controls. After lentivirus transduction of RIG-I overexpression and DUSP6 RNA interference stable transduction, ( C ) KYSE450 cells were exposed to different doses (0, 2, 4, 6, and 8 Gy, 2 Gy/min) and cultured for 14 days to measure radiosensitivity using colony formation assay; cell survival curve was fitted by the single-hit multi-target model. ( D ) Cell cycle distribution of KYSE450 cells with or without irradiation was assessed using flow cytometry. ( E ) G2/M phase DNA damage checkpoint signaling pathway-related proteins were detected in KYSE450 cells using Western blotting. GAPDH was used as an internal control. Empty vector + shRNA-control and vector-RIG-I + shRNA-control were used as controls. Data are shown as mean ± SD from three independent experiments. KYSE510 cells stably transfected with shRNA-RIG-I and shRNA-control were subcutaneously injected into the hind limbs of nude mice (n = 5 per group), and yshctrl + 6Gy and shRIG-I + 6Gy group were irradiated with 6Gy ionizing radiations 2 weeks after injection. ( F ) Transplanted tumors (tumor volume was measured every 7 days). ( G ) Tumor weight (g). The weight values of each tumor are displayed with dot, triangle, inverted triangle and block. ( H , I ) Immunohistochemical analysis of RIG-I, DUSP6, and Ki67 in RIG-I-silenced KYSE510 tumor-bearing nude mice with or without irradiation (40× magnification). Scale bar = 50 µm. The mean density of staining (IOD)was shown as dot, triangle, inverted triangle and block was on the histogram. Data are shown as mean ± SD, ns: not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The same membrane was stripped and reprobed with mouse anti-GAPDH, anti-β-actin, and anti-α-tubulin monoclonal antibodies (Proteintech Group Inc.) as internal controls.

Techniques: In Vivo, In Vitro, Knockdown, Western Blot, Quantitative RT-PCR, Transduction, Over Expression, Cell Culture, Colony Assay, Irradiation, Flow Cytometry, Control, Plasmid Preparation, shRNA, Stable Transfection, Transfection, Injection, Blocking Assay, Immunohistochemical staining, Staining