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ap2 gamma  (R&D Systems)


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    R&D Systems ap2 gamma
    a , Violin plots of BMP2/4/6 expression. N = 9862 single cells. b-c , Immunofluorescence images and quantification of primed hESCs cultured in control conditions (n = 2825), 25 ng/ml BMP2 (n = 2184), 25 ng/ml BMP4 (n = 1681), 50 ng/ml BMP6 (n = 889), or 50 ng/ml BMP6 + 200 nM LDN193189 (n = 1406). d-e , Bayesian coefficients ± credible interval for counts presented in Fig. ( d ) and Fig. ( e ) . f , Immunofluorescence images of mouse embryos recovered at E3.5 and cultured in vitro for 48 h in control (N = 19), 50 ng/ml BMP6 (N = 11), or 200 nM LDN193189 (N = 13) conditions. g-h , Quantification of the number epiblast ( g ) and primitive endoderm/visceral endoderm ( h ) from f . i , Immunofluorescence images of mouse embryos recovered at E5.0 and cultured in vitro for 36 h in control (N = 8), BMP6 (N = 10) or LDN193189 (N = 8) conditions. j , Quantification of the proportion of embryos that successfully made an egg cylinder and have a Cer1-positive DVE/AVE. k-l , Quantification of the epiblast length ( k ) and area ( l) . m , Immunofluorescence images and quantification of hESCs treated for 48 hours with control conditions (PXGL n = 2336, primed n = 7796) or 200 nM LDN193189 (PXGL n = 2398, primed n = 6500, N = 2 experiments). n , Pearson regression correlation coefficients for ID1 and ID3 expression with naïve pluripotency markers. o , Immunofluorescence and quantification of AP2γ in hESCs (LDN: n = 1695 untreated cells; n = 1426 LDN treated cells; N = 2 experiments). p , Immunofluorescence and quantification of Otx2 in mESCs cultured in either 2iLif naïve conditions (n = 1570 cells), N2B27 (n = 3602 cells), or N2B27 + LDN (n = 1064 cells). N = 3 experiments. q , Immunofluorescence images and quantification of pSMAD1.5.9 in YSLCs (control: n = 1150; LDN: n = 827; N = 2 experiments). For box plots, the box encompasses the 25 th -75 th quartiles with the median marked by the central line. The minimum and maximum are denoted by the whiskers. For violin plots, the mean is marked by the red line. Statistical tests: ( c, k-l, p ) One-way ANOVA with Tukey-Kramer post-hoc; ( e-f ) two-sided Mann-Whitney test, ( m, o, q ) two-sided unpaired T-test; ( o ) Coefficients were tested (cor.test; two-tailed) and corrected for multiple hypothesis testing with the Benjamini-Hochberg method; ****p < 0.0001. ***p < 0.001. **p < 0.01. *p < 0.05. Unmarked pairwise comparisons are not significant (ns). Exact p-values presented in Supplemental Table 8. Scale bars: ( b, m, o-q ) 50 µm, ( f, i ) 100 µm.
    Ap2 Gamma, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Distinct pathways drive anterior hypoblast specification in the implanting human embryo"

    Article Title: Distinct pathways drive anterior hypoblast specification in the implanting human embryo

    Journal: Nature Cell Biology

    doi: 10.1038/s41556-024-01367-1

    a , Violin plots of BMP2/4/6 expression. N = 9862 single cells. b-c , Immunofluorescence images and quantification of primed hESCs cultured in control conditions (n = 2825), 25 ng/ml BMP2 (n = 2184), 25 ng/ml BMP4 (n = 1681), 50 ng/ml BMP6 (n = 889), or 50 ng/ml BMP6 + 200 nM LDN193189 (n = 1406). d-e , Bayesian coefficients ± credible interval for counts presented in Fig. ( d ) and Fig. ( e ) . f , Immunofluorescence images of mouse embryos recovered at E3.5 and cultured in vitro for 48 h in control (N = 19), 50 ng/ml BMP6 (N = 11), or 200 nM LDN193189 (N = 13) conditions. g-h , Quantification of the number epiblast ( g ) and primitive endoderm/visceral endoderm ( h ) from f . i , Immunofluorescence images of mouse embryos recovered at E5.0 and cultured in vitro for 36 h in control (N = 8), BMP6 (N = 10) or LDN193189 (N = 8) conditions. j , Quantification of the proportion of embryos that successfully made an egg cylinder and have a Cer1-positive DVE/AVE. k-l , Quantification of the epiblast length ( k ) and area ( l) . m , Immunofluorescence images and quantification of hESCs treated for 48 hours with control conditions (PXGL n = 2336, primed n = 7796) or 200 nM LDN193189 (PXGL n = 2398, primed n = 6500, N = 2 experiments). n , Pearson regression correlation coefficients for ID1 and ID3 expression with naïve pluripotency markers. o , Immunofluorescence and quantification of AP2γ in hESCs (LDN: n = 1695 untreated cells; n = 1426 LDN treated cells; N = 2 experiments). p , Immunofluorescence and quantification of Otx2 in mESCs cultured in either 2iLif naïve conditions (n = 1570 cells), N2B27 (n = 3602 cells), or N2B27 + LDN (n = 1064 cells). N = 3 experiments. q , Immunofluorescence images and quantification of pSMAD1.5.9 in YSLCs (control: n = 1150; LDN: n = 827; N = 2 experiments). For box plots, the box encompasses the 25 th -75 th quartiles with the median marked by the central line. The minimum and maximum are denoted by the whiskers. For violin plots, the mean is marked by the red line. Statistical tests: ( c, k-l, p ) One-way ANOVA with Tukey-Kramer post-hoc; ( e-f ) two-sided Mann-Whitney test, ( m, o, q ) two-sided unpaired T-test; ( o ) Coefficients were tested (cor.test; two-tailed) and corrected for multiple hypothesis testing with the Benjamini-Hochberg method; ****p < 0.0001. ***p < 0.001. **p < 0.01. *p < 0.05. Unmarked pairwise comparisons are not significant (ns). Exact p-values presented in Supplemental Table 8. Scale bars: ( b, m, o-q ) 50 µm, ( f, i ) 100 µm.
    Figure Legend Snippet: a , Violin plots of BMP2/4/6 expression. N = 9862 single cells. b-c , Immunofluorescence images and quantification of primed hESCs cultured in control conditions (n = 2825), 25 ng/ml BMP2 (n = 2184), 25 ng/ml BMP4 (n = 1681), 50 ng/ml BMP6 (n = 889), or 50 ng/ml BMP6 + 200 nM LDN193189 (n = 1406). d-e , Bayesian coefficients ± credible interval for counts presented in Fig. ( d ) and Fig. ( e ) . f , Immunofluorescence images of mouse embryos recovered at E3.5 and cultured in vitro for 48 h in control (N = 19), 50 ng/ml BMP6 (N = 11), or 200 nM LDN193189 (N = 13) conditions. g-h , Quantification of the number epiblast ( g ) and primitive endoderm/visceral endoderm ( h ) from f . i , Immunofluorescence images of mouse embryos recovered at E5.0 and cultured in vitro for 36 h in control (N = 8), BMP6 (N = 10) or LDN193189 (N = 8) conditions. j , Quantification of the proportion of embryos that successfully made an egg cylinder and have a Cer1-positive DVE/AVE. k-l , Quantification of the epiblast length ( k ) and area ( l) . m , Immunofluorescence images and quantification of hESCs treated for 48 hours with control conditions (PXGL n = 2336, primed n = 7796) or 200 nM LDN193189 (PXGL n = 2398, primed n = 6500, N = 2 experiments). n , Pearson regression correlation coefficients for ID1 and ID3 expression with naïve pluripotency markers. o , Immunofluorescence and quantification of AP2γ in hESCs (LDN: n = 1695 untreated cells; n = 1426 LDN treated cells; N = 2 experiments). p , Immunofluorescence and quantification of Otx2 in mESCs cultured in either 2iLif naïve conditions (n = 1570 cells), N2B27 (n = 3602 cells), or N2B27 + LDN (n = 1064 cells). N = 3 experiments. q , Immunofluorescence images and quantification of pSMAD1.5.9 in YSLCs (control: n = 1150; LDN: n = 827; N = 2 experiments). For box plots, the box encompasses the 25 th -75 th quartiles with the median marked by the central line. The minimum and maximum are denoted by the whiskers. For violin plots, the mean is marked by the red line. Statistical tests: ( c, k-l, p ) One-way ANOVA with Tukey-Kramer post-hoc; ( e-f ) two-sided Mann-Whitney test, ( m, o, q ) two-sided unpaired T-test; ( o ) Coefficients were tested (cor.test; two-tailed) and corrected for multiple hypothesis testing with the Benjamini-Hochberg method; ****p < 0.0001. ***p < 0.001. **p < 0.01. *p < 0.05. Unmarked pairwise comparisons are not significant (ns). Exact p-values presented in Supplemental Table 8. Scale bars: ( b, m, o-q ) 50 µm, ( f, i ) 100 µm.

    Techniques Used: Expressing, Immunofluorescence, Cell Culture, Control, In Vitro, MANN-WHITNEY, Two Tailed Test



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    a , Violin plots of BMP2/4/6 expression. N = 9862 single cells. b-c , Immunofluorescence images and quantification of primed hESCs cultured in control conditions (n = 2825), 25 ng/ml BMP2 (n = 2184), 25 ng/ml BMP4 (n = 1681), 50 ng/ml BMP6 (n = 889), or 50 ng/ml BMP6 + 200 nM LDN193189 (n = 1406). d-e , Bayesian coefficients ± credible interval for counts presented in Fig. ( d ) and Fig. ( e ) . f , Immunofluorescence images of mouse embryos recovered at E3.5 and cultured in vitro for 48 h in control (N = 19), 50 ng/ml BMP6 (N = 11), or 200 nM LDN193189 (N = 13) conditions. g-h , Quantification of the number epiblast ( g ) and primitive endoderm/visceral endoderm ( h ) from f . i , Immunofluorescence images of mouse embryos recovered at E5.0 and cultured in vitro for 36 h in control (N = 8), BMP6 (N = 10) or LDN193189 (N = 8) conditions. j , Quantification of the proportion of embryos that successfully made an egg cylinder and have a Cer1-positive DVE/AVE. k-l , Quantification of the epiblast length ( k ) and area ( l) . m , Immunofluorescence images and quantification of hESCs treated for 48 hours with control conditions (PXGL n = 2336, primed n = 7796) or 200 nM LDN193189 (PXGL n = 2398, primed n = 6500, N = 2 experiments). n , Pearson regression correlation coefficients for ID1 and ID3 expression with naïve pluripotency markers. o , Immunofluorescence and quantification of AP2γ in hESCs (LDN: n = 1695 untreated cells; n = 1426 LDN treated cells; N = 2 experiments). p , Immunofluorescence and quantification of Otx2 in mESCs cultured in either 2iLif naïve conditions (n = 1570 cells), N2B27 (n = 3602 cells), or N2B27 + LDN (n = 1064 cells). N = 3 experiments. q , Immunofluorescence images and quantification of pSMAD1.5.9 in YSLCs (control: n = 1150; LDN: n = 827; N = 2 experiments). For box plots, the box encompasses the 25 th -75 th quartiles with the median marked by the central line. The minimum and maximum are denoted by the whiskers. For violin plots, the mean is marked by the red line. Statistical tests: ( c, k-l, p ) One-way ANOVA with Tukey-Kramer post-hoc; ( e-f ) two-sided Mann-Whitney test, ( m, o, q ) two-sided unpaired T-test; ( o ) Coefficients were tested (cor.test; two-tailed) and corrected for multiple hypothesis testing with the Benjamini-Hochberg method; ****p < 0.0001. ***p < 0.001. **p < 0.01. *p < 0.05. Unmarked pairwise comparisons are not significant (ns). Exact p-values presented in Supplemental Table 8. Scale bars: ( b, m, o-q ) 50 µm, ( f, i ) 100 µm.
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    tfap2c gfp tagged  (OriGene)
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    OriGene tfap2c gfp tagged
    Fig. 1. Deptor was upregulated during the adipogenic differentiation but downregulated during the osteogenic differentiation of BMSCs. (a–d) Primary rat BMSCs were induced to differentiate into osteoblasts for 14 days and subjected to quantitative reverse transcriptase (qRT)-PCR analysis of the relative levels of osteocalcin (Ocn) mRNA (a), Western blot analysis of protein levels of Ocn and Deptor (b), alkaline phosphatase (ALP) staining (c), and qRT-PCR analysis of the relative levels of Deptor mRNA (d) on the indicated days. (e–g) Primary rat BMSCs were induced to differentiate into adipocytes for 7 days, and subjected to qRT-PCR analysis of the relative levels of fatty acid–binding protein <t>(aP2)</t> mRNA (e), Western blot analysis of protein levels of aP2 and Deptor (f), Oil red O staining to visualize lipid droplet formation (g), and qRT-PCR analysis of the relative levels of Deptor mRNA (h) on the indicated days. GAPDH served as an internal control. All experiments were repeated three times. Scale bar, 100 µm in (g). n = 6 in a, c, d, e, g and h. All data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student t test).
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    Image Search Results


    a , Violin plots of BMP2/4/6 expression. N = 9862 single cells. b-c , Immunofluorescence images and quantification of primed hESCs cultured in control conditions (n = 2825), 25 ng/ml BMP2 (n = 2184), 25 ng/ml BMP4 (n = 1681), 50 ng/ml BMP6 (n = 889), or 50 ng/ml BMP6 + 200 nM LDN193189 (n = 1406). d-e , Bayesian coefficients ± credible interval for counts presented in Fig. ( d ) and Fig. ( e ) . f , Immunofluorescence images of mouse embryos recovered at E3.5 and cultured in vitro for 48 h in control (N = 19), 50 ng/ml BMP6 (N = 11), or 200 nM LDN193189 (N = 13) conditions. g-h , Quantification of the number epiblast ( g ) and primitive endoderm/visceral endoderm ( h ) from f . i , Immunofluorescence images of mouse embryos recovered at E5.0 and cultured in vitro for 36 h in control (N = 8), BMP6 (N = 10) or LDN193189 (N = 8) conditions. j , Quantification of the proportion of embryos that successfully made an egg cylinder and have a Cer1-positive DVE/AVE. k-l , Quantification of the epiblast length ( k ) and area ( l) . m , Immunofluorescence images and quantification of hESCs treated for 48 hours with control conditions (PXGL n = 2336, primed n = 7796) or 200 nM LDN193189 (PXGL n = 2398, primed n = 6500, N = 2 experiments). n , Pearson regression correlation coefficients for ID1 and ID3 expression with naïve pluripotency markers. o , Immunofluorescence and quantification of AP2γ in hESCs (LDN: n = 1695 untreated cells; n = 1426 LDN treated cells; N = 2 experiments). p , Immunofluorescence and quantification of Otx2 in mESCs cultured in either 2iLif naïve conditions (n = 1570 cells), N2B27 (n = 3602 cells), or N2B27 + LDN (n = 1064 cells). N = 3 experiments. q , Immunofluorescence images and quantification of pSMAD1.5.9 in YSLCs (control: n = 1150; LDN: n = 827; N = 2 experiments). For box plots, the box encompasses the 25 th -75 th quartiles with the median marked by the central line. The minimum and maximum are denoted by the whiskers. For violin plots, the mean is marked by the red line. Statistical tests: ( c, k-l, p ) One-way ANOVA with Tukey-Kramer post-hoc; ( e-f ) two-sided Mann-Whitney test, ( m, o, q ) two-sided unpaired T-test; ( o ) Coefficients were tested (cor.test; two-tailed) and corrected for multiple hypothesis testing with the Benjamini-Hochberg method; ****p < 0.0001. ***p < 0.001. **p < 0.01. *p < 0.05. Unmarked pairwise comparisons are not significant (ns). Exact p-values presented in Supplemental Table 8. Scale bars: ( b, m, o-q ) 50 µm, ( f, i ) 100 µm.

    Journal: Nature Cell Biology

    Article Title: Distinct pathways drive anterior hypoblast specification in the implanting human embryo

    doi: 10.1038/s41556-024-01367-1

    Figure Lengend Snippet: a , Violin plots of BMP2/4/6 expression. N = 9862 single cells. b-c , Immunofluorescence images and quantification of primed hESCs cultured in control conditions (n = 2825), 25 ng/ml BMP2 (n = 2184), 25 ng/ml BMP4 (n = 1681), 50 ng/ml BMP6 (n = 889), or 50 ng/ml BMP6 + 200 nM LDN193189 (n = 1406). d-e , Bayesian coefficients ± credible interval for counts presented in Fig. ( d ) and Fig. ( e ) . f , Immunofluorescence images of mouse embryos recovered at E3.5 and cultured in vitro for 48 h in control (N = 19), 50 ng/ml BMP6 (N = 11), or 200 nM LDN193189 (N = 13) conditions. g-h , Quantification of the number epiblast ( g ) and primitive endoderm/visceral endoderm ( h ) from f . i , Immunofluorescence images of mouse embryos recovered at E5.0 and cultured in vitro for 36 h in control (N = 8), BMP6 (N = 10) or LDN193189 (N = 8) conditions. j , Quantification of the proportion of embryos that successfully made an egg cylinder and have a Cer1-positive DVE/AVE. k-l , Quantification of the epiblast length ( k ) and area ( l) . m , Immunofluorescence images and quantification of hESCs treated for 48 hours with control conditions (PXGL n = 2336, primed n = 7796) or 200 nM LDN193189 (PXGL n = 2398, primed n = 6500, N = 2 experiments). n , Pearson regression correlation coefficients for ID1 and ID3 expression with naïve pluripotency markers. o , Immunofluorescence and quantification of AP2γ in hESCs (LDN: n = 1695 untreated cells; n = 1426 LDN treated cells; N = 2 experiments). p , Immunofluorescence and quantification of Otx2 in mESCs cultured in either 2iLif naïve conditions (n = 1570 cells), N2B27 (n = 3602 cells), or N2B27 + LDN (n = 1064 cells). N = 3 experiments. q , Immunofluorescence images and quantification of pSMAD1.5.9 in YSLCs (control: n = 1150; LDN: n = 827; N = 2 experiments). For box plots, the box encompasses the 25 th -75 th quartiles with the median marked by the central line. The minimum and maximum are denoted by the whiskers. For violin plots, the mean is marked by the red line. Statistical tests: ( c, k-l, p ) One-way ANOVA with Tukey-Kramer post-hoc; ( e-f ) two-sided Mann-Whitney test, ( m, o, q ) two-sided unpaired T-test; ( o ) Coefficients were tested (cor.test; two-tailed) and corrected for multiple hypothesis testing with the Benjamini-Hochberg method; ****p < 0.0001. ***p < 0.001. **p < 0.01. *p < 0.05. Unmarked pairwise comparisons are not significant (ns). Exact p-values presented in Supplemental Table 8. Scale bars: ( b, m, o-q ) 50 µm, ( f, i ) 100 µm.

    Article Snippet: Primary antibodies used in this study are as follows: mouse monoclonal anti OCT3/4 (sc5279, Santa Cruz; 1:200 dilution), rat monoclonal anti SOX2 (14-19811-82, Thermo Fisher Scientific; 1:500 dilution), goat polyclonal anti NANOG (AF1997 R&D Systems; 1:500 dilution), rabbit monoclonal anti GATA6 (5851, Cell Signaling Technology; 1:2,000 dilution), goat polyclonal anti GATA6 (AF1700, R&D Systems; 1:200 dilution), mouse anti monoclonal Cdx2 (MU392-UC, Biogenex; 1:200 dilution), goat polyclonal anti CER1 (AF1075, R&D Systems; 1:250 dilution), rat monoclonal anti Cerebus1 (MAB1986, R&D Systems; 1:200 dilution), rabbit monoclonal anti Phospho-Smad1(Ser463/465)/Smad5(Ser463/465)/Smad9(Ser465/467) (13820T, Cell Signaling Technology; 1:200 dilution), rabbit monoclonal anti Smad2.3 (8685T, Cell Signaling Technology; 1:200 dilution), rabbit monoclonal anti-cleaved caspase 3 (9664, Cell Signaling Technology; 1:200 dilution), mouse monoclonal anti Podocalyxin (MAB1658, R&D Systems; 1:500 dilution), goat polyclonal anti Brachyury (AF2085, R&D Systems; 1:500 dilution), rat monoclonal anti GATA4 (14-9980-82, Thermo Fisher Scientific; 1:500 dilution), goat polyclonal anti AP2-gamma (AF5059, R&D Systems; 1:500 dilution), goat polyclonal anti Otx2 (AF1979, R&D Systems; 1:1,000 dilution) and Alexa Flour 594 Phalloidin ( A12381 , Thermo Fisher Scientific; 1:500 dilution).

    Techniques: Expressing, Immunofluorescence, Cell Culture, Control, In Vitro, MANN-WHITNEY, Two Tailed Test

    Affymetrix microarray gene expression analysis performed with RNA extracted from #1- ctrl ; #1- Tfap2c −/− and #2- ctrl ; #2- Tfap2c −/− ESCs and PGCLCs. (A) Hierarchical clustering. The red bars cluster the ESCs, green bars the Tfap2c −/− PGCLCs and blue bars ctrl PGCLCs, respectively. The shorter the horizontal bar that connects two branches the closer are the populations. (B) Heat map was performed with the probes whose range or variation across all samples was at least 3. Color bar on top codifies the gene expression in log2 scale. Red and blue indicate higher and lower relative expression. (C-D) Pairwise scatter plot of global gene expression in ctrl versus Tfap2c −/− PGCLCs (C) and ESCs (D). Black lines indicate 1.5 fold-change in log2 scale of gene expression levels between paired PGCLCs and ESCs. Color bars on the side display the scattering density with light blue indicating lower and blue higher scatter density. Genes upregulated in Tfap2c −/− samples are shown as red dots; genes downregulated are shown as green dots. R 2 = Fisher’s correlation coefficient. (E) Venn diagram; in PGCLCs 455 genes are deregulated; in ESCs 26 genes are deregulated. The intersection part show the commonly deregulated genes (n = 13) by TFAP2C in PGCLCs and ESCs (Fold-change >1.5 in log2 scale).

    Journal: PLoS ONE

    Article Title: Transcription Factor TFAP2C Regulates Major Programs Required for Murine Fetal Germ Cell Maintenance and Haploinsufficiency Predisposes to Teratomas in Male Mice

    doi: 10.1371/journal.pone.0071113

    Figure Lengend Snippet: Affymetrix microarray gene expression analysis performed with RNA extracted from #1- ctrl ; #1- Tfap2c −/− and #2- ctrl ; #2- Tfap2c −/− ESCs and PGCLCs. (A) Hierarchical clustering. The red bars cluster the ESCs, green bars the Tfap2c −/− PGCLCs and blue bars ctrl PGCLCs, respectively. The shorter the horizontal bar that connects two branches the closer are the populations. (B) Heat map was performed with the probes whose range or variation across all samples was at least 3. Color bar on top codifies the gene expression in log2 scale. Red and blue indicate higher and lower relative expression. (C-D) Pairwise scatter plot of global gene expression in ctrl versus Tfap2c −/− PGCLCs (C) and ESCs (D). Black lines indicate 1.5 fold-change in log2 scale of gene expression levels between paired PGCLCs and ESCs. Color bars on the side display the scattering density with light blue indicating lower and blue higher scatter density. Genes upregulated in Tfap2c −/− samples are shown as red dots; genes downregulated are shown as green dots. R 2 = Fisher’s correlation coefficient. (E) Venn diagram; in PGCLCs 455 genes are deregulated; in ESCs 26 genes are deregulated. The intersection part show the commonly deregulated genes (n = 13) by TFAP2C in PGCLCs and ESCs (Fold-change >1.5 in log2 scale).

    Article Snippet: Scrambled and TFAP2C siRNA were obtained from Origene (TFAP2C SR304789, Origene Technologies, Rockville, USA).

    Techniques: Microarray, Expressing

    (A) Quantitative RT-PCR of a subset of markers with RNA isolated from ctrl and Tfap2c −/− PGCLCs. Expression levels were normalized to βActin and expression level of ctrl PGCLCs were set to 1. qRT-PCR was performed in biological triplicates. Error bars indicate standard deviation. (B) Quantitative RT-PCR of a subset of markers with RNA isolated from TCam-2 cells after siRNA mediated knockdown of TFAP2C. Expression levels were normalized to GAPDH and expression levels of scrambled-siRNA-transfection were set to 1. qRT-PCR was performed in biological triplicate. Error bars indicate standard deviation. (C) ChIP/qPCR analysis for Tfap2c was performed with four biological replicates of PGCLCs. The qPCR results were calculated with the percentage input method and ChIP analyses with IgG antibody served as control and were set to 1. Error bars indicate standard deviation. ChIP analysis demonstrates increased binding of TFAP2C at indicated loci. (A) – (C) Marker genes were grouped in categories: somatic differentiation (category I), germ cell maintenance and maturation (category II), cell cycle regulation (category III), pluripotency (category IV) and epigenetic modification (category V).

    Journal: PLoS ONE

    Article Title: Transcription Factor TFAP2C Regulates Major Programs Required for Murine Fetal Germ Cell Maintenance and Haploinsufficiency Predisposes to Teratomas in Male Mice

    doi: 10.1371/journal.pone.0071113

    Figure Lengend Snippet: (A) Quantitative RT-PCR of a subset of markers with RNA isolated from ctrl and Tfap2c −/− PGCLCs. Expression levels were normalized to βActin and expression level of ctrl PGCLCs were set to 1. qRT-PCR was performed in biological triplicates. Error bars indicate standard deviation. (B) Quantitative RT-PCR of a subset of markers with RNA isolated from TCam-2 cells after siRNA mediated knockdown of TFAP2C. Expression levels were normalized to GAPDH and expression levels of scrambled-siRNA-transfection were set to 1. qRT-PCR was performed in biological triplicate. Error bars indicate standard deviation. (C) ChIP/qPCR analysis for Tfap2c was performed with four biological replicates of PGCLCs. The qPCR results were calculated with the percentage input method and ChIP analyses with IgG antibody served as control and were set to 1. Error bars indicate standard deviation. ChIP analysis demonstrates increased binding of TFAP2C at indicated loci. (A) – (C) Marker genes were grouped in categories: somatic differentiation (category I), germ cell maintenance and maturation (category II), cell cycle regulation (category III), pluripotency (category IV) and epigenetic modification (category V).

    Article Snippet: Scrambled and TFAP2C siRNA were obtained from Origene (TFAP2C SR304789, Origene Technologies, Rockville, USA).

    Techniques: Quantitative RT-PCR, Isolation, Expressing, Standard Deviation, Transfection, Binding Assay, Marker, Modification

    (A) Quantitative RT-PCR with RNA isolated from E12.5 genital ridges of wt and Tfap2c +/− embryos was performed. Expression levels of Tfap2c and p21 were normalized to Gapdh . qRT-PCR was performed in biological duplicates. Error bars indicate standard deviation. (B) Percentage of total tumor incidence in Tfap2c heterozygous 129S2/Sv male mice. The seventh generation in 129S2/Sv shows 82% (n = 51) testicular tumors. Red bar: bilateral cases (35%), blue bar: unilateral tumors (47%). (C) Gross pathology of testicular teratoma in Tfap2c +/− male mice. (D–G) HE-staining of testicular teratomas of 3–6 month old mice. Tumors show immature glia (D), mature cartilage, muscle (E), respiratory epithelium (F) and squamous epithelium (G). Scale bars: 200 µm.

    Journal: PLoS ONE

    Article Title: Transcription Factor TFAP2C Regulates Major Programs Required for Murine Fetal Germ Cell Maintenance and Haploinsufficiency Predisposes to Teratomas in Male Mice

    doi: 10.1371/journal.pone.0071113

    Figure Lengend Snippet: (A) Quantitative RT-PCR with RNA isolated from E12.5 genital ridges of wt and Tfap2c +/− embryos was performed. Expression levels of Tfap2c and p21 were normalized to Gapdh . qRT-PCR was performed in biological duplicates. Error bars indicate standard deviation. (B) Percentage of total tumor incidence in Tfap2c heterozygous 129S2/Sv male mice. The seventh generation in 129S2/Sv shows 82% (n = 51) testicular tumors. Red bar: bilateral cases (35%), blue bar: unilateral tumors (47%). (C) Gross pathology of testicular teratoma in Tfap2c +/− male mice. (D–G) HE-staining of testicular teratomas of 3–6 month old mice. Tumors show immature glia (D), mature cartilage, muscle (E), respiratory epithelium (F) and squamous epithelium (G). Scale bars: 200 µm.

    Article Snippet: Scrambled and TFAP2C siRNA were obtained from Origene (TFAP2C SR304789, Origene Technologies, Rockville, USA).

    Techniques: Quantitative RT-PCR, Isolation, Expressing, Standard Deviation, Staining

    (A–C) IHC staining of E16.5 embryonal testis. (A) SSEA1, (B) OCT3/4 and (C) TFAP2C. Scale bars: 50 µm. (B) Black arrow show cells with large, highly condensated nuclei as well as clearly visible nucleoli. (D) RT-PCR from RNA of microdissected SSEA-1 positive foci of E16.5 testes detecting Nanog , Sox2 and c-Kit . Comparison of microdissected SSEA-1 positive tissue (T) and SSEA-1 negative tissue (wt). Gapdh served as control.

    Journal: PLoS ONE

    Article Title: Transcription Factor TFAP2C Regulates Major Programs Required for Murine Fetal Germ Cell Maintenance and Haploinsufficiency Predisposes to Teratomas in Male Mice

    doi: 10.1371/journal.pone.0071113

    Figure Lengend Snippet: (A–C) IHC staining of E16.5 embryonal testis. (A) SSEA1, (B) OCT3/4 and (C) TFAP2C. Scale bars: 50 µm. (B) Black arrow show cells with large, highly condensated nuclei as well as clearly visible nucleoli. (D) RT-PCR from RNA of microdissected SSEA-1 positive foci of E16.5 testes detecting Nanog , Sox2 and c-Kit . Comparison of microdissected SSEA-1 positive tissue (T) and SSEA-1 negative tissue (wt). Gapdh served as control.

    Article Snippet: Scrambled and TFAP2C siRNA were obtained from Origene (TFAP2C SR304789, Origene Technologies, Rockville, USA).

    Techniques: Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction

    Black arrows indicate pathways transactivated and induced by TFAP2C and black lines with terminal bars indicate pathways repressed by TFAP2C during development of primordial germ cells. Genes listed in respective pathways indicate direct regulation as demonstrated by ChIP analyses.

    Journal: PLoS ONE

    Article Title: Transcription Factor TFAP2C Regulates Major Programs Required for Murine Fetal Germ Cell Maintenance and Haploinsufficiency Predisposes to Teratomas in Male Mice

    doi: 10.1371/journal.pone.0071113

    Figure Lengend Snippet: Black arrows indicate pathways transactivated and induced by TFAP2C and black lines with terminal bars indicate pathways repressed by TFAP2C during development of primordial germ cells. Genes listed in respective pathways indicate direct regulation as demonstrated by ChIP analyses.

    Article Snippet: Scrambled and TFAP2C siRNA were obtained from Origene (TFAP2C SR304789, Origene Technologies, Rockville, USA).

    Techniques:

    Fig. 1. Deptor was upregulated during the adipogenic differentiation but downregulated during the osteogenic differentiation of BMSCs. (a–d) Primary rat BMSCs were induced to differentiate into osteoblasts for 14 days and subjected to quantitative reverse transcriptase (qRT)-PCR analysis of the relative levels of osteocalcin (Ocn) mRNA (a), Western blot analysis of protein levels of Ocn and Deptor (b), alkaline phosphatase (ALP) staining (c), and qRT-PCR analysis of the relative levels of Deptor mRNA (d) on the indicated days. (e–g) Primary rat BMSCs were induced to differentiate into adipocytes for 7 days, and subjected to qRT-PCR analysis of the relative levels of fatty acid–binding protein (aP2) mRNA (e), Western blot analysis of protein levels of aP2 and Deptor (f), Oil red O staining to visualize lipid droplet formation (g), and qRT-PCR analysis of the relative levels of Deptor mRNA (h) on the indicated days. GAPDH served as an internal control. All experiments were repeated three times. Scale bar, 100 µm in (g). n = 6 in a, c, d, e, g and h. All data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student t test).

    Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

    Article Title: DEPTOR exacerbates bone-fat imbalance in osteoporosis by transcriptionally modulating BMSC differentiation.

    doi: 10.1016/j.biopha.2022.113164

    Figure Lengend Snippet: Fig. 1. Deptor was upregulated during the adipogenic differentiation but downregulated during the osteogenic differentiation of BMSCs. (a–d) Primary rat BMSCs were induced to differentiate into osteoblasts for 14 days and subjected to quantitative reverse transcriptase (qRT)-PCR analysis of the relative levels of osteocalcin (Ocn) mRNA (a), Western blot analysis of protein levels of Ocn and Deptor (b), alkaline phosphatase (ALP) staining (c), and qRT-PCR analysis of the relative levels of Deptor mRNA (d) on the indicated days. (e–g) Primary rat BMSCs were induced to differentiate into adipocytes for 7 days, and subjected to qRT-PCR analysis of the relative levels of fatty acid–binding protein (aP2) mRNA (e), Western blot analysis of protein levels of aP2 and Deptor (f), Oil red O staining to visualize lipid droplet formation (g), and qRT-PCR analysis of the relative levels of Deptor mRNA (h) on the indicated days. GAPDH served as an internal control. All experiments were repeated three times. Scale bar, 100 µm in (g). n = 6 in a, c, d, e, g and h. All data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student t test).

    Article Snippet: The membranes were then incubated with specific antibodies to Ocn (Abcam, 1:1500, #AB93876), DEPTOR (Santa Cruz Biotechnology,1:200, #sc-398169), aP2 (Proteintech, 1:2000, #12802-1-AP), PPAR-γ (Proteintech, 1:1500, #16643-1-AP), TAZ (Proteintech,1:2000, #23306-1-AP), Runx2 (Santa Cruz Biotechnology, 1:200, #sc-390351), and GAPDH (Proteintech, 1:2000, #60004-1-Ig 4) and visualized by enhanced chemiluminescence (ECL Kit, Amersham Biosciences).

    Techniques: Reverse Transcription, Quantitative RT-PCR, Western Blot, Staining, Binding Assay, Control

    Fig. 2. Deptor was involved in bone–fat imbalance in osteoporosis. Representative images of micro-CT analyses (a) and quantitative analysis (b) of the trabecular bone microstructure of metaphyseal bone in proximal tibia from OVX and Sham mice (n = 5 per group). Scale bar, 500 µm. Representative images of H&E staining (c) and quantification of bone and adipocyte area (d) in the tibia; n = 6, scale bar = 200 µm. (e–j) Primary BMSCs were obtained from OVX and Sham mice. The cells were induced to differentiate into adipocytes and subjected to the western blot analysis of aP2 expression (e) and Oil red O staining (f) (n = 6). Scale bar, 100 µm. The cells were induced to differentiate into osteoblasts and subjected to the Western blot analysis of Ocn expression (g) and ALP staining (h) (n = 6). The cells were expanded in vitro and subjected to qPCR (i) (n = 6) or Western blot analysis (j) of Deptor expression. GAPDH served as an internal control. All experiments were repeated three times. All data are shown as the mean ± SD. **P < 0.01, ***P < 0.001, ns = not significant (Student t test).

    Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

    Article Title: DEPTOR exacerbates bone-fat imbalance in osteoporosis by transcriptionally modulating BMSC differentiation.

    doi: 10.1016/j.biopha.2022.113164

    Figure Lengend Snippet: Fig. 2. Deptor was involved in bone–fat imbalance in osteoporosis. Representative images of micro-CT analyses (a) and quantitative analysis (b) of the trabecular bone microstructure of metaphyseal bone in proximal tibia from OVX and Sham mice (n = 5 per group). Scale bar, 500 µm. Representative images of H&E staining (c) and quantification of bone and adipocyte area (d) in the tibia; n = 6, scale bar = 200 µm. (e–j) Primary BMSCs were obtained from OVX and Sham mice. The cells were induced to differentiate into adipocytes and subjected to the western blot analysis of aP2 expression (e) and Oil red O staining (f) (n = 6). Scale bar, 100 µm. The cells were induced to differentiate into osteoblasts and subjected to the Western blot analysis of Ocn expression (g) and ALP staining (h) (n = 6). The cells were expanded in vitro and subjected to qPCR (i) (n = 6) or Western blot analysis (j) of Deptor expression. GAPDH served as an internal control. All experiments were repeated three times. All data are shown as the mean ± SD. **P < 0.01, ***P < 0.001, ns = not significant (Student t test).

    Article Snippet: The membranes were then incubated with specific antibodies to Ocn (Abcam, 1:1500, #AB93876), DEPTOR (Santa Cruz Biotechnology,1:200, #sc-398169), aP2 (Proteintech, 1:2000, #12802-1-AP), PPAR-γ (Proteintech, 1:1500, #16643-1-AP), TAZ (Proteintech,1:2000, #23306-1-AP), Runx2 (Santa Cruz Biotechnology, 1:200, #sc-390351), and GAPDH (Proteintech, 1:2000, #60004-1-Ig 4) and visualized by enhanced chemiluminescence (ECL Kit, Amersham Biosciences).

    Techniques: Micro-CT, Staining, Western Blot, Expressing, In Vitro, Control

    Fig. 4. Deptor impaired osteogenic and facilitated adipogenic differentiation of BMSCs in vivo and in vitro. (a) Representative IHC images of Ocn in bone sections from ovariectomized control and Deptor-cKO mice; scale bar = 100 µm. (b) Quantification of Ocn-positive cells (N.Ocn+) on the bone surface per bone perimeter (/B.Pm). n = 7. (c) Representative images of calcein labels of femurs from ovariectomized control and Deptor-cKO mice; scale bar = 20 µm. (d) Quan tification of the mineral apposition rate (MAR) in c. n = 5. (e–j) Rat BMSCs were infected with lentivirus expressing shRNA targeting Deptor or Deptor-encoding plasmids. The cells were induced to differentiate into osteoblasts and subjected to qPCR (e) and Western blot (f) analysis of Deptor and Ocn expression and ALP staining (g). The cells were induced to differentiate into adipocytes and subjected qPCR (h) and Western blot (i) analysis of Deptor and aP2 expression and Oil red O staining (j). Scale bar = 100 µm. n = 6. All experiments were repeated three times. All data are shown as the mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student t test).

    Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

    Article Title: DEPTOR exacerbates bone-fat imbalance in osteoporosis by transcriptionally modulating BMSC differentiation.

    doi: 10.1016/j.biopha.2022.113164

    Figure Lengend Snippet: Fig. 4. Deptor impaired osteogenic and facilitated adipogenic differentiation of BMSCs in vivo and in vitro. (a) Representative IHC images of Ocn in bone sections from ovariectomized control and Deptor-cKO mice; scale bar = 100 µm. (b) Quantification of Ocn-positive cells (N.Ocn+) on the bone surface per bone perimeter (/B.Pm). n = 7. (c) Representative images of calcein labels of femurs from ovariectomized control and Deptor-cKO mice; scale bar = 20 µm. (d) Quan tification of the mineral apposition rate (MAR) in c. n = 5. (e–j) Rat BMSCs were infected with lentivirus expressing shRNA targeting Deptor or Deptor-encoding plasmids. The cells were induced to differentiate into osteoblasts and subjected to qPCR (e) and Western blot (f) analysis of Deptor and Ocn expression and ALP staining (g). The cells were induced to differentiate into adipocytes and subjected qPCR (h) and Western blot (i) analysis of Deptor and aP2 expression and Oil red O staining (j). Scale bar = 100 µm. n = 6. All experiments were repeated three times. All data are shown as the mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student t test).

    Article Snippet: The membranes were then incubated with specific antibodies to Ocn (Abcam, 1:1500, #AB93876), DEPTOR (Santa Cruz Biotechnology,1:200, #sc-398169), aP2 (Proteintech, 1:2000, #12802-1-AP), PPAR-γ (Proteintech, 1:1500, #16643-1-AP), TAZ (Proteintech,1:2000, #23306-1-AP), Runx2 (Santa Cruz Biotechnology, 1:200, #sc-390351), and GAPDH (Proteintech, 1:2000, #60004-1-Ig 4) and visualized by enhanced chemiluminescence (ECL Kit, Amersham Biosciences).

    Techniques: In Vivo, In Vitro, Control, Infection, Expressing, shRNA, Western Blot, Staining

    Fig. 5. Deptor coordinated the transcriptional activities of Runx2 and PPARγ to determine the cell fates of BMSCs. (a) Endogenous Deptor co- immunoprecipitated endogenous Runx2. Anti-Deptor and anti-Runx2 immunoprecipitates were prepared from C3H10T1/2 cells and analyzed for Runx2 and Dep tor, respectively. (b) Binding of Runx2 to the promoter sequence of Ocn in vivo, determined by a ChIP assay using Deptor knockdown and overexpression C3H10T1/2 cells and anti-Runx2 antibody or IgG. The ChIP samples were then subjected to qPCR with the Ocn promoter primers. The percentage of the input of the sample using anti-Runx2 antibody in the control cells was normalized to 100. (c) Schematic representation of the structural features of p6OSE2-Luciferase. (d) C3H10T1/2 cells infected with lentivirus expressing shRNA targeting Deptor or Deptor-encoding plasmids were cotransfected with p6OSE2-Lucferase plasmid. The cell lysates were analyzed for Deptor expression and luciferase reporter activity (n = 6). (e) Endogenous Deptor co-immunoprecipitated endogenous PPARγ. Anti-Deptor and anti- PPARγ immunoprecipitates were prepared from C3H10T1/2 cells and analyzed for PPARγ and Deptor, respectively. (f) Binding of PPARγ to the promoter sequence of aP2 in vivo, determined by a ChIP assay using Deptor knockdown and overexpression C3H10T1/2 cells and anti- PPARγ antibody or IgG. The ChIP samples were then subjected to qPCR with the aP2 promoter primers. The percentage of the input of the sample using anti-Runx2 antibody in the control cells was normalized to 100 (n = 6). (g) Schematic representation of structural features of the aP2-Lucferase plasmid. (h) C3H10T1/2 cells infected with lentivirus expressing shRNA targeting Deptor or Deptor-encoding plasmids were cotransfected with aP2-Lucferase plasmid. The cell lysates were then analyzed for Deptor expression and luciferase reporter activity (n = 6). All experiments were repeated three times. All data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student t test).

    Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

    Article Title: DEPTOR exacerbates bone-fat imbalance in osteoporosis by transcriptionally modulating BMSC differentiation.

    doi: 10.1016/j.biopha.2022.113164

    Figure Lengend Snippet: Fig. 5. Deptor coordinated the transcriptional activities of Runx2 and PPARγ to determine the cell fates of BMSCs. (a) Endogenous Deptor co- immunoprecipitated endogenous Runx2. Anti-Deptor and anti-Runx2 immunoprecipitates were prepared from C3H10T1/2 cells and analyzed for Runx2 and Dep tor, respectively. (b) Binding of Runx2 to the promoter sequence of Ocn in vivo, determined by a ChIP assay using Deptor knockdown and overexpression C3H10T1/2 cells and anti-Runx2 antibody or IgG. The ChIP samples were then subjected to qPCR with the Ocn promoter primers. The percentage of the input of the sample using anti-Runx2 antibody in the control cells was normalized to 100. (c) Schematic representation of the structural features of p6OSE2-Luciferase. (d) C3H10T1/2 cells infected with lentivirus expressing shRNA targeting Deptor or Deptor-encoding plasmids were cotransfected with p6OSE2-Lucferase plasmid. The cell lysates were analyzed for Deptor expression and luciferase reporter activity (n = 6). (e) Endogenous Deptor co-immunoprecipitated endogenous PPARγ. Anti-Deptor and anti- PPARγ immunoprecipitates were prepared from C3H10T1/2 cells and analyzed for PPARγ and Deptor, respectively. (f) Binding of PPARγ to the promoter sequence of aP2 in vivo, determined by a ChIP assay using Deptor knockdown and overexpression C3H10T1/2 cells and anti- PPARγ antibody or IgG. The ChIP samples were then subjected to qPCR with the aP2 promoter primers. The percentage of the input of the sample using anti-Runx2 antibody in the control cells was normalized to 100 (n = 6). (g) Schematic representation of structural features of the aP2-Lucferase plasmid. (h) C3H10T1/2 cells infected with lentivirus expressing shRNA targeting Deptor or Deptor-encoding plasmids were cotransfected with aP2-Lucferase plasmid. The cell lysates were then analyzed for Deptor expression and luciferase reporter activity (n = 6). All experiments were repeated three times. All data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student t test).

    Article Snippet: The membranes were then incubated with specific antibodies to Ocn (Abcam, 1:1500, #AB93876), DEPTOR (Santa Cruz Biotechnology,1:200, #sc-398169), aP2 (Proteintech, 1:2000, #12802-1-AP), PPAR-γ (Proteintech, 1:1500, #16643-1-AP), TAZ (Proteintech,1:2000, #23306-1-AP), Runx2 (Santa Cruz Biotechnology, 1:200, #sc-390351), and GAPDH (Proteintech, 1:2000, #60004-1-Ig 4) and visualized by enhanced chemiluminescence (ECL Kit, Amersham Biosciences).

    Techniques: Immunoprecipitation, Binding Assay, Sequencing, In Vivo, Knockdown, Over Expression, Control, Luciferase, Infection, Expressing, shRNA, Plasmid Preparation, Activity Assay

    Fig. 7. DEPTOR regulated BMSC differentiation by inhibiting the transactivation properties of TAZ. (a) C3H10T1/2 cells undergoing osteogenic and adi pogenic differentiation were subjected to the immunofluorescence staining of TAZ; scale bar = 10 µm. (b) C3H10T1/2 cells infected with Deptor-encoding plasmids or lentivirus expressing shRNA targeting Deptor were subjected to immunofluorescence staining of TAZ; scale bar = 10 µm. (c) Representative IHC images of TAZ in bone sections from ovariectomized control and Deptor-cKO mice; scale bar = 200 µm. (d) Quantification of cells with TAZ expressed in the nucleus (nTAZ+) relative to total TAZ-positive cells (tTAZ+) (n = 6). (e) HEK 293/T cells expressing DEP domain or full-length DEPTOR were transfected with TAZ reporter plasmid. mCherry signals were observed to assess the transcriptional activity of TAZ in the cells (n = 5). Scale bar = 100 µm. (f–m) C3H10T1/2 cells infected with Deptor-encoding plasmids were further transfected with TAZ encoding plasmid. The cell lysates were analyzed for Deptor and TAZ expression (f) and p6OSE2-luciferase reporter activity (g) (n = 6), and subjected to qPCR analysis of Ocn (h) (n = 6) and ALP staining (i) (n = 6) after being induced to differentiate into osteoblasts. The cells were further analyzed for Deptor and TAZ expression (j) and aP2-luciferase reporter activity (k) (n = 6), and subjected to the qPCR analysis of aP2 (l) (n = 6) and Oil Red O staining (m) (n = 6) after being induced to adipogenic differentiation. Scale bar = 100 µm. All data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant (Student t test).

    Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

    Article Title: DEPTOR exacerbates bone-fat imbalance in osteoporosis by transcriptionally modulating BMSC differentiation.

    doi: 10.1016/j.biopha.2022.113164

    Figure Lengend Snippet: Fig. 7. DEPTOR regulated BMSC differentiation by inhibiting the transactivation properties of TAZ. (a) C3H10T1/2 cells undergoing osteogenic and adi pogenic differentiation were subjected to the immunofluorescence staining of TAZ; scale bar = 10 µm. (b) C3H10T1/2 cells infected with Deptor-encoding plasmids or lentivirus expressing shRNA targeting Deptor were subjected to immunofluorescence staining of TAZ; scale bar = 10 µm. (c) Representative IHC images of TAZ in bone sections from ovariectomized control and Deptor-cKO mice; scale bar = 200 µm. (d) Quantification of cells with TAZ expressed in the nucleus (nTAZ+) relative to total TAZ-positive cells (tTAZ+) (n = 6). (e) HEK 293/T cells expressing DEP domain or full-length DEPTOR were transfected with TAZ reporter plasmid. mCherry signals were observed to assess the transcriptional activity of TAZ in the cells (n = 5). Scale bar = 100 µm. (f–m) C3H10T1/2 cells infected with Deptor-encoding plasmids were further transfected with TAZ encoding plasmid. The cell lysates were analyzed for Deptor and TAZ expression (f) and p6OSE2-luciferase reporter activity (g) (n = 6), and subjected to qPCR analysis of Ocn (h) (n = 6) and ALP staining (i) (n = 6) after being induced to differentiate into osteoblasts. The cells were further analyzed for Deptor and TAZ expression (j) and aP2-luciferase reporter activity (k) (n = 6), and subjected to the qPCR analysis of aP2 (l) (n = 6) and Oil Red O staining (m) (n = 6) after being induced to adipogenic differentiation. Scale bar = 100 µm. All data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant (Student t test).

    Article Snippet: The membranes were then incubated with specific antibodies to Ocn (Abcam, 1:1500, #AB93876), DEPTOR (Santa Cruz Biotechnology,1:200, #sc-398169), aP2 (Proteintech, 1:2000, #12802-1-AP), PPAR-γ (Proteintech, 1:1500, #16643-1-AP), TAZ (Proteintech,1:2000, #23306-1-AP), Runx2 (Santa Cruz Biotechnology, 1:200, #sc-390351), and GAPDH (Proteintech, 1:2000, #60004-1-Ig 4) and visualized by enhanced chemiluminescence (ECL Kit, Amersham Biosciences).

    Techniques: Immunofluorescence, Staining, Infection, Expressing, shRNA, Control, Transfection, Plasmid Preparation, Activity Assay, Luciferase