Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The expression and significance of IDH1 and p53 in osteosarcoma

doi: 10.1186/1756-9966-29-43

Figure Lengend Snippet: The immunocytochemistry of IDH1 in MG63 and U2OS . IDH1 is specifically detected in the cytoplasm of both osteosarcoma cell lines MG63 and U2OS.(A) Expression of IDH1 in U2OS, × 200; (B) Expression of IDH1 in MG63,× 200; (C) Expression of IDH1 in U2OS,× 400; (D) Expression of IDH1 in MG63,× 400.

Article Snippet: They were incubated in anti-IDH1 mAb (protein technology group, USA) or anti-p53 mAb (Santa Cruz, CA, USA) for 1 h at room temperature, followed by secondary antibody and peroxidase-conjugated strepavidin-biotin complex (Santa Cruz, CA, USA) at 37°C for 30 min. Immunoreactivity was visualized with diaminobenzidine (DAB) (Zymed, South San Francisco, CA).

Techniques: Immunocytochemistry, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The expression and significance of IDH1 and p53 in osteosarcoma

doi: 10.1186/1756-9966-29-43

Figure Lengend Snippet: The mRNA levels of IDH1 in MG63 and U2OS (on fold) . The mRNA levels of IDH1 is higher in U2OS than in MG63( P < 0.01).

Article Snippet: They were incubated in anti-IDH1 mAb (protein technology group, USA) or anti-p53 mAb (Santa Cruz, CA, USA) for 1 h at room temperature, followed by secondary antibody and peroxidase-conjugated strepavidin-biotin complex (Santa Cruz, CA, USA) at 37°C for 30 min. Immunoreactivity was visualized with diaminobenzidine (DAB) (Zymed, South San Francisco, CA).

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The expression and significance of IDH1 and p53 in osteosarcoma

doi: 10.1186/1756-9966-29-43

Figure Lengend Snippet: The protein expression levels of IDH1 and p53 in U2OS and MG63 . MG63 demonstrates no detectable p53 while U2OS cells demonstrates a high expressed p53. IDH1 expresses higher in U2OS than in MG63 at the protein level( P < 0.01).

Article Snippet: They were incubated in anti-IDH1 mAb (protein technology group, USA) or anti-p53 mAb (Santa Cruz, CA, USA) for 1 h at room temperature, followed by secondary antibody and peroxidase-conjugated strepavidin-biotin complex (Santa Cruz, CA, USA) at 37°C for 30 min. Immunoreactivity was visualized with diaminobenzidine (DAB) (Zymed, South San Francisco, CA).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The expression and significance of IDH1 and p53 in osteosarcoma

doi: 10.1186/1756-9966-29-43

Figure Lengend Snippet: The expression of IDH1 and p53 in low histological Rosen grade biopsy . IDH1 expresses at high level accompanying with high expressed p53 in Low histological Rosen grade biopsy.(A) Expression of IDH1 in low histological Rosen grade biopsy, × 100;(B) Expression of p53 in low histological Rosen grade biopsy, × 100; (C) Expression of IDH1 in low histological Rosen grade biopsy, × 200;(D) Expression of p53 in low histological Rosen grade biopsy, × 200.

Article Snippet: They were incubated in anti-IDH1 mAb (protein technology group, USA) or anti-p53 mAb (Santa Cruz, CA, USA) for 1 h at room temperature, followed by secondary antibody and peroxidase-conjugated strepavidin-biotin complex (Santa Cruz, CA, USA) at 37°C for 30 min. Immunoreactivity was visualized with diaminobenzidine (DAB) (Zymed, South San Francisco, CA).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The expression and significance of IDH1 and p53 in osteosarcoma

doi: 10.1186/1756-9966-29-43

Figure Lengend Snippet: The expression of IDH1 and p53 in high histological Rosen grade biopsy . IDH1 expresses at low level accompanying with low expressed p53 in high histological Rosen grade biopsy.(A) Expression of IDH1 in high histological Rosen grade biopsy, × 100;(B) Expression of p53 in high histological Rosen grade biopsy, × 100; (C) Expression of IDH1 in high histological Rosen grade biopsy, × 200;(D) Expression of p53 in high histological Rosen grade biopsy, × 200.

Article Snippet: They were incubated in anti-IDH1 mAb (protein technology group, USA) or anti-p53 mAb (Santa Cruz, CA, USA) for 1 h at room temperature, followed by secondary antibody and peroxidase-conjugated strepavidin-biotin complex (Santa Cruz, CA, USA) at 37°C for 30 min. Immunoreactivity was visualized with diaminobenzidine (DAB) (Zymed, South San Francisco, CA).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The expression and significance of IDH1 and p53 in osteosarcoma

doi: 10.1186/1756-9966-29-43

Figure Lengend Snippet: The expression of IDH1 and P53 in osteosarcoma biopsies

Article Snippet: They were incubated in anti-IDH1 mAb (protein technology group, USA) or anti-p53 mAb (Santa Cruz, CA, USA) for 1 h at room temperature, followed by secondary antibody and peroxidase-conjugated strepavidin-biotin complex (Santa Cruz, CA, USA) at 37°C for 30 min. Immunoreactivity was visualized with diaminobenzidine (DAB) (Zymed, South San Francisco, CA).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The expression and significance of IDH1 and p53 in osteosarcoma

doi: 10.1186/1756-9966-29-43

Figure Lengend Snippet: The immunostaining percentages of IDH1 and p53 in low Rosen grade vs. high Rosen grade . IDH1 expresses higher in Low histological Rosen grade compare with high histological Rosen grade at the level of the immunostaining percentages ( P < 0.01), so does p53 ( P < 0.01).

Article Snippet: They were incubated in anti-IDH1 mAb (protein technology group, USA) or anti-p53 mAb (Santa Cruz, CA, USA) for 1 h at room temperature, followed by secondary antibody and peroxidase-conjugated strepavidin-biotin complex (Santa Cruz, CA, USA) at 37°C for 30 min. Immunoreactivity was visualized with diaminobenzidine (DAB) (Zymed, South San Francisco, CA).

Techniques: Immunostaining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The expression and significance of IDH1 and p53 in osteosarcoma

doi: 10.1186/1756-9966-29-43

Figure Lengend Snippet: The immunostaining scores of IDH1 and p53 in low Rosen grade vs. high Rosen grade . IDH1 expresses higher in Low histological Rosen grade compare with high histological Rosen grade at the level of the immunostaining scores ( P < 0.05), so does p53 ( P < 0.01).

Article Snippet: They were incubated in anti-IDH1 mAb (protein technology group, USA) or anti-p53 mAb (Santa Cruz, CA, USA) for 1 h at room temperature, followed by secondary antibody and peroxidase-conjugated strepavidin-biotin complex (Santa Cruz, CA, USA) at 37°C for 30 min. Immunoreactivity was visualized with diaminobenzidine (DAB) (Zymed, South San Francisco, CA).

Techniques: Immunostaining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The expression and significance of IDH1 and p53 in osteosarcoma

doi: 10.1186/1756-9966-29-43

Figure Lengend Snippet: The relationship between IDH1 and survival . The IDH1 high expression group represents the osteosarcoma patients with >50% IDH1 positive staining. Patients with ≤ 50% IDH1 positive staining are recorded as low-expression group. The survival time in the χ -axis was given as years. There is no significant correlation between IDH1 expression and overall survival ( P = 0.342).

Article Snippet: They were incubated in anti-IDH1 mAb (protein technology group, USA) or anti-p53 mAb (Santa Cruz, CA, USA) for 1 h at room temperature, followed by secondary antibody and peroxidase-conjugated strepavidin-biotin complex (Santa Cruz, CA, USA) at 37°C for 30 min. Immunoreactivity was visualized with diaminobenzidine (DAB) (Zymed, South San Francisco, CA).

Techniques: Expressing, Staining

Journal: eLife

Article Title: Experiments from unfinished Registered Reports in the Reproducibility Project: Cancer Biology

doi: 10.7554/eLife.73430

Figure Lengend Snippet: ( A ) HEK293T cells transfected with wild-type (WT) or the indicated mutant of IDH1 or IDH2, or empty vector, were analyzed for intracellular metabolites 72 hr after transfection by gas chromatography–mass spectrometry (GC–MS). Quantitation of 2HG single intensity relative to glutamate was determined using total ion chromatograms (TIC) for each biological repeat (n = 6). Box and whisker plot with median represented as the line through the box and whiskers representing values within 1.5 interquartile range (IQR) of the first and third quartiles. Means as black dot and bold error bars represent 95% CI. Data estimated from the representative experiment reported in Figure 1B of is displayed as a single point (red circle) for comparison. Statistical analysis was performed on log 10 -transformed data generated during this replication attempt. One-way analysis of variance (ANOVA) of IDH1 and IDH2 groups: F (3, 20) = 56.8, p = 5.73 × 10 −10 . Planned contrasts between IDH1 WT and IDH1 R132H: t (20) = 8.33, uncorrected p = 6.23 × 10 −8 , Bonferroni corrected p = 1.25 × 10 −7 , Cohen’s d = 4.81, 95% CI [2.42, 7.15]; IDH2 WT and IDH2 R172K: t (20) = 9.35, uncorrected p = 9.71 × 10 −9 , Bonferroni corrected p = 1.94 × 10 −8 , Cohen’s d = 5.40, 95% CI [2.79, 7.96]. ( B ) Western blot analysis for HEK293T cells transfected with WT or the indicated mutant of IDH1 or IDH2, or empty vector. Methylation status from histone extracts of the indicated markers were normalized to H3 and then to vector for each biological repeat (n = 6). Box and whisker plot with median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartiles. For each marker a one-way analysis of variance (ANOVA; or Kruskal–Wallis) was performed on IDH1 and IDH2 groups and the correlation with 2HG levels in A was performed. H3K4me2: H(3) = 0.367, uncorrected p = 0.947, Bonferroni corrected p > 0.99; correlation with 2HG: t (12) = 1.12, uncorrected p = 0.275, Bonferroni corrected p > 0.99. H3K9me2: F (3,20) = 0.793, uncorrected p = 0.512, Bonferroni corrected p > 0.99; correlation with 2HG: t (12) = 1.53, uncorrected p = 0.140, Bonferroni corrected p = 0.699. H3K9me3: F (3,20) = 0.515, uncorrected p = 0.676, Bonferroni corrected p > 0.99; correlation with 2HG: t (12) = 1.35, uncorrected p = 0.191, Bonferroni corrected p = 0.957. H3K36me3: F (3,20) = 0.248, uncorrected p = 0.862, Bonferroni corrected p > 0.99; correlation with 2HG: t (12) = 0.725, uncorrected p = 0.476, Bonferroni corrected p > 0.99. H3K79me2: H(3) = 1.55, uncorrected p = 0.672, Bonferroni corrected p > 0.99; correlation with 2HG: t (12) = 0.752, uncorrected p = 0.460, Bonferroni corrected p > 0.99. Planned contrasts for H3K9me2 between IDH1 WT and IDH1 R132H: t (20) = −0.238, uncorrected p = 0.815, Bonferroni corrected p > 0.99; IDH2 WT and IDH2 R172K: t (20) = 1.31, uncorrected p = 0.203, Bonferroni corrected p > 0.99. ( C ) 3T3-L1 cells were transduced to express the indicated proteins and analyzed and presented in the same way as A . Number of independent biological repeats (n = 5). Data estimated from the representative experiment reported in Figure 2A of is displayed as a single point (red circle) for comparison. Statistical analysis was performed on log 10 -transformed data generated during this replication attempt. Student’s t -test of IDH2 WT and IDH2 R172K: t (8) = 14.3, p = 5.49 × 10 −7 , Cohen’s d = 9.06, 95% CI [4.53, 13.57]. Additional details can be found at https://osf.io/vfsbo/ .

Article Snippet: Antibody , rabbit anti-IDH1 , Proteintech , cat# 12332-1-AP; RRID: AB_2123159 , 1:1500 dilution.

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Gas Chromatography, Mass Spectrometry, Gas Chromatography-Mass Spectrometry, Quantitation Assay, Whisker Assay, Transformation Assay, Generated, Western Blot, Methylation, Marker

Journal: eLife

Article Title: Experiments from unfinished Registered Reports in the Reproducibility Project: Cancer Biology

doi: 10.7554/eLife.73430

Figure Lengend Snippet: ( A ) This is the same experiment as in . Representative Western blots (experiment performed independently six times) of histone extractions from HEK293T cells transfected with the indicated plasmids. Membranes were probed with the indicated antibodies (total H3 served as loading control). ( B ) This is the same experiment as in . Representative total ion chromatograms (TIC) from samples harvested 72 hr after transfection for the indicated transfected HEK293T cells. The detection of 2HG and glutamate are shown and based on spectra of derivatized commercial standards. ( C ) This is the same experiment as in . Representative Western blots (experiment performed independently six times) of histone extractions from 3T3-L1 cells transduced to express the indicated proteins. Membranes were probed with IDH1 and IDH2. ( D ) This is the same experiment as in . Representative TIC from samples harvested 7 days after transduction to express the indicated proteins in 3T-L1 cells. The detection of 2HG and glutamate are shown and based on spectra of derivatized commercial standards. Additional details can be found at https://osf.io/vfsbo/ .

Article Snippet: Antibody , rabbit anti-IDH1 , Proteintech , cat# 12332-1-AP; RRID: AB_2123159 , 1:1500 dilution.

Techniques: Western Blot, Transfection, Transduction

Journal: eLife

Article Title: Experiments from unfinished Registered Reports in the Reproducibility Project: Cancer Biology

doi: 10.7554/eLife.73430

Figure Lengend Snippet: Meta-analysis of each effect reported in . Effect size and 95 % confidence intervals are presented for , this replication study (Reproducibility Project: Cancer Biology, RP:CB), and a random-effects meta-analysis of those two effects. The effect size r is a standardized measure of the correlation (strength and direction) of the association between two variables and Cohen’s d is a standardized difference between the two indicated measurements where a larger value indicates a difference in expression between the two conditions. ( A ) Meta-analysis p values: H3K4me3 expression ( p = 0.235), H3K9me2 expression ( p = 0.117), H3K9me3 expression ( p = 0.135), H3K36me3 expression ( p = 0.133), and H3K79me2 expression ( p = 0.090). ( B ) Meta-analysis p values: H3K9me2 expression between IDH1 WT and IDH1 R132H ( p = 0.398), and H3K9me2 expression between IDH2 WT and IDH2 R172K ( p = 0.202). ( C ) Meta-analysis p values: correlation of H3K4me3 expression and 2HG ( p = 0.122), correlation of H3K9me2 expression and 2HG ( p = 0.092), correlation of H3K9me3 expression and 2HG ( p = 0.081), correlation of H3K36me3 expression and 2HG ( p = 0.181), and correlation of H3K79me2 expression and 2HG ( p = 0.185). Sample sizes used in and RP:CB are reported under the study name. Additional details can be found at https://osf.io/vfsbo/ .

Article Snippet: Antibody , rabbit anti-IDH1 , Proteintech , cat# 12332-1-AP; RRID: AB_2123159 , 1:1500 dilution.

Techniques: Expressing

Journal: eLife

Article Title: Experiments from unfinished Registered Reports in the Reproducibility Project: Cancer Biology

doi: 10.7554/eLife.73430

Figure Lengend Snippet:

Article Snippet: Antibody , rabbit anti-IDH1 , Proteintech , cat# 12332-1-AP; RRID: AB_2123159 , 1:1500 dilution.

Techniques: Software, Infection, Recombinant, Plasmid Preparation