Journal: Scientific Reports

Article Title: Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method

doi: 10.1038/s41598-020-69471-4

Figure Lengend Snippet: The clinicopathological characteristics of the patients.

Article Snippet: Rabbit anti-HER2 antibody (clone EP3) was purchased from ZSGB-BIO (Beijing, China).

Techniques:

Journal: Scientific Reports

Article Title: Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method

doi: 10.1038/s41598-020-69471-4

Figure Lengend Snippet: Correlation of HER2 levels measured with 4B5 and EP3 antibodies. A total of 332 breast cancer FFPE tissues in 2 × 5 µm slices were provided by a local hospital. MCF-7 and BT474 cell lysates were used as internal controls. FFPE tissue lysates (about 0.5 µg/unit) and cell lysates (about 0.3 µg/unit) were applied onto the QDB plates at 2 µl/unit in triplicate for the QDB measurements with clone EP3 and 4B5 respectively. A set of serially diluted HER2 recombinant protein were included in each plate to develop plate-specific standard curve. All results were averaged from three independent experiments, with each sample in triplicate. The correlation of HER2 levels measured with 4B5 and EP3 was analyzed with Pearson’s correlation coefficient analysis using Graphpad software, r = 0.963, p < 0.0001.

Article Snippet: Rabbit anti-HER2 antibody (clone EP3) was purchased from ZSGB-BIO (Beijing, China).

Techniques: Recombinant, Software

Journal: Scientific Reports

Article Title: Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method

doi: 10.1038/s41598-020-69471-4

Figure Lengend Snippet: Distribution of all 332 samples. HER2 levels in all 332 breast cancer FFPE sample lysates were measured with the QDB method using EP3 antibody. The lysates were diluted to about 0.25 µg/µl, and then 2 µl lysate was used for each sample. ( a ) the distribution of HER2 levels among 332 samples. HER2 levels ranged from 0 (chemiluminescence readings less than two fold over the background) to 31.310 nmol/g. ( b ) All samples were grouped by their IHC scores provided by local hospital. The distributions of HER2 levels in each IHC group were recorded as following: 0, 0–0.205 nmol/g, mean = 0.045 ± 0.006 nmol/g, n = 77; 1 +, 0 –0.410 nmol/g, mean = 0.049 ± 0.008 nmol/g, n = 65; 2+ , 0–7.250 nmol/g, mean = 0.537 ± 0.122 nmol/g, n = 108; and 3+ , 0.329–31.310 nmol/g, mean = 7.120 ± 0.773 nmol/g, n = 82. The intra- and inter-CV were 8.98% and 9.89% respectively.

Article Snippet: Rabbit anti-HER2 antibody (clone EP3) was purchased from ZSGB-BIO (Beijing, China).

Techniques:

Journal: Scientific Reports

Article Title: Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method

doi: 10.1038/s41598-020-69471-4

Figure Lengend Snippet: Evaluation of QDB results with those of IHC and FISH analyses using ROC analysis. Samples were separated into negative (HER2−) and positive (HER2+) groups based on the recommendations from ASCO/CAP. In ( a ), samples were grouped based on their IHC scores, with 142 samples in the negative group (IHC 0 and 1+), and 82 samples in the positive group (IHC 3+). Absolute HER2 levels from these samples were used for ROC analysis with Graphpad Prism7.0 software. The ROC curve of QDB results was obtained with area under the Curve (AUC) at 0.9998 ± 0.0002; 95% CI 0.9994–1; p < 0.0001. In ( b ), samples were grouped based on both IHC and FISH results, with 214 samples as negative (HER2−) group and 105 samples as positive (HER2+) group. 6 equivocal cases and 7 cases with missing FISH results were excluded in the analysis. Absolute HER2 levels from these samples were used for ROC analysis with Graphpad Prism7.0 software. The area under the curve (AUC) was at 0.9942 ± 0.0031, with 95% CI at 0.9881–1; p < 0.0001. ( c ) The samples were grouped by IHC scores, and the suggested cutoff values from ROC analyses in ( a ) at 0.267 nmol/g (solid line), and in ( b ) at 0.261 nmol/g (dashed line) were shown to demonstrate the effectiveness of these cutoff values to separate samples from HER2+ to HER2− groups. HER2 levels were plotted in log scale to better demonstrate the distribution of QDB results among these samples. For those samples with undetectable HER2 level, a value of 0.001 nmol/g was arbitrarily entered to avoid omitting any sample in the log scale graph.

Article Snippet: Rabbit anti-HER2 antibody (clone EP3) was purchased from ZSGB-BIO (Beijing, China).

Techniques: Software

Journal: Scientific Reports

Article Title: Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method

doi: 10.1038/s41598-020-69471-4

Figure Lengend Snippet: Assessing HER2 levels by histologic grade. The FFPE specimens (300 out of 332) were grouped according to their Nottingham histologic scores into Grades I, II, and III. The HER2 levels of each grade were used for column statistics analysis with Graphpad Prism7.0 software. The mean ± SD of the HER2 levels were 0.791 ± 0.555 nmol/g for Grade I (n = 36), 1.554 ± 0.330 nmol/g for Grade II (n = 145), and 3.271 ± 0.535 nmol/g for Grade III (n = 119). The statistical difference was assessed with two-tailed Student’s t-test, with p < 0.05 between Grades I and III, and p = 0.005 between Grades II and III. There was no statistical difference between Grade I and Grade II samples.

Article Snippet: Rabbit anti-HER2 antibody (clone EP3) was purchased from ZSGB-BIO (Beijing, China).

Techniques: Software, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Prostaglandin E2 alters Wnt-dependent migration and proliferation in neuroectodermal stem cells: implications for autism spectrum disorders

doi: 10.1186/1478-811X-12-19

Figure Lengend Snippet: Expression of EP receptors’ mRNA and protein in NE-4C cells. (A) Real-time PCR was used to determine the RQ value for EP1, EP2, EP3α, EP3β, EP3γ and EP4 receptors, which was found to be 2, 16, 1, 2, 46 and 46 respectively. The error bars represent + SEM. (B) Western blot analysis of the EP1, EP2, EP3 and EP4 receptors expression (65, 68, 62 and 53 kDa, respectively). β-Actin was used to indicate equal loading. (C) Immunocytochemistry revealed the subcellular localization of EP1-4 receptors with specific organelles visualized through the use of anti-PDI endoplasmic reticulum marker, anti-Lamin A + C nuclear envelope marker, β -Actin cell membrane marker, and anti-58 K Golgi marker. The scale bar represents 10 μm.

Article Snippet: Primary antibodies used for EP expression levels include rabbit polyclonal anti-EP1, −EP2, −EP3, −EP4 (1:200; Santa Cruz Biotechnology).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunocytochemistry, Marker, Membrane