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tissue microarray small cell lung cancer tmas  (TissueArray.com LLC)
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TissueArray.com LLC tissue microarray small cell lung cancer tmas
(A) Scatter plot shows an upregulation of the PP2A-A subunit in the tumor samples (p=0.0144). A Mann-Whitney U test was used for comparison between the normal and <t>SCLC</t> samples. (B) IHC for PP2A was conducted on TMA tissue sections and images were captured at 4x or 20x using a 3D-Histech PANNORAMIC SCAN whole slide scanner (3D-Histech, Budapest, Hungary). PP2A subunit A positively immunostained the cytoplasm and nucleus of normal lung and tumor tissue but was highly upregulated in tumor tissue. <t>TMAs</t> were scored in normal (n=24) and tumor (n=79) cores on a scale from 0 (no staining/no protein expression) to 3+ (strong staining/high protein expression). (C) Summary bar graph of the average PP2A subunit staining. IHC staining intensity of normal and tumor cores. There was a statistically significant difference between normal and tumor tissue (***, p<0.001). Student’s t test was used for comparison between the normal and SCLC samples. (D) In order to compare the expression of PP2A subunits A and C, cell lysates from seven SCLC cell lines and HBEC 3KT (non-malignant cell line) were subjected to western blotting (n=3 biological replicates). (E) PP2A activity was determined using a serine/threonine phosphatase activity assay (Millipore) after 24 h exposure to cantharidin (10 µM) and LB100 (5 µM) (n=3 biological replicates). ***, p<0.001, results were analyzed by ANOVA with Tukey post-test. (F) The inset showed reduction of PP2A subunit Aα in H524 cells as well as inhibition of cell proliferation due to PP2A subunit Aα knockdown (n=3 biological replicates). p<0.05, Student’s t test was used for comparison between the groups. LB100 alone or in combination with carboplatin inhibited proliferation and colony formation in SCLC cells. The Cell Counting Kit-8 assay detected cell H524 and H69 cell viability. (n=3 biological replicates). (G, H) Cells were treated with LB100, carboplatin and etoposide, as a single treatment or in combination, at constant ratio. The combination index (CI) was calculated using Chou-Talalay method to find synergism between LB100 with carboplatin and etoposide (CompuSyn software: www.combosyn.com). **, p<0.01, ANOVA with Tukey post-test was used for comparison between LB100, LB100/carboplatin and LB100/etoposide. Colony formation assays were used to count the ability of H524 (I) and H69 (J) cells to form colonies. Drug concentrations are listed for two assays with H524 and H69 respectively: LB100 (2.5 µM; 20 µM), carboplatin (4 µM; 20 µM), etoposide (3 µM; 30 µM), LB100/carboplatin (2.5&4 µM; 20&20 µM) and LB100/etoposide (2.5&3 µM; 20&30 µM). Representative images of colonies at 4x are shown under the graph (n=2). *p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Results were analyzed by ANOVA with Tukey post-test.
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(A) Scatter plot shows an upregulation of the PP2A-A subunit in the tumor samples (p=0.0144). A Mann-Whitney U test was used for comparison between the normal and SCLC samples. (B) IHC for PP2A was conducted on TMA tissue sections and images were captured at 4x or 20x using a 3D-Histech PANNORAMIC SCAN whole slide scanner (3D-Histech, Budapest, Hungary). PP2A subunit A positively immunostained the cytoplasm and nucleus of normal lung and tumor tissue but was highly upregulated in tumor tissue. TMAs were scored in normal (n=24) and tumor (n=79) cores on a scale from 0 (no staining/no protein expression) to 3+ (strong staining/high protein expression). (C) Summary bar graph of the average PP2A subunit staining. IHC staining intensity of normal and tumor cores. There was a statistically significant difference between normal and tumor tissue (***, p<0.001). Student’s t test was used for comparison between the normal and SCLC samples. (D) In order to compare the expression of PP2A subunits A and C, cell lysates from seven SCLC cell lines and HBEC 3KT (non-malignant cell line) were subjected to western blotting (n=3 biological replicates). (E) PP2A activity was determined using a serine/threonine phosphatase activity assay (Millipore) after 24 h exposure to cantharidin (10 µM) and LB100 (5 µM) (n=3 biological replicates). ***, p<0.001, results were analyzed by ANOVA with Tukey post-test. (F) The inset showed reduction of PP2A subunit Aα in H524 cells as well as inhibition of cell proliferation due to PP2A subunit Aα knockdown (n=3 biological replicates). p<0.05, Student’s t test was used for comparison between the groups. LB100 alone or in combination with carboplatin inhibited proliferation and colony formation in SCLC cells. The Cell Counting Kit-8 assay detected cell H524 and H69 cell viability. (n=3 biological replicates). (G, H) Cells were treated with LB100, carboplatin and etoposide, as a single treatment or in combination, at constant ratio. The combination index (CI) was calculated using Chou-Talalay method to find synergism between LB100 with carboplatin and etoposide (CompuSyn software: www.combosyn.com). **, p<0.01, ANOVA with Tukey post-test was used for comparison between LB100, LB100/carboplatin and LB100/etoposide. Colony formation assays were used to count the ability of H524 (I) and H69 (J) cells to form colonies. Drug concentrations are listed for two assays with H524 and H69 respectively: LB100 (2.5 µM; 20 µM), carboplatin (4 µM; 20 µM), etoposide (3 µM; 30 µM), LB100/carboplatin (2.5&4 µM; 20&20 µM) and LB100/etoposide (2.5&3 µM; 20&30 µM). Representative images of colonies at 4x are shown under the graph (n=2). *p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Results were analyzed by ANOVA with Tukey post-test.

Journal: Molecular cancer therapeutics

Article Title: Protein phosphatase 2A as a therapeutic target in small cell lung cancer

doi: 10.1158/1535-7163.MCT-21-0013

Figure Lengend Snippet: (A) Scatter plot shows an upregulation of the PP2A-A subunit in the tumor samples (p=0.0144). A Mann-Whitney U test was used for comparison between the normal and SCLC samples. (B) IHC for PP2A was conducted on TMA tissue sections and images were captured at 4x or 20x using a 3D-Histech PANNORAMIC SCAN whole slide scanner (3D-Histech, Budapest, Hungary). PP2A subunit A positively immunostained the cytoplasm and nucleus of normal lung and tumor tissue but was highly upregulated in tumor tissue. TMAs were scored in normal (n=24) and tumor (n=79) cores on a scale from 0 (no staining/no protein expression) to 3+ (strong staining/high protein expression). (C) Summary bar graph of the average PP2A subunit staining. IHC staining intensity of normal and tumor cores. There was a statistically significant difference between normal and tumor tissue (***, p<0.001). Student’s t test was used for comparison between the normal and SCLC samples. (D) In order to compare the expression of PP2A subunits A and C, cell lysates from seven SCLC cell lines and HBEC 3KT (non-malignant cell line) were subjected to western blotting (n=3 biological replicates). (E) PP2A activity was determined using a serine/threonine phosphatase activity assay (Millipore) after 24 h exposure to cantharidin (10 µM) and LB100 (5 µM) (n=3 biological replicates). ***, p<0.001, results were analyzed by ANOVA with Tukey post-test. (F) The inset showed reduction of PP2A subunit Aα in H524 cells as well as inhibition of cell proliferation due to PP2A subunit Aα knockdown (n=3 biological replicates). p<0.05, Student’s t test was used for comparison between the groups. LB100 alone or in combination with carboplatin inhibited proliferation and colony formation in SCLC cells. The Cell Counting Kit-8 assay detected cell H524 and H69 cell viability. (n=3 biological replicates). (G, H) Cells were treated with LB100, carboplatin and etoposide, as a single treatment or in combination, at constant ratio. The combination index (CI) was calculated using Chou-Talalay method to find synergism between LB100 with carboplatin and etoposide (CompuSyn software: www.combosyn.com). **, p<0.01, ANOVA with Tukey post-test was used for comparison between LB100, LB100/carboplatin and LB100/etoposide. Colony formation assays were used to count the ability of H524 (I) and H69 (J) cells to form colonies. Drug concentrations are listed for two assays with H524 and H69 respectively: LB100 (2.5 µM; 20 µM), carboplatin (4 µM; 20 µM), etoposide (3 µM; 30 µM), LB100/carboplatin (2.5&4 µM; 20&20 µM) and LB100/etoposide (2.5&3 µM; 20&30 µM). Representative images of colonies at 4x are shown under the graph (n=2). *p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Results were analyzed by ANOVA with Tukey post-test.

Article Snippet: Tissue Microarray Small cell lung cancer TMAs were from US Biomax Inc. (Rockville, MD; LC818).

Techniques: MANN-WHITNEY, Comparison, Staining, Expressing, Immunohistochemistry, Western Blot, Activity Assay, Phosphatase Assay, Inhibition, Knockdown, Cell Counting, Software

(A) Significant changes were observed for signal transduction and metabolic pathways. (B) MicroArray analysis showed that overnight treatment with 20 µM treatment with LB100 inhibited utilization of carbon substrate sources. Table includes 10 carbon sources affected by LB100 (n=3). (C) LB100 significantly inhibited two carbon substrates utilization by H69 cells. P < 0.001 (***) for control (untreated cells) vs. LB100. Results were analyzed by ANOVA with Tukey post-test. (D) Amplex Red Glucose/Oxidase assay kit was used to measure glucose level in cell culture media. Glucose level was significantly higher in cell culture medium from cells treated with LB100 (20 µM). Glucose concentration detected in initial medium and counted as 100%. Subtracting final medium level of glucose from initial glucose medium concentration yielded % glucose in the medium with cells. Level of glucose dropped in control with cells and in LB100 treated groups (p < 0.0001 (****). Results were analyzed by ANOVA with Tukey post-test, (n=3 biological replicates). Effect of LB100 on MET phosphorylation. (E) H524 and H69 cells were treated overnight with LB100 (H524 – 5 µM and H69 – 20 µM) following by stimulation with 100 ng/ml HGF in 10 min. Cells were collected and lysed for WB analysis with pMET and total MET antibody. Pan-actin was used as loading control (n=3 biological replicates). (F) H524 cell lysates (control, LB100, carboplatin and combination (LB100/carboplatin) were analyzed by western blots to check phosphorylation status of MET at Ser985 and Tyr1234/1235. Actin was used as a loading control (n=3 biological replicates).

Journal: Molecular cancer therapeutics

Article Title: Protein phosphatase 2A as a therapeutic target in small cell lung cancer

doi: 10.1158/1535-7163.MCT-21-0013

Figure Lengend Snippet: (A) Significant changes were observed for signal transduction and metabolic pathways. (B) MicroArray analysis showed that overnight treatment with 20 µM treatment with LB100 inhibited utilization of carbon substrate sources. Table includes 10 carbon sources affected by LB100 (n=3). (C) LB100 significantly inhibited two carbon substrates utilization by H69 cells. P < 0.001 (***) for control (untreated cells) vs. LB100. Results were analyzed by ANOVA with Tukey post-test. (D) Amplex Red Glucose/Oxidase assay kit was used to measure glucose level in cell culture media. Glucose level was significantly higher in cell culture medium from cells treated with LB100 (20 µM). Glucose concentration detected in initial medium and counted as 100%. Subtracting final medium level of glucose from initial glucose medium concentration yielded % glucose in the medium with cells. Level of glucose dropped in control with cells and in LB100 treated groups (p < 0.0001 (****). Results were analyzed by ANOVA with Tukey post-test, (n=3 biological replicates). Effect of LB100 on MET phosphorylation. (E) H524 and H69 cells were treated overnight with LB100 (H524 – 5 µM and H69 – 20 µM) following by stimulation with 100 ng/ml HGF in 10 min. Cells were collected and lysed for WB analysis with pMET and total MET antibody. Pan-actin was used as loading control (n=3 biological replicates). (F) H524 cell lysates (control, LB100, carboplatin and combination (LB100/carboplatin) were analyzed by western blots to check phosphorylation status of MET at Ser985 and Tyr1234/1235. Actin was used as a loading control (n=3 biological replicates).

Article Snippet: Tissue Microarray Small cell lung cancer TMAs were from US Biomax Inc. (Rockville, MD; LC818).

Techniques: Transduction, Microarray, Control, Glucose Oxidase Assay, Cell Culture, Concentration Assay, Western Blot