Journal: Epigenetics

Article Title: PTPRT epigenetic silencing defines lung cancer with STAT3 activation and can direct STAT3 targeted therapies.

doi: 10.1080/15592294.2019.1676597

Figure Lengend Snippet: Figure 2. (a) Methylation of PTPRT promoter region in NSCLC tumours. NSCLC tumours samples from Stage 1A/1B patients were processed to extract DNA followed by bisulphite conversion to determine PTPRT promoter methylation by Real Time PCR. Out of the 12 tumour samples, six tumours had detectable promoter region methylation (Patient #s 2, 4, 6, 8, 9 and 11) whereas the remaining 6 tumours demonstrated very low methylation of the PTPRT promoter region (Patient #s 1, 3, 5, 7, 10 and 12), which compared well with the methylated beta values. (b&c) Association of PTPRT promoter methylation to STAT3 target gene expression in lung tumours. Lung tumours (Patient #s 1, 2 and 3) were assessed for STAT3 target gene expression by RT-PCR. Cyclin D1 (Figure 2(b)) and Bcl-XL (Figure 2(c)) mRNA expression was found to associate with PTPRT methylation status. (d&e) Association of PTPRT promoter methylation and Bcl-XL and Cyclin D1 mRNA expression. Scatter plots of DNA promoter methylation of PTPRT and Bcl-XL and Cyclin D1 mRNA expression in TCGA adenocarcinoma showed an increase in Bcl-XL (Figure 2(d)) and Cyclin D1 (Figure 2(e)) mRNA expression with increase in PTPRT methylation.

Article Snippet: To determine pSTAT3Tyr705, STAT3 and STAT3 targets such as Cyclin D1 and Bcl-XL protein expressions, 40 μg/lane were separated by 10% SDSPAGE and probed with rabbit anti-pSTAT3Tyr705 or mouse anti-STAT3 monoclonal antibodies (Cell Signalling Technology, Boston, MA), rabbit anti- human Cyclin D1 polyclonal or mouse anti-human Bcl-XL monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and developed using the Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Massachusetts, MA, USA).

Techniques: Methylation, Real-time Polymerase Chain Reaction, Targeted Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Epigenetics

Article Title: PTPRT epigenetic silencing defines lung cancer with STAT3 activation and can direct STAT3 targeted therapies.

doi: 10.1080/15592294.2019.1676597

Figure Lengend Snippet: Figure 4. (a) Knockdown of PTPRT mRNA expression in H520 cells by PTPRT siRNA. PTPRT knockdown by siRNA in H520 cells which demonstrated PTPRT mRNA expression in our study resulted in decreased expression of PTPRT mRNA. H520 cells were treated with PTPRT siRNA in presence of OPTI-MEM and after 48h, cells were replaced with complete media and at the end of 72h, RNA was extracted. RT-PCR data showed that H520 cells transfected with PTPRT siRNA had significantly lower 48% (p < 0.05) PTPRT mRNA expression compared to cells treated with vehicle. PRPRT mRNA expression in H520 cells transfected with scrambled siRNA did not show any significant decrease when compared to cells treated with vehicle. Results are from three triplicate wells in one experiment. (b & c) Increased expression of Cyclin D1 and Bcl-XL upon PTPRT knockdown in H520 cells. H520 cells which demonstrated unmethylation of the PTPRT promoter region were transfected with PTPRT siRNA. After 72h, the cells showed increased mRNA expression of the STAT3 target genes such as Cyclin D1 and Bcl-XL compared to the cells treated with vehicle or scrambled siRNA as determined by RT-PCR. Results are from three triplicate wells in one experiment. (d & e) Increased pSTAT3Tyr705 along with STAT3 target gene expression upon PTPRT knockdown by siRNA. H520 cells transfected with PTPRT siRNA caused increased pSTAT3Tyr705 along with Cyclin D1 and Bcl-XL protein expression compared to the cells treated with vehicle or scrambled siRNA at the end of 72h and 96h as determined by western analyses. The results are from triplicate wells from one experiment.

Article Snippet: To determine pSTAT3Tyr705, STAT3 and STAT3 targets such as Cyclin D1 and Bcl-XL protein expressions, 40 μg/lane were separated by 10% SDSPAGE and probed with rabbit anti-pSTAT3Tyr705 or mouse anti-STAT3 monoclonal antibodies (Cell Signalling Technology, Boston, MA), rabbit anti- human Cyclin D1 polyclonal or mouse anti-human Bcl-XL monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and developed using the Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Massachusetts, MA, USA).

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Targeted Gene Expression, Western Blot

Journal: Epigenetics

Article Title: PTPRT epigenetic silencing defines lung cancer with STAT3 activation and can direct STAT3 targeted therapies.

doi: 10.1080/15592294.2019.1676597

Figure Lengend Snippet: Figure 5. (a, b, c & d) Increased PTPRT promoter methylation is associated with increased sensitivity to STAT3 inhibition. Lung cancer cell lines which are methylated (H23 and H1703) or unmethylated (H520) in the PTPRT promoter region were treated with increasing concentrations of the three STAT3 inhibitors SID 864,669, SID 4,248,543 and UPCDC 10,205 in complete media. At the end of 72h, MTT assay was done to determine % kill which increased with increasing concentrations of SID 864,669 (Figure 5(a)), SID 4,248,543 (Figure 5(b)) and UPCDC 10,205 (Figure 5(c)) and was more pronounced in H23 and H1703 which are methylated in the PTPRT promoter region in conjunction with high pSTAT3Tyr705 expression. The inactive compound UPCDC 10,381 did not have an effect in any cell lines tested (Figure 5(d)). Results are from four separate experiments. (e, f & g) Increased PTPRT promoter methylation was accompanied with increased sensitivity to STAT3 inhibition as evident from the reduction of STAT3 target gene expression. H23, H1703 and H520 cells were treated with varying concentrations of SID 864,669 and at the end of 24h, cells were harvested for RNA extraction and RT-PCR was performed to determine the mRNA expression of the STAT3 target genes. The STAT3 inhibitor SID 864,669 caused a reduction in Cyclin D1 and Bcl-XL mRNA expression in H23 (Figure 5(e), * indicates p < 0.05) and H1703 (Figure 5(f), * indicates p < 0.05) cells which showed PTPRT promoter methylation with concomitant expression of pSTAT3Tyr705. In H520 cells, there was no effect of SID 864,669 on the mRNA expression of STAT3 target genes (Figure 5(g)).

Article Snippet: To determine pSTAT3Tyr705, STAT3 and STAT3 targets such as Cyclin D1 and Bcl-XL protein expressions, 40 μg/lane were separated by 10% SDSPAGE and probed with rabbit anti-pSTAT3Tyr705 or mouse anti-STAT3 monoclonal antibodies (Cell Signalling Technology, Boston, MA), rabbit anti- human Cyclin D1 polyclonal or mouse anti-human Bcl-XL monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and developed using the Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Massachusetts, MA, USA).

Techniques: Methylation, Inhibition, MTT Assay, Expressing, Targeted Gene Expression, RNA Extraction, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular and cellular endocrinology

Article Title: DUAL INHIBITION OF ERK1/2 AND AKT PATHWAYS IS REQUIRED TO SUPPRESS THE GROWTH AND SURVIVAL OF ENDOMETRIOTIC CELLS AND LESIONS

doi: 10.1016/j.mce.2018.12.011

Figure Lengend Snippet: Details of the antibody used.

Article Snippet: Anti-human rabbit polyclonal Cyclin E2 , Cell Signaling , 4132 , 1:1000.

Techniques: Concentration Assay

Journal: Molecular and cellular endocrinology

Article Title: DUAL INHIBITION OF ERK1/2 AND AKT PATHWAYS IS REQUIRED TO SUPPRESS THE GROWTH AND SURVIVAL OF ENDOMETRIOTIC CELLS AND LESIONS

doi: 10.1016/j.mce.2018.12.011

Figure Lengend Snippet: Details of the antibody used.

Article Snippet: Anti-human rabbit polyclonal Cyclin D2 , Cell Signaling , 2924 , 1:1000.

Techniques: Concentration Assay