Journal: bioRxiv

Article Title: A novel cis regulatory element regulates human XIST in CTCF-dependent manner

doi: 10.1101/871178

Figure Lengend Snippet: (A) Semi-quantitative RT-PCR for mature and premature XIST using cDNA prepared from HEK293T (female), NT2/D1 (male) and DLD1 (male) cells. 18s rRNA and β-ACTIN serve as controls. (B) qRT-PCR depicting a decrease in the levels of OCT4 and NANOG and increase in PAX6 levels upon RA-mediated differentiation of NT2/D1 cells for 6 days. X-axis represents the differentiation time-point and Y-axis represents the fold change normalized to 18s rRNA. Each point on the graph represents values from 5 independent experiments and error bar represents ±S.E.M. (C) Immunoblotting showing a decrease in OCT4, SOX2 and NANOG levels upon RA-mediated differentiation of NT2/D1 cells for 6 days. γ-TUBULIN serves as an equal loading control. (D) qRT-PCR depicting an increase in the levels of XIST upon RA-mediated differentiation of NT2/D1 cells for 6 days. X-axis represents the differentiation time-point and Y-axis represents the fold change normalized to 18s rRNA. Each point on the graph represents values from 3 independent experiments and error bar represents ±S.E.M. (E) X chromosome paint for metaphase spread and interphase nuclei from undifferentiated (0 day) and 5day RA-treated NT2/D1cells. RNA FISH for mature XIST for undifferentiated (0 day) and differentiating (5day, RA) NT2/D1 cells. Arrowheads indicate the FISH signal. (F) Quantification for the RNA FISH signals in 0 day and 5day RA treated NT2/D1 cells. N=3, 200 nuclei were counted for each replicate and statistical significance was ascertained by Student’s T-test.

Article Snippet: CTCF antibody (sc-21298) purchased from Santacruz Biotechnologies (Dallas, Texas, USA) was used for immunoblotting, Normal rabbit IgG (12–370) and Normal mouse IgG (12–371) for ChIP were purchased from Millipore (Billerica, Massachusetts, USA), β-ACTIN (VMA00048) and γ-TUBULIN primary antibodies, mouse-HRP and rabbit-HRP secondary antibodies were purchased from BioRad Laboratories (Hercules, California, USA).

Techniques: Quantitative RT-PCR, Western Blot, Control

Journal: bioRxiv

Article Title: A novel cis regulatory element regulates human XIST in CTCF-dependent manner

doi: 10.1101/871178

Figure Lengend Snippet: (A) Immunoblotting to determine the knockdown efficiencies of OCT4, SOX2 and NANOG in NT2/D1 cells. β-ACTIN serves as an equal loading control. (B) qRT-PCR for PAX6 upon siRNA mediated knockdown of OCT4, SOX2, NANOG in NT2/D1 cells. X-axis represents siRNA transfected and Y-axis represents the fold change normalized to 18s rRNA. Each bar represents values from 3 independent experiments. Error bar represents the ±S.E.M. Asterisks represent the significance over vector control as per Student’s T-test (**P value < 0.01, *P value < 0.05, ns=non-significant). (C) qRT-PCR for mature XIST upon siRNA mediated knockdown of OCT4, SOX2, NANOG in NT2/D1 cells. X-axis represents siRNA transfected and Y-axis represents the fold change normalized to 18s rRNA. Each bar represents values from 3 independent experiments. Error bar represents the ±S.E.M. Asterisks represent the significance over vector control as per Student’s T-test (**P value < 0.01, *P value < 0.05, ns=non-significant). (D) Experimental scheme to overexpress OCT4, SOX2, NANOG in NT2/D1 cells differentiated for 4 days. (E) Immunoblotting for FLAG to confirm the over-expression of OCT4, SOX2, NANOG in NT2/D1 cells differentiated for 4 days. γ-TUBULIN serves as an equal loading control. (F) qRT-PCR showing a significant reduction in mature XIST upon over-expression of pluripotency factors in NT2/D1 cells differentiated for 4 days. X-axis represents transfected DNA and Y-axis represents the fold change normalized to 18s rRNA. Each point on the graph represents values from 3 independent experiments and error bar represents ±S.E.M. Asterisks represent the significance over vector control as per Student’s T-test (****P value < 0.0001, P value < 0.001, P value < 0.05). (G) Immunoblotting for FLAG to confirm the over-expression of OCT4, SOX2, NANOG in HEK293T cells. γ-TUBULIN serves as an equal loading control. (H) qRT-PCR showing a significant reduction in mature and premature XIST upon over-expression of pluripotency factors in HEK293T cells. X-axis represents the mature or premature XIST and Y-axis represents the fold change normalized to 18s rRNA. Each point on the graph represents values from 5 independent experiments and error bar represents ±S.E.M. Asterisks represent the significance over vector control as per Student’s T-test (****P value < 0.0001, ***P value < 0.001, *P value < 0.05). (I) ChIP-qPCR showing occupancies of OCT4, SOX2, NANOG on the exon 1 (+4.5 Kb) site upon their over-expression in HEK293T cells. X-axis represents the transfected DNA and Y-axis represents the enrichment calculated as percent input. Each point on the graph represents values from 2 independent experiments and error bar represents ±S.E.M. Asterisks represent the significance over vector control as per Student’s T-test (*P value < 0.05).

Article Snippet: CTCF antibody (sc-21298) purchased from Santacruz Biotechnologies (Dallas, Texas, USA) was used for immunoblotting, Normal rabbit IgG (12–370) and Normal mouse IgG (12–371) for ChIP were purchased from Millipore (Billerica, Massachusetts, USA), β-ACTIN (VMA00048) and γ-TUBULIN primary antibodies, mouse-HRP and rabbit-HRP secondary antibodies were purchased from BioRad Laboratories (Hercules, California, USA).

Techniques: Western Blot, Knockdown, Control, Quantitative RT-PCR, Transfection, Plasmid Preparation, Over Expression, ChIP-qPCR