RIPK1 and BECN1 gene expression.

<p>(A) Gene expression of RIPK1 and (B) BECN1 in ALL cells treated with 1μM Dex, and 10μM Etop individually or in combination for 24h in the presence or absence of CM. The data is representative of at least three independent experiments. Error bars represent SEM. P-value of less than or equal to 0.05 is indicated by *.</p

Microenvironment and chemotherapy effects on ALL cell fate.

<p>(A) Cells were treated with CM (48 hours), 1μM Dex (36 hours) and 10μM Etop (24 hours) in varying combinations. Percentage of cell death was obtained using FACS analysis of PI-stained CEM-C1-15 and CEM-C7-14 cells treated as above. SubG1 phase is shown. (B) Caspase-8 activity was measured in ALL CEM-C7-14 and CEM-C1-15 cells treated as indicated. CEM-C1-15 cells are represented by dark grey bars, CEM-C7-14 by light grey bars. (C) RIPK1 high molecular weight forms were analysed in cells treated with CM and BIRC3 inhibitor AT406 (10μM for 48 hours). Western blot analysis was carried out as described above. The data is representative of at least three independent experiments. Error bars represent SEM. P-value of less than or equal to 0.05 is indicated by *.</p

Control of BIRC3 gene expression by GR in ALL.

<p>(A) mRNA expression of BIRC3 in ALL cells treated with Dex, Etop and CM. (B) ChIP analysis was carried out in CEM-C1-15 and (C) CEM-C7-14 cells treated with 1μM Dex for 24hrs. Occupancy on one of the GREs by total GR, and GR phosphorylated on S211 and S226 was analysed. The data is representative of at least three independent experiments. Error bars represent SEM. P-value of less than or equal to 0.05 is indicated by *.</p

Differential recruitment of GR and its phosphoisoforms on the RIPK1 and BECN1 promoters.

<p>Chromatin immuniprecipitation (ChIP) analysis was carried out in CEM-C1-15 and CEM-C7-14 cells treated with 1μM Dex for 24hrs. We identified potential GR binding sites on the (A) RIPK1 and (B) BECN1 promoters and analysed total GR, and GR phosphorylated on S211 and S226 on a subset of sites. The data is representative of at least three independent experiments. Error bars represent SEM. P-value of less than or equal to 0.05 is indicated by *.</p

Clusters, profiles, and GO groupings of CEM-C7-14 cells.

<p>CEM-C7-14 cells were grown in the absence and presence of CM or standard RPMI media for 48h and treated with Dex (1μM) and Etop (10μM) individually or in combination for 24h. Cells were treated with vehicle (1), CM (2), Dex (3), Etop (4), Dex and Etop (5) or Dex, Etop and CM (6). Identified genes were assigned to one of eight distinct clusters using k-means clustering algorithms. On the left, the data for each cluster are represented as a profile of the z-transformed, log₂ values for the mean of each experimental group/condition. The most significantly overrepresented GO terms are shown for the genes within each cluster.</p

RIPK1, BECN1 and Caspase-3 protein levels in ALL cells.

<p>(A) CEM-C7-14 and CEM-C1-15 cells were grown in the absence and presence of CM or standard RPMI media for 48h and treated with Dex (1μM) and Etop (10μM) individually or in combination for 24h. Cells were lysed and analysed by SDS PAGE followed by western blot. Blots were probed with antibodies specific for RIPK1, BECN1 and cleaved caspase 3. Actin was used as a loading control. Western blots (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178606#pone.0178606.g002" target="_blank">Fig 2A</a> and data not shown) were densitometrically scanned, normalised to actin and presented as bar charts. (B) RIPK1 protein expression; (C) BECN1 protein expression. The data is representative of at least three independent experiments. Error bars represent SEM. P-value of less than or equal to 0.05 is indicated by *. C is control; CM is conditioned media; D is Dex; DCM is Dex and CM; E is Etop; ECM is Etop and CM; DE is Dex and Etop; DECM is Dex, Etop and CM.</p

Dex, Etop and the microenvironment affect GR phosphorylation in ALL.

<p>(A) CEM-C1-15 and CEM-C7-14 cells were cultured in CM or standard RPMI media for 48h and treated with Dex (1μM) and Etop (10μM) individually or in combination for 24h. Treatments were as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178606#pone.0178606.g002" target="_blank">Fig 2</a>. Cells were lysed and protein extracts were subjected to western blot analysis. Total and phosphorylated GR was detected using specific antibodies against these proteins. Actin was used as loading control. Western blots (Fig 4A and data not shown) were densitometrically scanned, normalised to actin and presented as bar charts. For phosphorylation experiments, phosphorylation blot data was scanned and normalised to the actin-normalised total GR. (B) Total GR protein expression. (C) S226-phosphorylated GR protein expression. (D) S211-phosphorylated GR protein expression. The data is representative of at least three independent experiments. Error bars represent SEM. P-value of less than or equal to 0.05 is indicated by *.</p

Further clustering according to the percent of different cellular processes assigned by EASE analysis.

<p>Further clustering according to the percent of different cellular processes assigned by EASE analysis.</p