Immunoblot examining Pvr, phospho-histone H3, and total histone H3 after two days with no treatment or two days of Pvr or GFP dsRNA treatment. Samples of equal numbers of cells were loaded.
Summary of dsRNA-mediated knockdown efficiencies assessed by quantitative real time PCR (qRT-PCR) or immunoblot.
Plotted (y-axis) is the level of Pvf2 transcript remaining in Kc cells treated with dsRNA targeting Akt relative to a dsRNA targeting GFP. For normalization, Ribosomal protein L32 was used as a reference gene.
Live/dead cell counting performed after silencing of 22 Pvr Suppressors or insulin stimulation in combination with Pvr, and compared to Pvr and GFP (control) knockdown.
Immunoblot examining Pvr after treatment of Kc and Kcp35 cells with 0.01 ug/ml 20HE for three days.
Plotted (y-axis) is the level of rpr or E93 transcript remaining in Kc cells treated with dsRNA targeting Pvr or EcR relative to a dsRNA targeting GFP. For normalization, Ribosomal protein L32 was used as a reference gene. Two non-overlapping qPCR primers for each gene were used. No significant changes were observed.
Live hemocyte counts of embryos and larvae at the indicated times after egg laying (AEL), grown at 25C. UAS-Stinger; Pxn-GAL4 was crossed to w1118 (control), or UAS-EcRA dn, respectively. Note that around the time of hatching (grey bar), hemocyte numbers have dropped to about 60% of embryonic counts. Hemocyte-specific expression of UAS-EcRA dn does not significantly protect hemocytes from the decline, as indicated for 22h AEL.
Immunoblot confirming knockdown of Pvr and EcR (top panels) after two days dsRNA treatment in Kc cells used for phosphoproteomic analysis by mass spectrometry.
Amplicons of primary Pvr modifier screen with resulting ZDiff > = 2 or Pvr Enhancers, Suppressors, and Upstream Regulators are indicated.
Verification screen amplicons, Z scores of all replicates with resulting ZDiff values. Homologs and Scores predicted using DIOPT—DRSC Integrative Ortholog Prediction Tool (www.flyrnai.org/cgi-bin/DRSC_orthologs.pl)
Final scores ZDiffFinal resulting from equally averaging all amplicons of a gene in the primary and secondary screen.
Phosphosites identified in Kc cells, a condition of unaltered Pvr activity (‘high Pvr’), in combination with EcR knockdown (‘low EcR’) or insulin stimulation (‘high InR’). Duplicate samples were examined.
Phosphosites identified in Pvr RNAi cells, in combination with EcR knockdown (‘low EcR’) or insulin stimulation (‘high InR’). Duplicate samples were examined.
Phosphosites identified in direct comparison of Kc cells treated with control or Pvr dsRNAs, in combination with EcR knockdown (‘low EcR’) or insulin stimulation (‘high InR’).
Phosphosites predicted to be targeted by both Pvr or InR, based on their common directionality of change under conditions of ‘high Pvr, low InR’ and ‘low Pvr, high InR’.
Phosphosites predicted to be targeted by either Pvr or InR specifically, based on their directionality of change under ‘high Pvr, low InR’ and ‘low Pvr, high InR’ conditions.