DEV025999 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1-S2 -

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• Supplemental Figure S1 -

  Fig. S1. miR-7Δ¹ GSC clones marked by the absence of GFP exhibit normal division kinetics as compared with heterozygous GSCs. Clones of GSC were generated by heat shock during larval/pupal stages and flies were dissected after 8 days. Adducin (Add), red; DAPI, blue; GFP, green.

• Supplemental Figure S2 -

  Fig. S2. Cyclin B (CycB) expression in GSCs from control hsFLP;;FRT82B/FRT82BGFP flies day 8 after heat shock. DAPI, blue; CycB, red; GFP, green. Yellow arrowheads indicate CycB-positive GSCs; white arrowhead indicates a CycB-negative GSC. (A,A′) A GSC with strong positive CycB staining (3.1× background). (B,B′) A GSC with moderate positive CycB (2.3×) and a GSC negative for CycB staining (0.9×). Three separate regions were used to determine the ‘speckled’ pattern of background staining. GSCs counted positive were 1.2× above average background intensity. White box in B′ shows an example of background staining. As an additional form of analysis, CycB levels in the GSCs were compared with positive CycB staining in follicle cells (Jordan et al., 2006).

• Supplemental Figure S3 -

  Fig. S3. CycE expression in control GSCs (hsFLP;; FRT82B/FRT82BGFP) day 8 after heat shock. Cyclin E (CycE) expression in control GSCs (hsFLP;; FRT82B/FRT82BGFP) at day 8 after heat shock. The germaria were stained with DAPI (blue), antibodies against CycE (red) and GFP (green). The yellow arrowhead indicates a GSC with strong CycE staining that is scored as positive for CycE (3.4× over background); the white arrowhead indicates a GSC with a low level of CycE (1.9× over background) that is scored as negative for CycE expression. Since low levels of CycE accumulate in all the nuclei in region 1, we chose three separate nuclei to determine the average background intensity for this antibody.

• Supplemental Figure S4 -

  Fig. S4. Expression of dap-5gm. dap-5gm is revealed by anti-Myc antibodies in (A) control and (B) mutant (hsFLP;dap5gm/+;FRT82B InR^ex52.1/FRT82BGFP) GSCs 9 days after heat shock. Germaria were stained with antibodies against Myc (red) and GFP (green). GSCs are outlined by white dashed lines. The lower GSCs are considered positive for the dap-5gm Myc-tag signal (4.6× and 2.1×, respectively; white arrows), whereas the upper GSCs are negative for Myc signal. The Myc-tag expression frequency is the same in InR-deficient GSCs as in neighboring heterozygous control GSCs.

• Supplemental Figure S5 -

  Fig. S5. Normalized luciferase activity with expression of miRNAs. miR-1 was the control. A combination of miR-7, miR-278 and miR-309 repressed the dap 3′UTR more than miR-278 alone. Mean±s.e. of at least three repeats. Student's t-test; *P<0.05.