Fig. 2

Apontic is a downstream target of STAT. a-b Optical sections of testes from males of the specified genotypes in a PTTapt ^CC01186 genetic background, stained with antibodies directed against Zfh-1 (magenta) and GFP (white, to report Apt) and DAPI to label nuclei (blue). Insets display Apt-GFP expression alone. Arrowheads indicate CySCs. For all optical sections, a blue line outlines the hub perimeter, the first tier of Zfh-1+ or Tj+ cells around the hub were considered CySCs, and the scale bar = 20 μm. a Apt reporter expression in CySCs is wild type in the PTTapt/+ control, and is lower when stat is reduced in CySCs as in (b) c587-Gal4; PTTapt; UAS-statRNAi ^TRiP.JF01265 (compare arrowheads). c Apt-GFP expression is significantly reduced in CySCs nuclei when stat is lowered in these cells, measured by pixel intensity relative to the no driver controls or PTTapt alone, normalized to DAPI (see Methods). Statistical analysis was performed between experimental and the no Gal4 driver controls, unless indicated by bar. P-values for graphs (c, f, i, l) are from two-tailed t-tests where *p<0.05; **p<0.01; ***p<0.005; ****p<0.0001. For graphs, “n” represents total testes analyzed; number of cells measured is in parentheses. d-e Single section images of testes, stained with antibodies specific for Apt (red) and NCad (green, hub) and GFP (green, (d)), counterstained with DAPI (blue). Expression of a constitutively active JAK mutant in the CySCs and early cyst cells via Tj-Gal4 heightens nuclear Apt protein levels (e), relative to the GFP control (d). Insets show Apt expression. f Quantification of Apt, as in (c): Apt protein levels are significantly increased when JAK/STAT is activated in the soma. g-h Optical sections of testes stained with antibodies directed against Apt (magenta) and Tj (white) and counterstained with DAPI (blue). Insets display Apt alone. g Testis from a control male (UAS-updRNAi/+, no Gal4 driver, incubated at 30 °C) shows wild-type levels of Apt.  h Apt expression is decreased in CySCs when upd is reduced in the hub of adult males (upd-Gal4; updRNAi; tsGal80-RNAi-ON, 30 °C, restrictive temperature for tsGal80). i Apt is significantly reduced in CySCs when upd is disrupted in the adult hub, relative to controls. With the exception of upd-Gal4; updRNAi; tsGal80-RNAi-OFF flies, all adult males were incubated at 30 °C for 4 days prior to dissection. j-k Optical sections of testes from males bearing upd-Gal4, updRNAi, and tub-tsGal80 (to regulate RNAi expression temporally) transgenes in a PTTapt genetic background, stained with antibodies, as in (a-b). Insets display Apt-GFP alone. j Testis from a control male (RNAi-OFF, 25 °C, permissive for tsGal80 repression) shows wild-type levels of Apt-GFP (white). k Apt (white) expression is decreased in CySCs when upd is reduced in the hub of adult males (RNAi-ON, same genotype as (j), 30 °C, restrictive temperature for tsGal80). l Apt-GFP is significantly reduced in CySCs when upd is disrupted in the adult hub, relative to control males