Cooley, (School of Medicine, University, CT, USA). old females (see Materials and methods); DNA blue (DAPI), F-actin red (phalloidin), size bar is 20m.(TIF) pone.0147631.s001.tif (5.2M) GUID:?9CEB61FD-4573-4B42-9941-30DF2299BB05 S2 Fig: Zfrp8 regulates RpS2, but does not affect expression of Venus or RpS18. (A-B) driven expression of Venus (green) reflects the expression pattern in control ovaries. (C-D) In KD ovaries the Venus expression pattern was altered reflecting the phenotype, but the levels of expression were not reduced. (E-H) driven expression of GFP-RpS18 (green) was not reduced in KD, but changed concurrently to the phenotype (G-H). (I-L) Levels of mCherry-RpS2 (KD ovaries (compare I-J and K-L DNA blue (DAPI), size bar is 20m.(TIF) pone.0147631.s002.tif (3.0M) GUID:?C1849A3D-2D2B-49F6-B01E-D0D3763AD2F2 S3 Fig: Effect of on protein expression levels in ovaries. Examples of GFP-trap protein expression in control and ovaries. (A-B) Zn72D (green) was seen in the nuclei of germ line cells (arrows) and follicle cells (small nuclei). (C-D) Levels and distribution of Zn72D were unaffected in Zfrp8 KD ovaries. (E-F) At early stages of oogenesis Me31B (green) is highly enriched in oocytes (arrowheads), and present in lower levels in all ovarian cells. In egg chambers after stage 6C7 3-Hydroxyglutaric acid Me31B is also strongly increased in the nurse cells and oocytes (not-shown). (G-H) KD egg chambers do not develop beyond stage 4 and showed defects in oocyte specification (G-H, and ). However, in KD germaria (G, arrow), Me31B was expressed at similar levels as in control (E). (I-J) CycB (green) is present in GSCs and cystoblasts (arrows) and is also observed in most follicle cells. (K-L) The numbers of GSCs and cystoblasts expressing CycB and the level of the protein was similar in control and KD germaria (O, S, arrows) and control (M, Q), but the levels of both proteins are decreased in KD egg chambers (O, S, arrowheads). DNA blue (DAPI), size bar 20m.(TIF) pone.0147631.s003.tif (6.0M) GUID:?5E0CB9BA-84F9-4762-98D5-7D35EB7C9531 S4 Fig: Zfrp8 affects cytoplasmic localization of select transcripts. (A-B) Levels and localization 3-Hydroxyglutaric acid of transcripts (FISH, red) are low in control ovaries and are significantly increased in the cytoplasm of ovaries (C-D arrows show GSCs). In ovaries (driven by nos-GAL4), levels of RNA was increased in both nuclei and cytoplasm of germ line cells (E-F, arrow). Increase in RNA was also observed in follicle cells, suggesting that Zfrp8 may also have non-cell autonomous effect on regulation . (G-R) FISH with (G-J), (K-N) and (O-R) probes. Levels of RNA are somewhat elevated and showed significant nuclear accumulation (G-J, arrows) in ovaries. transcript levels and localization were not changed in KD ovaries (K-N, arrows). transcripts showed increased nuclear accumulation in KD ovaries (O-R, arrows). To visualize cellular compartments ovaries were counterstained with anti-RanGAP antibodies, green (A, C, E, O and Q, cytoplasm and nuclear envelope), anti-Baz antibodies, green, (G, I, K and 3-Hydroxyglutaric acid M, cytoplasm and apical-lateral membrane of follicle cells) and DAPI, blue (DNA).(TIF) pone.0147631.s004.tif (8.9M) GUID:?18C6928F-9EB3-401D-9AB7-F1226343D5ED S5 Fig: Quantification of mRNA fluorescence* in the nuclei and cytoplasm of stage Rabbit polyclonal to Src.This gene is highly similar to the v-src gene of Rous sarcoma virus.This proto-oncogene may play a role in the regulation of embryonic development and cell growth.The protein encoded by this gene is a tyrosine-protein kinase whose activity can be inhibited by phosphorylation by c-SRC kinase.Mutations in this gene could be involved in the malignant progression of colon cancer.Two transcript variants encoding the same protein have been found for this gene. 4C5 nurse cells. (A) RNA is increased in both nuclei and cytoplasm of and ovaries compared to wild type controls. The ratio 3-Hydroxyglutaric acid of nuclear to cytoplasmic levels (n/c) is increased in KD. (B) 3-Hydroxyglutaric acid and (D) mRNAs also show increased nuclear accumulation and elevated n/c ratio in ovaries, while the levels and nuclear accumulation of mRNA (C) and mRNA (E) remain unchanged. Y axis shows mRNA fluorescence* (fluorescence intensity, see below), error bars represent standard deviation. n/c shows average ratio between nuclear and cytoplasmic mRNA fluorescence, p values were calculated using Student t test and n/c ratios from individual cells. *To allow for accurate measurements of mRNA fluorescence for each probe, crosses, ovary dissection, processing (FISH), and imaging were done in parallel. Images were captured using a Leica TSC SP5 laser scanning confocal microscopes (objective 63 oil) with the same microscope settings, scanner and laser.