Fig 1: Tctp, Foxo, or 14-3-3ε affects endoreplication in larval salivary glands.a–e Salivary glands are labeled with DAPI and BrdU (DNA replication marker). a–a'' Control salivary gland cells show high levels of BrdU in the nuclei. b–b'' Compared to control, Tctp RNAi leads to a lower level of BrdU in the nuclei. c–c'' Overexpressing Foxo results in a small salivary gland. BrdU is strongly reduced in the nuclei. d–d'' 14-3-3ζ knockdown does not affect the gland size or the BrdU levels in the nuclei. e–e'' 14-3-3ε RNAi causes a strong reduction in gland size and nuclear BrdU staining. f–j Salivary glands are immunostained with anti-CycE and DAPI. f–f'' The control gland shows high levels of CycE in the nuclei. g–g'' Salivary glands with Tctp RNAi show CycE mislocalized to the cytoplasm. h–h'' Overexpression of Foxo results in severe loss of nuclear CycE. i–i'' The reduction of 14-3-3ζ does not affect the nuclear localization of CycE. j–j'' Knockdown of 14-3-3ε leads to the near absence of CycE in the nuclei. The white box at the bottom right of each panel is a magnification of the small white box in the same panel. Scale bars, 150 μm (a', b', d', f', g', and i'), 75 µm (c', e', h', and j).
Fig 2: Endogenous Foxo is enriched in the cytoplasm by Tctp knockdown.a–b Immunocytochemical effects of Tctp knockdown on Foxo levels. S2 cells were stained with DAPI and the anti-Foxo antibody. a–a'' Endogenous Foxo is mostly found in the cytoplasm. b–b'' Tctp knockdown increases Foxo levels in the cytoplasm. For Tctp knockdown, S2 cells were treated with Tctp dsRNA twice a day for 3 days. Scale bar, 10 µm. c Effects of Tctp RNAi on the level of cytoplasmic Foxo in salivary glands. Foxo is undetectable in two control strains (AB1/+ and AB1 > GFP) but is increased by Tctp RNAi (AB1 > Tctp i). Asterisk indicates a non-specific band. d Effects of Tctp knockdown on the Foxo level in S2 cells. The cytoplasmic extracts from S2 cells treated with Tctp dsRNA show an enhanced level of Foxo compared with untreated cells. e Effects of human TCTP knockdown on hFOXO1. Endogenous hTCTP was knocked down by siRNA treatment in HeLa cells for 72 h. Immunoblotting shows that hTCTP siRNA leads to an increase in the hFOXO1 level. ß-Tubulin shows a loading control.
Fig 3: Knockdown of Tctp increases endogenous Foxo levels in the eye disc.a–b Eye discs from third instar larvae were stained with DAPI and antibodies for Arm and Foxo. a–a''' ey > RFP control. The eye field with photoreceptor clusters is indicated by an arrow, and the morphogenic furrow is marked by an arrowhead. b–b''' ey > Tctp i. Tctp RNAi reduces the size of the eye field as indicated by fewer ommatidial arrays (b'') compared with the control (a''). Foxo staining is increased in Tctp RNAi discs. Scale bar, 10 µm. c Quantification of the relative eye field based on the column number of photoreceptor clusters. (n = 8 discs for ey > + and 7 discs for ey > Tctp RNAi).
Fig 4: Tctp RNAi, 14-3-3e RNAi, or Foxo overexpression reduces salivary gland size by affecting cell size.a–e Salivary glands of late 3rd instar larvae were stained with DAPI and antibodies for Arm. a–a''' AB1 > RFP control. The gland is normal in size and the pattern of Arm staining (a'') and RFP (a''') expression. b–b''' AB1 > RFP, Tctp i. Tctp knockdown shows a thin salivary gland. c–c''' AB1 > RFP, foxoORF. Foxo overexpression induces a striking reduction of tissue and nucleus size in the salivary gland. d–d''' AB1 > RFP,14-3-3? i. Knockdown of 14-3-3? has negligible effect on salivary gland development. e–e''' AB1 > RFP, 14-3-3e i. 14-3-3e RNAi leads to a strong reduction in gland size. f Quantification of cell numbers based on the counting of the DAPI-stained nuclei. AB1 > RFP, n = 18; AB1 > RFP, Tctp i, n = 7; AB1 > RFP, foxoORF, n = 4; AB1 > RFP, 14-3-3? i, n = 10; AB1 > RFP, 14-3-3e i, n = 16 glands. g The relative cell size from cell boundaries stained with anti-Arm antibody. Scale bar, 100 µm in a'-e'; Error bars in (g) indicate standard deviation (SD); AB1 > RFP, n = 17 cells from three glands; AB1 > RFP, Tctp i, n = 12 from four glands; AB1 > RFP, foxoORF, n = 23 from four glands; AB1 > RFP, 14-3-3? i, n = 6 from two glands; AB1 > RFP, 14-3-3e i, n = 12 from four glands; ***<0.001; **<0.01; *<0.05; n.s. (not significant); N = 2.
Fig 5: Tctp negatively regulates ectopic Foxo.a–d Eye/antenna discs from 3rd instar larvae were stained with DAPI, anti-Arm, and anti-Foxo antibodies as indicated. a–a''' ey > RFP control shows normal arrays of ommatidial clusters labeled by Arm staining posterior to the morphogenetic furrow (MF, white arrowheads) (a''). Foxo is ubiquitously expressed at a low level and is slightly elevated along the furrow. b–b''' ey > foxoORF. Foxo overexpression causes a considerable reduction in the eye disc size and the number of photoreceptor clusters (b''). The disc shows a high level of Foxo staining due to foxoORF overexpression (b'''). c–c''' ey > Tctp, foxoORF. The inhibition of photoreceptor differentiation by foxoORF overexpression is partially suppressed by Tctp co-expression, and Foxo levels are reduced by Tctp overexpression in 70% of eye discs examined (n = 27). d–d''' ey > Tctp shows no significant differences to control. Anterior is to the right. Scale bar, 20 μm.
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