<i>Ago-1</i> over expression induces apoptosis in <i>Drosophila</i> eye.

<p><b>I.</b> (<b>A)</b> Western blot analysis showing the expression of proteins isolated from adult fly heads of wild type and <i>Ago-1</i> over expressed stocks. (<b>B)</b> Graphical presentation of AGO1 band intensity relative to β-Actin loading control. The average from three independent experiments was taken and plotted. <b>II.</b> (<b>A1-A2, B1-B2, C1-C2)</b> Acridine Orange (AO) staining showing more apoptotic population (marked by arrow head)in <i>Ago-1</i> over expressed 3^rd instar larval eye imaginal disc compared to wild type and down regulated group. (<b>D1 and D2</b>) AO positive cells reduced significantly in <i>Ago-1</i> over expressed eye disc upon introduction of baculovirus p35 trans-gene in fly eye. (<b>E1 and E2</b>) Over expression of transgenic control protein (GFP) in developing eye can’t increase AO positive cells in developing fly eye. (<b>A’, B’, C’)</b> Scanning electron micrograph of adult eyes showing reduced number of normal ommatidia in <i>Ago-1</i> over expressed line. (<b>D’</b>) baculovirus p35 protein can inhibit <i>Ago-1</i> over expression induced apoptotic phenotype in fly eye (<b>E’</b>) GFP over expression can’t induce <i>Ago-1</i> over expression like eye phenotype (<b>III.)</b> <i>Drosophila</i> JNK pathway is triggered by <i>Ago-1</i> over expression. <b>(A)</b> Adult eye phenotype of <i>Ago-1</i> over expressed fly under Scanning Electron Microscope (SEM). <b>(A’)</b> Ommatidia structure of same fly. <b>(III. B.)</b> Adult eye phenotype of <i>tak1</i> deficient mutant, <b>(B’)</b> same in higher magnification. <b>(C)</b> Adult eye phenotype of dominant negative mutation of <i>basket</i>, <b>(C’)</b> magnified view of same showing complete recovery of ommatidia morphology. <b>(D)</b> Adult eye phenotype of <i>bsk</i> deficient with <i>Ago-1</i> over expression showing partial recovery <b>(D’)</b> ommatidia morphology. <b>(E)</b> Mutation in <i>hid</i> gene (<i>hid</i>^<i>H109</i>) prevents <i>Ago-1</i> over expression induced apoptotic phenotype in adult fly eye as well as <b>(E’)</b> Ommatidia structure of the same.</p

<i>Ago-1</i> regulates miR-14 expression to get a better grip on the control of apoptosis.

<p><b>I.</b> micro RNA micro array analysis clearly showed a significant down regulation of both miR-14 variant as a result of <i>Ago-1</i> over expression. <b>II.</b> Up regulation of miR-14 in <i>Ago-1</i> mutant and strong down regulation in <i>Ago-1</i> over expression was noticed in quantitative PCR analysis. <b>III.</b> Ectopic expression of miR-14 successfully rescued the <i>GMR-GAL4</i> driven <i>Ago-1</i> over expressed phenotype of fruit eye. (<b>A, A’</b>) Control eye, (<b>B, B’</b>) <i>Ago-1</i> over expressed eye, (<b>C, C’</b>) <i>Ago-1</i> and miR-14 co-expressed ectopically in fly eye.</p

Level of JNK phosphorylation and consequent effector caspase activation with <i>Ago-1</i> expression.

<p><b>(A-F’)</b> Control group showing normal level of (A, A’) <i>Ago-1</i> expression; (C,C’) JNK phosphorylation and (E-E’) Dcp-1 activation. <b>(G-L’)</b> <i>Ago-1</i> mutant group expressing low level of (G,G’) <i>Ago-1</i>, (I,I’) JNK phosphorylation and (K,K’) active Dcp-1 level. <b>(M-R’)</b> <i>Ago-1</i> over expressed group showing high level of (M,M’) <i>Ago-1</i> expression along with (O,O’) elevated JNK phosphorylation and subsequent activation of (Q,Q’) effector caspase, Dcp-1. <b>(S,T</b> and <b>U)</b> Diagram showing the over expressing area of discs for (S) AGO1, (T) p-JNK and (U) active Dcp-1.</p

<i>Ago-1</i> over expression results in elevated levels of JNK phosphorylation.

<p><b>I.</b> Western blot analysis showing the increased level of JNK phosphorylation in <i>Ago-1</i> over expressed (<i>UAS Ago-1/GMR GAL4</i>) and reduced level in mutant background (<i>Ago-1</i>^<i>72</i><i>/Ago-1</i>^<i>45</i> and <i>GMR GAL4</i>; <i>TRiP</i>.<i>HM04006</i>). Histogram presents mean relative band density (±SEM). <b>II. (A)</b> <i>Ago-1</i> over expression in 3^rd instar larval eye/antennal disc by imaginal discs specific GAL4 [<i>P{w[+mW</i>.<i>hs] = GawB}32B</i>]. <b>(A’)</b> <i>Ago-1</i> over expression in the antennal part of the same disc, <b>(A”)</b> Retinal part, <b>(A”‘)</b> Preproneural part. <b>(B-B”‘)</b> JNK phosphorylated areas of the disc. <b>(C-C”‘)</b> <i>Ago-1</i> over expression co localizes with JNK phosphorylated area of the disc. <b>(D-D”‘)</b> DAPI stained part of the same disc. <b>(E-E”‘)</b> Merged figure. <b>III.</b> <i>Ago-1</i> over expression in 3^rd instar larval eye disc by eye discs specific GAL4 (<i>GMR GAL4</i>). <b>(A-A”)</b> <i>Ago-1</i> over expression in the eye disc, <b>(B-B”)</b> JNK phosphorylated areas of the disc. <b>(C-C”)</b> <i>Ago-1</i> over expression co localizes with JNK phosphorylated area of the disc. <b>(D-D”‘)</b> DAPI staining indicates nuclear region of eye disc cells <b>(E-E”)</b> Merged image.</p

Schematic presentation showing the action mechanism of <i>Ago-1</i> to induce apoptosis via JNK pathway.

<p>Schematic presentation showing the action mechanism of <i>Ago-1</i> to induce apoptosis via JNK pathway.</p

Apoptotic inhibitors and caspases are important for <i>Ago-1</i> regulated apoptotic cell death I.

<p>Real time PCR analysis showing increased level of expression of pro apoptotic and caspase genes in <i>Ago-1</i> over expressed line whereas the same line showing a decreased expression level of <i>th</i> gene transcript. II. (<b>A</b>) Western blot bands showing activation of both initiator (DRONC) and effector (ICE and DCP1) caspases upon <i>Ago-1</i> over expression and reduced level activation was observed in mutant line (<b>B</b>) Graphical presentation of western blot band intensities (±SEM) <b>III. (A, A’)</b> <i>GMR GAL4</i> driven <i>UAS GFP</i> carrying adult eye phenotype of <i>D</i>. <i>melanogaster</i> <b>(C, C’)</b> <i>UAS Ago-1/GMR GAL4</i> adult eye phenotype <b>(C, C’)</b> <i>th</i> encodes an apoptotic inhibitor, DIAP-1. Co expression of <i>th</i> with high level of <i>Ago-1</i> inhibits the apoptotic effect of <i>Ago-1</i>. <b>(D, D’)</b> Hetero allelic mutation of apical <i>Drosophila</i> caspase, <i>Dronc</i> with <i>Ago-1</i> over expression recovers adult eye phenotype towards wild type. <b>(E, E’)</b> <i>Ark (Dark)</i> is an ortholog of mammalian Apaf1; deficient mutation of <i>Ark</i> reduces the effect of over expressed <i>Ago-1</i>. <b>(F, F’)</b> Single copy deficient mutations of effector caspase <i>Drice</i> (<i>ice</i>) cannot rescue <i>Ago-1</i> induced eye phenotype significantly. <b>(G, G’)</b> Deficient mutation of another effector caspase <i>Dcp-1</i> can inhibit <i>Ago-1</i> induced apoptosis; but, fails to recover normal phenotype.</p