RETRACTED: DNA-PKcs-PIDDosome: A Nuclear Caspase-2-Activating Complex with Role in G2/M Checkpoint Maintenance

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).This article has been retracted at the request of the Authors.Our paper reported the identification of a nuclear protein complex comprising DNA-PKcs, PIDD, and caspase-2 and characterization of its role in G2/M checkpoint maintenance, thereby providing insight into the functional significance of nuclear caspase-2. We recently identified errors affecting several figure panels where original data were processed inappropriately such that the figure panels do not accurately report the original data. We believe that the most responsible course of action is to retract the paper. We sincerely apologize to the scientific community for any inconvenience this might cause

MET Is Required for the Maximal Action of 20-Hydroxyecdysone during <em>Bombyx</em> Metamorphosis

<div><p>Little is known about how the putative juvenile hormone (JH) receptor, the bHLH-PAS transcription factor MET, is involved in 20-hydroxyecdysone (20E; the molting hormone) action. Here we report that two MET proteins found in the silkworm, <em>Bombyx mori</em>, participate in 20E signal transduction. <em>Met</em> is 20E responsive and its expression peaks during molting and pupation, when the 20E titer is high. As found with results from RNAi knockdown of <em>EcR</em>-<em>USP</em> (the ecdysone receptor genes), RNAi knockdown of <em>Met</em> at the early wandering stage disrupts the 20E-triggered transcriptional cascade, preventing tissue remodeling (including autophagy, apoptosis and destruction of larval tissues and generation of adult structures) and causing lethality during the larval-pupal transition. MET physically interacts with EcR-USP. Moreover, MET, EcR-USP and the 20E-response element (EcRE) form a protein-DNA complex, implying that MET might modulate 20E-induced gene transcription by interacting with EcR-USP. In conclusion, the 20E induction of MET is required for the maximal action of 20E during <em>Bombyx</em> metamorphosis.</p> </div

Physical interaction between MET and EcR-USP.

<p>(A) The CytoTrap yeast two-hybrid analyses revealed direct associations among MET1, MET2, EcR and USP. Strong associations between bait and prey proteins led to more yeast colonies. (B) When the <i>HA-EcR</i>, <i>FLAG-USP</i>, and <i>V5-Met1</i> constructs were co-transfected into human HEK 293 cells, 20E treatment for 6 hr at a final concentration of 1 µM had little or no stimulating effects on the physical interactions between MET and EcR-USP. In the immunoprecipation experiments, the bottom Western blot is input. IP, immunoprecipitate; Blot, Western blot. (C) The <i>HA-EcR</i>, <i>FLAG-USP</i>, <i>V5-Met1</i>, and <i>cMyc-Met2</i> constructs were co-transfected into the human HEK 293 cells. After nuclear extracts were bound with biotin-labeled EcRE, the protein-DNA complexes were separated on a 5% native PAGE gel followed by EMSA. Addition of the HA or FLAG antibody resulted in a shift of EcRE. In (C) and (D), the shift was indicated by a black arrow in comparison with a gray arrow. (D) The <i>HA-EcR</i>, <i>FLAG-USP</i>, <i>V5-Met1</i>, and <i>cMyc-Met2</i> constructs were co-transfected into human HEK 293 cells. After nuclear extracts were bound with biotin-labeled EcRE, the protein-DNA complexes were separated 5% native PAGE followed by EMSA. When the V5 or cMyc antibody was added, binding of EcR-USP-EcRE was shifted by MET1 and MET2 in EMSA showing that MET, EcR-USP and EcRE form a protein-DNA complex. (E) <i>Met1</i> RNAi and transfection were simultaneously conducted in <i>Bombyx</i> DZNU-Bm-12 cells for 48 hr, followed by 20E treatment for 6 hr at a final concentration of 1 µM, and measurements of EcRE-driven luciferase activity were done. MET is required for 20E function to induce gene expression via the ecdysone receptor and EcRE. The bars labeled with different lowercase letters are significantly different (P<0.05, ANOVA).</p

Lethal and defective phenotypes caused by <i>Met</i> RNAi in silkworms.

<p>dsRNA (10 µg per larva) was injected into selected larva during initiation of the early wandering stage. <i>egfp</i> dsRNA was used as a control. (A–C) Typical <i>Met1</i> RNAi and <i>Met2</i> RNAi treated silkworms died during the wandering stage (A) or during pupation (B), while some were arrested at the mid-pupal stage (C). The pictures (A–C) show the dying animals after <i>Met</i> RNAi. (D, E) <i>Met</i> RNAi affected adult structure formation. The surviving <i>Met1</i> RNAi and <i>Met2</i> RNAi treated pupae did not fully develop legs and wings during the late pupal stage (D). Many of the surviving <i>Met1</i> RNAi adults failed to shed the pupal cuticle attached to their head or abdomen, exhibiting shortened and distorted legs or unexpanded wings (E).</p

<i>Met</i> RNAi results in lethality during the larval-pupal-adult metamorphosis.

<p>dsRNA (10 µg per larva) was injected into selected larvae during initiation of the early wandering stage. Lethality was scored at the larval, prepupal, and pupal stages to compare the effects of <i>egfp</i>, <i>Met1</i>, <i>Met2</i>, and <i>Met1</i>+<i>2</i> dsRNAs.</p

Two <i>Met</i> genes in the <i>Bombyx</i> genome.

<p>Three biological replicates were used, one of which is represented. In each biological replicate, more than 10 larvae were used (A–C). (A) From day 2 of the 4^th instar to day 2 of the prepupal stage, <i>Met1</i> and <i>Met2</i> mRNA expression in fat body was determined by qPCR. The developmental profiles show expression peaks during molting and pupation. 4L2D, day 2 of the fourth instar, and so on; M, molting; W, the wandering stage; PP, the prepupal stage. (B) <i>Met1</i> and <i>Met2</i> mRNA levels (left panel), and MET1 protein level (right panel) were increased by 20E treatment <i>in vivo</i>. 20E (1 µg per larva) was injected into selected larvae on day 2 of the fifth instar, and fat body was explanted for qPCR analysis and Western blots 6 hr after 20E treatment. Tubulin was used as a loading control. (C) <i>Met1</i> and <i>Met2</i> mRNA levels (left panel), and MET1 protein level (right panel) were decreased by <i>EcR</i> RNAi and <i>USP</i> RNAi <i>in vivo</i>. dsRNA (10 µg per larva) was injected into larvae during the initiation of the early wandering stage, and fat body was explanted for qPCR analysis and Western blots 24 hr after RNAi treatment. Tubulin was used as a loading control. (D) Simultaneous addition of 1 µM 20E and 10 µg/ml cycloheximide (CHX) to <i>Bombyx</i> DZNU-Bm-12 cells for 2 hr revealed that <i>Met1</i> and <i>Met2</i> are 20E primary- and secondary-response genes, respectively.</p