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1 Supplementary Information Sung Bae Lee, Sunhong Kim, Jiwoon Lee, Jeehye Park, Gina Lee, Yongsung Kim, Jin-Man Kim, and Jongkyeong Chung This section contains: 1) Additional Information for Main Figures and Supplementary Methods 2) Supplementary Figure Legends (Supplementary Figure S1-S8) 3) Supplementary References 1

2 1) Additional Information for Main Figures and Supplementary Methods Quantitative real time RT-PCR (qpcr) For qpcr, total RA was extracted by the Easy-Blue System (Intron, Korea), and reverse-transcribed using Oligo-dT Reverse Transcription Kit (Promega). Then, PCR was performed using SYBR Premix Ex Taq (Takara) on icycler iq Multicolor Real-Time PCR Detection System (Bio-Rad). rp49 (Drosophila) or β-actin (HEK 293T) was measured for an internal control. Results are presented as fold change compared to the first indicated sample. Primers used for qpcr The primer sequences used for qpcr were as follows: DmATG1-specific primers (5 -CTACATAGTCTGCTGGACACAG-3 for 5 and 5 -GTTCAGCTTGGACAATGTCT CG-3 for 3 ), human ATG1α-specific primers (5' CAAGTTCGAGTTCTCCCGCAAGG- 3 for 5 and 5 -CTTCAGGATTTTGATTTCCTTCCC-3 for 3 ), human ATG1β-specific primers (5'-AGTCTGCAGCCCTGGATGAGATG-3 for 5 and 5 -CACACGGTTGCGGT GCTATGG-3 for 3 ). Determination of lethal phase of DmATG1 1 mutants Homozygous mutant flies of DmATG1 1 showed highly reduced viability after pupal period and about 70% of the mutant flies died before pharate adult stage (Fig 2A). Survived adults exhibited locomotive defects and complete sterility, and most of them died within 1 week after eclosion from pupae (data not shown). Examination of autophagy Semi-thin sections of the fat body of third instar larvae were stained with a toluidine blue-azure II mixture as described previously (Juhasz et al., 2003) to observe autophasic vesicle (autophagosome). The fat body of third instar larvae was processed for 2

3 electron microscopy as described previously (Juhasz et al., 2003). The fat body cells of wild type (w1118) third instar larvae showed a dramatic increase of autophagosomes in starved conditions (supplementary Fig S2C, dark circular azurophilic granules), while those of the larvae in feeding period did not (supplementary Fig S2B). However, the starved DmATG1 1 homozygous larvae almost completely lacked autophagosomes in the fat body cells (supplementary Fig S2D). To further confirm these results, we analyzed ultra-structural morphology of the fat body cells using transmission electron microscopy (TEM). Starved wild type (w1118) flies had many electron dense vesicles containing subcellular organelles such as mitochondria, which is a hallmark of autophagosome, in the fat body cells (supplementary Fig S2G), while fed wild type (w1118, supplementary Fig S2F) and starved DmATG1 1 homozygous flies (supplementary Fig S2H) did not, in accordance with the above toluidine blue-azure II staining results. Conditions for fly culture and sample preparation In Fig 1 and 2A, eggs were collected for 6 hr on agar plates supplemented with grape juice and dried yeast, and incubated at 25 C for 1 day. Fifteen larvae of each genotype were transferred to a new media plate to observe their growth and developmental defects. The black-red color (Fig 1C) shown mainly in the posterior region of larvae were from the color of food (wine-colored grape juice used for determining feeding behavioral defects of larvae). For comparison of the lipid vesicles in the larval fat body (Fig 1D), samples were collected at 4 th day after egg laying (AEL) for dtor mutants and dtor, DmATG1 double mutants and 3 rd day AEL for wild type (w1118). For salivary gland staining and quantification (Fig 1E, F), samples were collected at the 6 th day AEL for dtor mutants and dtor, DmATG1 double mutants and 4 th day AEL for wild type (w1118) to adjust growth stage at the third instar larval stage. Examination of lipid vesicle aggregation in the larval fat body Larval fat body was dissected in phosphate-buffered saline (PBS) and the samples 3

4 were then directly observed using a light microscope (Leica) at the magnification of ⅹ1,600 (Fig 1D). Histochemical analysis of larval salivary glands (Fig 1E, F) Drosophila salivary glands were dissected, fixed in 4% paraformaldehyde in PBS, and washed with PBS-T (PBS + 0.1% Triton X-100). The samples were treated with Hoechst33342 (Sigma, 1:1,000 dilution) and phalloidin-tritc (Sigma, 1:400 dilution) for 20 min at ambient temperature. The stained samples were observed using an LSM510 laser confocal microscope (Carl Zeiss) at the magnification of ⅹ400. Quantification of cell and nucleus sizes of larval salivary glands The cell and nucleus sizes of five different salivary glands of each genotype were measured using Adobe Photoshop program. For dtor and DmATG1 double mutants, we selectively picked the rescued ones (about 30% of larvae, Fig 1C) and observed their salivary glands. For measuring the cell size of dtor mutants and the double mutants, the size of the whole salivary gland was measured and then the value was divided by the number of nuclei within the measured area. The relative cell and nucleus sizes were determined by comparing the averages of the measured pixel numbers acquired from the mutants to those acquired from the control (Fig 1F). Immunoblot analysis of ds6k phosphorylation in Drosophila As previously described (Khurana et al. 2006; Patel and Tamanoi 2006), we examined ds6k activity in Drosophila by immunoblot analysis using an anti-phosphospecific ds6k T398 antibody (Fig 2B, C). Fifteen third instar larvae or pupae of each genotype were collected and ground in 300 µl of lysis solution (1% Triton X-100, 50 mm Tris ph7.4, 500 mm acl, 7.5 mm MgCl 2, 0.2 mm EDTA, 1 mm avo 4, 50 mm β-glycerophosphate, 1 mm DTT, 25% glycerol). Each 50 µg of protein was loaded on SDS-gel and transferred onto nitrocellulose membrane. Before blocking, the membrane 4

5 was boiled for 5 min in PBS. The membrane was then incubated overnight at 4 C with a primary antibody [Rabbit anti-phosphospecific ds6k T398 antibody (Cell Signaling, 1:500 dilution) or mouse anti-tubulin antibody (E7, DSHB, 1:1,000 dilution)]. Examination of mitotic defects in the mitotic tissues of DmATG1 mutants To examine mitosis in the larval mitotic tissues such as imaginal discs and brains of DmATG1 mutants at late third instar larval stage, we labeled those tissues with rabbit anti-phospho-histone H3 antibody (PH3, Upstate Biotechnology, 1:400 dilution) and mouse anti-tubulin antibody (E7, DSHB, 1:50 dilution) to mark mitotic chromosomes and spindles, respectively (supplementary Fig S3A, B). Examination of metaphase chromosomes of the larval neuroblast cells in DmATG1 mutants (supplementary Fig S3C) To check gross chromosomal abnormalities in DmATG1 mutants, we conducted squashing of the third instar larval brain of DmATG1 mutants as described previously (Gatti et al., 1994). To obtain well-spread metaphase figures of chromosomes, we prepared samples using both hypotonic and colchicine treatments (Gatti et al., 1994). The metaphase chromosomes of neuroblast cells were observed using a light microscope (Leica). Characterization of other autophagy-related genes (DmATG6 and UVRAG) and their mutants The mutants for DmATG6 and UVRAG (CG6116) showed defects in autophagy in a starved condition (Scott et al., 2004, supplementary Fig S4). As a mutant allele of DmATG6, DmATG allele (Scott et al., 2004) was used. To reduce the gene dosage of UVRAG, we used Df(2L)ED784, a deficiency allele containing a deleted chromosomal region spanning from 34A4 to 34B6 where dtor and UVRAG genes are located, because the chromosomal locations of UVRAG (34B4) and dtor (34A4) were too close to make a 5

6 double mutant allele by genetic recombination technique. The fly lines for DmATG , KG04163 (UVRAG 1 ), and Df(2L)ED784 were obtained from the Bloomington Stock Center. Sample preparation for salivary gland staining For staining of the salivary gland (supplementary Fig S5C), samples were collected at 6 th day AEL for dtor mutants and the double mutants for dtor and other autophagy-related genes (ATG6 and UVRAG), and 4 th day AEL for wild type (w1118) to adjust growth stage at third instar larval stage. Cell culture and transfection HEK 293T cells were grown in Dulbecco s Modified Eagle s medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen) at 37 C in a humidified atmosphere with 5% CO 2, and were transiently transfected using the standard calcium phosphate protocol or Lipofectamine Plus reagent (Invitrogen). sira experiments sira targeting human ATG1α and ATG1β (sigeome SMART pool, catalog number M for ATG1α, M for ATG1β) and control sira (sicotrol, catalog number D ) were purchased from Dharmacon. One hundred nm sira was transfected by Lipofectamine 2000 (Invitrogen). After 72 hr incubation, the cells were subjected to qpcr and immunoprecipitation. Preparation of cell lysates, immunoprecipitation, and immunoblots Cell stimulation was terminated by washing cells with ice-cold PBS. Cell lysates were prepared in Buffer A and subjected to immunoprecipitation and immunoblots as described previously (Kim et al., 2001). The immunoblot results shown are representative of three independent experiments. 6

7 Determination of autophagy using a GFP-ATG8/LC3 system (Kerscher et al. 2006) Induction of autophagy was determined either by the formation of cytoplasmic aggregation of ATG8/LC3 (supplementary Fig S7A) or by the formation of lipid-conjugated form of LC3 (LC3-II, supplementary Fig S7B, C). Immunocytochemistry HEK 293T cells were grown on poly-l-lysine (Sigma)-coated coverslips and transiently transfected with pegfp-c1 (Clontech) and sira. Anti-phosphospecific S6 S235/236 antibody and Alexa 568-conjugated anti-rabbit antibody (Invitrogen) were used for cell staining according to the manufacturer s instructions. The samples were observed by a laser scanning confocal microscope (Carl Zeiss). Kinase activity-dependent modifications of ATG1 WT ATG1 moves slower than its kinase dead form in a gel (supplementary Fig S6A, compare lane 1 to lane 2 for ATG1α and lane 5 to lane 6 for ATG1β, respectively), suggesting the posttranslational modification of ATG1 in its kinase activity dependent manner. Therefore, we tested whether this slow migrating form resulted from phosphorylation. As a result, we found that the slow migrating form of WT ATG1α and ATG1β was significantly reduced by CIAP treatment (supplementary Fig S6A, compare lane 1 to lane 3 and lane 5 to lane 7, respectively), demonstrating that ATG1 is phosphorylated in its kinase activity-dependent manner. otably, we obtained similar results by increasing the amount of CIAP and the reaction time twice as much (data not shown) and using a different protein phosphatase, λ protein phosphatase (EB), for the reaction (data not shown). Meanwhile, although CIAP treatment increased ATG1-WT gel mobility, CIAP-treated ATG1-WT still migrated slower than ATG1-KT (supplementary Fig S6A), suggesting the possibility of a second modification(s) of ATG1 in addition to 7

8 phosphorylation. However, we could not find any evidence of ubiquitination, glycosylation, and acetylation of ATG1 (data not shown). Thus, further studies are required to clearly elucidate the exact nature of the posttranslational modifications of ATG1. Protein dephosphorylation by calf intestinal alkaline phosphatase (CIAP) ATG1 immune complex was mixed with a reaction mixture containing CIAP reaction buffer and 20 units of CIAP (KOSCHEM, Korea), and then incubated at 37 C for 1 hr. The samples were washed once with PBS and subjected to immunoblot analysis. 8

9 2) Supplementary Figure Legends (Supplementary Figure S1-S8) Supplementary Fig S1 Sequence alignment of ATG1 family members of human (hatg1α), C. elegans (UC-51), Drosophila (DmATG1), and yeast (ATG1). Supplementary Fig S2 Defects in autophagosome formation in DmATG1 1 mutants. (A) A schematic diagram of the domain structure of ATG1 family members of human, Drosophila, C. elegans, and yeast. (B-I) Autophagosomes were visualized by toluidine blue-azure II staining (B-E) and TEM analysis (F-I). Many autophagosomes were present in the cytoplasm of the fat body cells of starved wild type (w1118) larvae (small dark dots in C and electron dense vesicles containing mitochondria in G), compared to fed wild type larvae (B, F). Autophagosomes were completely absent in the cytoplasm of the fat body cells of DmATG1 1 homozygous larvae (D, H). Reduction of ds6k gene dosage did not rescue the autophagy defect of DmATG1 1 homozygous larvae (E, I). marks the nucleus. Supplementary Fig S3 Examination of mitosis in the mitotic tissues of DmATG1 mutants. (A, B) Images of immunostained mitotic tissues of the late third instar larvae of denoted genotypes. (A) Larval brains (top), eye (middle), and wing (bottom) imaginal discs were stained with anti-phospho-histone H3 (PH3) antibody and anti-tubulin antibody. HS and VG represent hemisphere and ventral ganglion regions of larval brain, respectively (top). White arrowhead indicates the morphogenetic furrow of eye imaginal disc (middle). P, A, D, V represent posterior, anterior, dorsal and ventral regions of imaginal disc, respectively (middle and bottom). (B) Magnified images of the immunostained wing imaginal discs of denoted genotypes. (C) Images of metaphase chromosomes of the larval neuroblast cells of denoted genotypes. Supplementary Fig S4 Defects in autophagosome formation in UVRAG and ATG6 mutants. (A) Genomic structure of CG6116, which encodes a sole Drosophila orthologue 9

10 of UVRAG. The P-element insertion site of KG04163 (UVRAG 1 ) is denoted. (B) The transcriptional levels of UVRAG in the third instar larvae were analyzed by RT-PCR. Ribosomal protein 49 (rp49) was used as an internal control. (C) Autophagosomes in the larval fat body cells were visualized by toluidine blue-azure II staining (top) and LysoTracker staining (bottom). LysoTracker staining was performed as described previously (Scott et al., 2004). Supplementary Fig S5 Genetic interaction analysis between dtor and UVRAG or ATG6. (A) Images of the larvae of denoted genotypes at 3 days (top) and 6 days (bottom) after AEL. (B) Quantification of the larvae developed into mid/late third instar stage of each genotype in supplementary Fig S5A. The quantified results were the average values from three independent experiments ± S.D. Fifteen larvae of each genotype were examined in each experiment. (C) Images of the larval salivary glands of denoted genotypes. Hoechst33342 (pseudo-colored green) and phalloidin-tritc (red) were used to visualize nucleus and cell boundary of the larval salivary gland cells, respectively. Supplementary Fig S6 Inhibition of S6K phosphorylation by ATG1β. (A) Examination of auto-phosphorylation of ATG1. HEK 293T cells were transfected with denoted plasmids. The immune complexes containing wild type (WT) or kinase dead form (KI for ATG1α, KT for ATG1β) of ATG1α or ATG1β were incubated with CIAP or buffer alone, and then, subjected to immunoblot. The immune complexes were analyzed by immunoblot analyses using anti-ha antibody (top panel). Anti-HA blot was also completed from the same cell lysates (bottom panel). (B) HEK 293T cells transfected with denoted plasmids were deprived of nutrients for 90 min, and then replenished with DMEM for 30 min. The immune complexes were analyzed by immunoblot analyses using appropriate antibodies (top three panels). Anti-FLAG (ATG1β) blot was completed from the same cell lysates (bottom panel). 10

11 Supplementary Fig S7 Inability of ATG1 overexpression to induce autophagy in MCF-7 and HEK 293T cells. (A-C) Autophagosome formation was examined using GFP-LC3 assay. (A) MCF-7 cells were transfected with indicated plasmids and fixed and stained with anti-ha antibody (red). Autophagosome formation was determined by the formation of cytoplasmic aggregation of GFP-LC3 (green). utrient-starved cells (nutrient deprivation for 90 min) were used as positive controls. (B-C) MCF-7 (B) or HEK 293T (C) cells transfected with indicated plasmids were lysed and subjected to immunoblot analysis using appropriate antibodies. Arrows indicate LC3-I (unmodified form) and LC3-II (lipid-conjugated form embedded in autophagosome). utrient-starved cells (nutrient deprivation for 90 min) were used as positive controls (B). In lane 7 of C, 2 µg/ml rapamycin (Rap) was treated for 6 hr. (D) HEK 293T cells transfected with denoted plasmids were deprived of nutrients for 90 min, and then replenished with DMEM for 30 min. The immune complexes harboring S6K were analyzed by immunoblot analyses using appropriate antibodies (top three panels). Anti-V5 (ATG6) and anti-flag (UVRAG) blots were completed from the same cell lysates (bottom two panels). Supplementary Fig S8 Expression levels of ATG1α (A) and ATG1β (B) transcripts in specific siras-transfected HEK 293T cells were analyzed by qpcr. β-actin was used as an internal control. n=3. Bars indicate mean ± S.D. 11

12 3) Supplementary References Gatti M, Bonaccorsi S, Pimpinelli S (1994) Looking at Drosophila mitotic chromosomes. Methods Cell Biol 44: Juhasz G, Csikos G, Sinka R, Erdelyi M, Sass M (2003) The Drosophila homolog of Aut1 is essential for autophagy and development. FEBS Lett 543: Kerscher O, Felberbaum R, Hochstrasser M (2006) Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu Rev Cell Dev Biol 22: Khurana V, Lu Y, Steinhilb ML, Oldham S, Shulman JM, Feany MB (2006) TOR-mediated cell-cycle activation causes neurodegeneration in Drosophila tauopathy model. Curr Biol 16: Kim S, Jee K, Kim D, Koh H, Chung J (2001) Cyclic AMP inhibits Akt activity by blocking the membrane localization of PDK1. J Biol Chem 276: Patel PH, Tamanoi F (2006) Increased Rheb-TOR signaling enhances sensitivity of the whole organism to oxidative stress. J Cell Sci 119:

13 Supplementary Fig S1 (continued)

14 Supplementary Fig S1

15 A 1 Human 1 Drosophila 1 C. elegans 1 S. cerevisiae 1050 Kinase Domain PS-rich Domain C-Domain 75% 19% 46% 835 Kinase Domain PS-rich Domain C-Domain 80% 25% 57% 856 Kinase Domain PS-rich Domain C-Domain 51% 23% 27% 897 Kinase Domain PS-rich Domain C-Domain X1,000 B F C G D H E I X20,000 Supplementary Fig S2

16 A Brain X150 HS w1118 DmATG1 1 DmATG1 3d αph3 VG HS αtubulin αph3 Eye disc X300 P A αtubulin αph3 Wing disc X200 B X4,000 A D V P w1118 X1,000 αtubulin αph3 DmATG1 1 C w1118 DmATG1 1 DmATG1 3d Supplementary Fig S3

17 A KG04163 (UVRAG 1 ) UVRAG (CG6116) Rep4 ATG 1 kb B UVRAG rp49 w1118 UVRAG 1 C w1118 UVRAG 1 Df(2L)ED784 /UVRAG 1 DmATG DmATG1 1 Hoechst LysoTracker Supplementary Fig S4

18 A 3 days AEL 6 days AEL w1118 dtor P1 dtor P1 ; DmATG /+ dtor P1 / Df(2L)ED784 B Mid/late 3rd instar larvae (%) w1118 dtor P1 dtor P1 ; DmATG /+ dtor P1 /Df(2L)ED784 C w1118 dtor P1 Salivary gland X400 dtor P1 ; DmATG /+ dtor P1 /Df(2L)ED784 Supplementary Fig S5

A αha IP:αHA WCL 160 120 αha HA- ATG1α HA- ATG1β WT KI

160 120 B IP:αMyc αpt229 αpt389 S6K WCL ATG1β Myc-S6K

19 A αha IP:αHA WCL αha HA- ATG1α HA- ATG1β WT KI WT KI CIP WT KT WT KT B IP:αMyc αpt229 αpt389 S6K WCL ATG1β Myc-S6K HA- ATG1β WT KT - WT KT DMEM Supplementary Fig S6

20 A LC3 ATG1α Merge B <MCF-7> - αgfp LC3 I LC3 II GFP-LC3 HA-ATG1α WT - (starvation) ATG1α (starvation) HA-ATG1α WT GFP-LC3 C αgfp <HEK 293T> LC3 I LC3 II D αpt229 IP:αMyc αflag αv5 - - FLAG-ATG1α KI FLAG-ATG1α WT GFP-LC3 ATG6-V5 FLAG-UVRAG - (Rap) αpt389 S6K ATG6 UVRAG Myc-S6K FLAG- UVRAG WCL ATG6-V DMEM Supplementary Fig S7

21 A Relative level sira: CO ATG1α B Relative level sira: CO ATG1β Supplementary Fig S8

Illegitimate translation causes unexpected gene expression from on-target out-of-frame alleles

Illegitimate translation causes unexpected gene expression from on-target out-of-frame alleles created by CRISPR-Cas9 Shigeru Makino, Ryutaro Fukumura, Yoichi Gondo* Mutagenesis and Genomics Team, RIKEN