Regulation of autophagic activity by ζ proteins associated with class III. phosphatidylinositol-3 kinase. Mercedes Pozuelo Rubio

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1 ONLINE SUPPORTING INFORMATION Regulation of autophagic activity by ζ proteins associated with class III phosphatidylinositol-3 kinase Mercedes Pozuelo Rubio entro Andaluz de Biología Molecular y Medicina Regenerativa, onsejo Superior de Investigaciones ientíficas. Sevilla. Spain. orrespondence to Mercedes Pozuelo Rubio, ABIMER (S4). Av. Américo Vespucio s/n, Sevilla-419, Spain. TEL: (34) ; FAX: (34) merce_pozo@yahoo.es Supplementary Figure S1. -ceramide 1 μm for 4 h does not induce cell death. Hela cells were left untreated or stimulated with -ceramide (1 μm for 4 h) or TRAIL (1 μg/ml) (generously provided by Dr. Lopez-Rivas) as a control of cell death. ells were analyzed for the DNA content by flow cytometry. The percentage of death cells with sub-g1 DNA content is shown and the values represent mean ± SD for three independent experiments. Supplementary Figure S. Over expression of ζ proteins block - ceramide cell death with autophagy. HeLa and HEK93T cells left untransfected or HeLa cells stably expressing GFP ζ and HEK93T cells transiently expressing GFP ζ were stimulated with -ceramide () (1 μm) for 3 or 5 days. The percentage of living cells (rystal violet) was expressed according to untreated cells and quantified (Quantification: N = 3, P=.148, P=.33, P=.19, P=.8, Student t- test).

2 Supplementary Figure S3. Downregulation of endogenous ζ sensitizes cells to autophagy. (A) MF-7 cells stably expressing GFP-L3 fusion protein were transfected either with sirna oligonucleotide targeting ζ or with a scrambled RNA oligonucleotide as described in Material ad Methods. After 48 hours, untransfected cells (ontrol) or cells transfected with either with sirna ζ (14-3-3ζ) or scrambled sirna (S) were analysed using green fluorescence GFP-L3 by microscopy. Bars = 1 μm. Quantification of number of GFP-L3 punctae per cell was performed by counting in a blinded experiment. olumns, average of three different experiments, set up at duplicated samples and counting 6 x 1 cells per sample (N=3, P=.4, P=.6 Student t-test ). (B) As in (A) extracts from untransfected cells (ontrol) or transfected either with sirna ζ (14-3-3ζ) or scrambled sirna (S) were harvested (after 48 hours) for immunoblot analysis of L3-I processing into L3-II, GFP-L3 processing into GFP, p6/sqstm1 and endogenous ζ isoform. Tubulin was used as a protein loading control. Endogenous L3-II/Tubulin levels were quantified and the ratio presented as arbitrary units (N=3, P=.6, P=.7, Student t-test). Supplementary Figure S4. Downregulation of endogenous ζ does not affect levels of expression of autophagy-related proteins. Extracts of HeLa cells (3 μg) untransfected () or transfected either with sirna ζ (14-3-3ζ) or scrambled sirna (S) for 48 hours were run on SDS/PAGE transferred to nitrocellulose membranes and probed with indicated antibodies. Supplementary Figure S5. Downregulation of endogenous σ and θ sensitizes cells to autophagy. (A) Extract of HeLa, MF-7 and HEK93T cells (3 μg) were run on SDS/PAGE transferred to nitrocellulose membranes and probed with indicated antibodies against different isoforms. The pan antibody recognized all of the different isoforms. GAPDH was used as a proteins loading control. (B) MF-7 cells expressing GFP-L3 were left untransfected (ontrol) or transfected either with sirna oligonucleotide targeting σ (14-3-3σ) or with a scrambled RNA oligonucleotide (S) as described in Material and Methods. After 48 hours, MF-7/GFP-L3 cells were analysed using green fluorescence GFP-L3 by microscopy. Quantification of number of GFP-L3 punctae per cell was performed by counting in a blinded experiment. olumns, average of

3 three different experiments, set up at duplicated samples and counting 6 x 1 cells per sample (N=3, P=,18, P=,6, Student t-test). () As in (B) MF-7 cells expressing GFP-L3 were left untransfected (ontrol) or transfected either with sirna oligonucleotide targeting θ (14-3-3θ) or with a scrambled RNA oligonucleotide (S). After 48 hours, cells were analysed using green fluorescence GFP-L3 by microscopy. Quantification of number of GFP-L3 punctae per cell was performed by counting in a blinded experiment. olumns, average of three different experiments, set up at duplicated samples and counting 6 x 1 cells per sample (N=3, P==,85, P=,31, Student t- test). (D) As in (B) extracts from MF-7/GFP-L3 untransfected cells () or transfected either with sirna σ (14-3-3σ) or scrambled sirna (S) for 48 hours were harvested for immunoblot analysis of L3-I processing into L3-II, GFP-L3 processing into GFP, p6/sqstm1 and endogenous isoforms. GAPDH was used as a protein loading control. Endogenous L3-II/GAPDH levels were quantified and the ratio presented as arbitrary units (N=3, P=.31, P=.4, Student t-test). (E) As in () extracts from MF-7/GFP-L3 untransfected cells () or transfected either with sirna θ ( θ) or scrambled sirna (S) for 48 hours were harvested for immunoblot analysis of L3- I processing into L3-II, GFP-L3 processing into GFP, p6/sqstm1 and endogenous isoforms. GAPDH was used as a protein loading control. Endogenous L3-II/GAPDH levels were quantified and the ratio presented as arbitrary units (N=3, P=.39, P=.65, Student t-test). Supplementary Figure S6. dissociates from and is activated during -ceramide-induced autophagy in HEK93T cell. HEK93T continuously growing in serum were left untreated or stimulated with -ceramide (1 μm) for 4 h to promote autophagy. (A) Extracts ( mg) from untreated (U) or -ceramide (1 μm for 4 h) () stimulated HEK93T cells were incubated with 1 μg of (Santa ruz) antibody bound to Protein-G-Sepharose. The washed immunoprecipitates were resolved using SDS-PAGE, transferred to nitrocellulose and probed with DIG ( overlay), (BD Biosciences) and (Zymed) antibodies. The washed immunoprecipitates were also used for in vitro kinase assay. Autoradiography shows levels of PI3P 3. (Quantification of in vitro kinase assay expressed as PI(3) fold change, N = 3, P=.5, Student t-test). (B) Extracts (1 mg) from untreated (U) or -ceramide (1 μm for 4 h) () stimulated HEK93T cells were incubated with 1 μg of

4 (Echelon Biosciences) antibody bound to Protein-G-Sepharose. The washed immunoprecipitates were used for in vitro kinase assay and also to analyse interaction with (DIG ) and presence of and in the immunoprecipitates (Quantification of in vitro kinase assay, N = 3, P=.64, Student t-test). A piece of the DIG shown corresponds to size. () Extracts (3 μg) from HEK93T cells continuously grown in serum or stimulated with -ceramide (1 μm) for 4 h to promote autophagy were run on SDS-PAGE, transferred to nitrocellulose membranes and probed with indicated antibodies. Supplementary Figure S7. Overexpression of ζ decreases activity meanwhile blocking -ceramide-dependent autophagy in HEK93T cells. HEK93T cells were transfected with GFP-control () or GFP ζ. (A) ontinuously serumgrown cell extracts (1 mg) were incubated with 1 μg of (Echelon Biosciences) antibody bound to Protein-G-Sepharose. The washed immunoprecipitates were used for in vitro kinase assay and also resolved to detect and proteins. ell extracts (3 μg) from both samples were run on SDS-PAGE, transferred to nitrocellulose membranes and probed with indicated antibodies (bottom panel) (Top panel: Quantification of in vitro kinase assay expressed as PI(3) fold change, N = 3, P=.6 Student t- test). (B) ell extracts (3 μg) from left untreated (U) or -ceramide (1 μm for 4h) stimulated () were run on SDS-PAGE, transferred to nitrocellulose membranes and probed with indicated antibodies. Endogenous L3-II/Tubulin levels were quantified and the ratio presented as arbitrary units (N=3, P=.45 Student t-test). () ell extracts ( mg) from left untreated (U) or -ceramide (1 μm for 4h) stimulated () cells were incubated with 1 μg of Belin-1 (Santa ruz) antibody bound to Protein-G-Sepharose. The washed immunoprecipitates were used for in vitro kinase assay and to analyse presence of and proteins in immonoprecipitates. (On the top: Quantifications of in vitro kinase assays, N = 3, P=.59, Student t-test). Supplementary Figure S8. Downregulation of endogenous ζ increase activity in MF-7 cells. MF-7 cells were transfected either with sirna oligonucleotide targeting ζ or with a scrambled RNA oligonucleotide as described in Material ad Methods. After 48 hours, extract of untransfected (ontrol) or transfected either with sirna ζ (14-3-3ζ) or scrambled sirna (S) MF-7 cells were incubated with

5 1 μg of (Echelon Biosciences) antibody bound to Protein-G-Sepharose. The washed immunoprecipitates were used for in vitro kinase assay and to analyse presence of protein in immonoprecipitates (Top panel: Quantification of in vitro kinase assay, N = 3, P=.5, P=.11, Student t-test). Extracts from untransfected cells () or transfected either with sirna ζ (14-3-3ζ) or scrambled sirna (S) were harvested for immunoblot analysis of ζ isoform. Tubulin was used as a protein loading control (bottom panel). Supplementary Figure S9. dissociates from and is activated during Starvation-induced autophagy in MF-7 cells. (A) MF-7 cells expressing GFP- L3 fusion protein and continuously growing in serum were left untreated or starved during 6h. ells were analysed using green fluorescence GFP-L3 by microscopy. Bars = 1 μm. Quantification of number of GFP-L3 punctae per cell was performed by counting in a blinded experiment. olumns, average of three different experiments, set up at duplicated samples and counting 6 x 1 cells per sample (N=3, P=.7, Student t-test ) (top panel). Untreated and starved MF-7/GFP-L3 cells were harvested for immunoblot analysis indicated proteins. Tubulin was used as a protein loading control (bottom panel). (B) MF-7/GFP-L3 cells were treated as in (A) and extracts from untreated and starved cells were incubated with 1 μg of (Echelon Biosciences) antibody bound to Protein- G-Sepharose. The washed immunoprecipitates were used for in vitro kinase assay and test presence of protein in immonoprecipitates and binding (DIG ) (Top panel: Quantification of in vitro kinase assay, N = 3, P=.35, Student t- test). Supplementary Figure S1. dissociates from during Etoposide and Rapamycin-induced autophagy. (A) HeLa continuously grown in serum () were left untreated or incubated in presence or absence of 3-methyladenine (3MA) (1 mm for 1 h) and then stimulated with Etoposide ( μm for 4 h) as indicated. ell extracts were incubated with 1 μg of (Echelon Biosciences) antibody bound to Protein-G- Sepharose. The washed immunoprecipitates were used to test presence of protein in immonoprecipitates and binding (DIG ) (Quantification of DIG overlay signal, N = 3, P=.81, P=.14, Student t-test). (B) HeLa/GFP-L3 cells treated as in (A) were analysed using green fluorescence GFP-L3 by microscopy.

6 Quantification of number of GFP-L3 punctae per cell was performed by counting in a blinded experiment. olumns, average of three different experiments, set up at duplicated samples and counting 6 x 1 cells per sample (N=3, P=,1, P=,9, Student t- test ). () HeLa cells treated as in (A) were harvested for immunoblot analysis of indicated proteins. GAPDH was used as a protein loading control. (Endogenous L3-II/GAPDH levels were quantified and the ratio presented as arbitrary units (N=3, P=.97, P=.7, Student t-test). (D) MF-7/GFP-L3 cells continuously grown in serum () were left untreated or incubated in presence or absence of 3-methyladenine (3MA) (1 mm for 1 h) and then stimulated with Rapamycin (5 μm for 4 h) as indicated. ell extracts were incubated with 1 μg of (Echelon Biosciences) antibody bound to Protein-G- Sepharose. The washed immunoprecipitates were used to test presence of protein in immonoprecipitates and binding (DIG ) (Quantification of DIG overlay signal, N = 3, P=.18, P=., Student t-test). (E) MF-7/GFP-L-3 cells treated as in (D) were analysed using green fluorescence GFP-L3 by microscopy. Quantification of number of GFP-L3 punctae per cell was performed by counting in a blinded experiment. olumns, average of three different experiments, set up at duplicated samples and counting 6 x 1 cells per sample (N=3, P=,14, P=,31, Student t-test). (F) MF-7/GFP-L-3 cells treated as in (D) were harvested for immunoblot analysis of indicated proteins. GAPDH was used as a protein loading control. (Endogenous L3- II/GAPDH levels were quantified and the ratio presented as arbitrary units (N=3, P=.45, P=.9, Student t-test). Supplementary Figure S11. from lung mouse tissue binds proteins. (A) Extract of lung mouse tissue and HeLa cells continuously growing in serum (3 μg) were run on SDS-PAGE, transferred to nitrocellulose membranes and probed with indicated antibodies. GAPDH was used as a protein loading control. (B) Extract of lung mouse tissue (3-5 mg of protein) were incubated for h with 5 μl of Sepharose, pellets were washed () and extracted with SDS-sample buffer (Pull down), run on SDS/PAGE and probed with antibodies against. Beside, lung mouse tissue extract (Ext) were run in term of comparison. () Extract of lung mouse tissue ( mg of protein) was incubated with 1 μg of (Echelon Biosciences) antibody bound to Protein-G- Sepharose. The washed immunoprecipitates, from extract (IP) or control (), were resolved

7 in SDS-PAGE, transferred to nitrocellulose and probed with DIG ( overlay) and (Zymed) antibodies. Supplementary Figure S1. Serum-free medium increases lipid kinase activity. ontinuously serum-grown HeLa cells were left untreated (U) or serum-starved (- S) for 16 h. ells extracts (1 mg) from both samples were incubated with 1 μg of antibody bound to Protein-G-Sepharose. The washed immunoprecipitates were used for in vitro kinase assay and also resolved to detect protein (Top panel: Quantification of in vitro kinase assay expressed as PI(3) fold change, N = 3, P=.9, Student t-test). Additionally, cell extract (3 μg) was used for immunoblot analysis of indicated proteins. Tubulin was used as a protein loading control. Supplementary Figure S13. binds proteins by a PMAphosphorylation-dependent mechanism in HEK93T cells. HEK93T cells growing in medium containing serum were treated with -ceramide (1 μm, 4 h) () or serumstarved for 16 h and where indicated, cells were incubated with H-7 (1 μm), PD ( μm) or no inhibitor for 1 h before stimulation with PMA (4 ng/ml) for a further 15 min. Extract from HEK93T cells were incubated with 1 μg of (Echelon Biosciences) antibody bound to Protein-G-Sepharose. The washed immunoprecipitates were used to detect protein and for interaction with proteins by analysing DIG ell extracts (3 μg) from both samples were run on SDS-PAGE, transferred to nitrocellulose membranes and probed with indicated antibodies (bottom panel). Supplementary Figure S14. Amino acid sequence of with the binding domains. The amino acid sequence of with the binding domains is shown. Length of deletion mutants MT1 (1-68 aa) and MT (1-39 aa) can be distinguish by asterisk (). Serines or Threonines mutated to Alanine by site-directed mutagenesis are shown in bold. Underlined sequences show Thr197 and S1 domains whose change to Alanine reduces its binding status.

8 Supplementary Figure S1 HeLa cells ell death percentage ontrol TRAIL

9 Supplementary Figure S HEK93T cells Living cells (%) HeLa cells 3 days 5 days -eramide Living cells (%) ONTROL GFP ζ ONTROL GFP ζ

10 Supplementary Figure S3 A sirna ζ MF-7/GFP-L3 cells No of GFP-L3 punctae per cells ontrol ζ S ontrol ζ S B sirna ζ MF-7/GFP-L3 cells ontrol ζ S L3-I L3-II GFP-L3 3 6 GFP p ζ Tubulin L3-II / Tubulin (A.U.) ζ S

11 Supplementary Figure S4 sirna ζ ζ S Atg1 hvps 34 PIST Atg1-Atg5 Atg 7 Atg 16 LAMP 1 LAMP ζ Tubulin

12 Supplementary Figure S5 A HeLa MF-7 HEK93T ζ θ B No of GFP-L3 punctae per cells sirna σ MF-7/GFP-L3 cells ontrol σ S σ ε sirna θ γ pan antibody GAPDH No of GFP-L3 punctae per cells MF-7/GFP-L3 cells ontrol θ S D sirna σ E sirna θ MF-7/GFP-L3 cells MF-7/GFP-L3 cells σ S θ S L3-I L3-II L3-I L3-II 48 GFP-L3 48 GFP-L3-I 3 GFP 3 GFP 6 p6 6 p σ θ θ σ ζ ζ GAPDH 36 GAPDH L3-II / GAPDH (A.U.) σ S L3-II / GAPDH (A.U.) θ S

13 Supplementary Figure S6 A B IP: PI(3)P fold change 3 1 HEK93T cells IP: PI(3)P fold change 3 1 HEK93T cells PI3P 3 PI3P 3 DIG DIG U U HEK93T cells U L3-I L3-II p6 P-p7S6K p7s6k Tubulin

14 Supplementary Figure S7 A B HEK93T cells PI(3)P fold change 1,5 1,5 HEK93T cells U GFP ζ L3-I L3-II p6 IP: PI3P 3 7 P-p7S6K 7 p7s6k 5 58 GFP GFP ζ 5 Tubulin 58 GFP ζ GFP ζ L3-II / Tubulin (A.U.) Tubulin PI(3)P fold change 3 1 HEK93T cells IP: 5 PI3P 3 U GFP ζ

15 Supplementary Figure S8 sirna ζ PI(3)P fold change 1,5 1,5 MF-7/GFP-L3 cells IP: PI3P ζ S ζ 5 Tubulin ζ S

16 Supplementary Figure S9 A B N o of GFP-L3 punctae per cell MF7/GFP-L3 cells PI(3)P fold change MF7 GFP-L3 cells IP: PI3P 3 DIG ONTROL STARVATION U PBS 48 U STARV GFP-L3 3 GFP 6 p6 7 P-p7S6K 7 p7s6k Tubulin

17 Supplementary Figure S1 D A IP: HeLa cells ETO 3MA+ ETO DIG IP: MF-7/GFP-L3 cells Ra 3MA+ Ra DIG Arbitrary units 1,5 1,5 5 Arbitrary units 1,5 1,5 B E MF-7/GFP-L3 cells N o of GFP-L3 punctae per cell HeLa/GFP-L3 cells N o of GFP-L3 punctae per cell MF-7/GFP-L3 cells F 48 Ra 3MA+ Ra GFP-L HeLa cells 3MA+ ETO ETO L3-I L3-II GFP L3-I L3-II p6 6 p6 7 P-p7S6K 7 p7s6k GAPDH L3-II / GAPDH (A.U.) 6 4 ETO 3MA+ETO L3-II / GAPDH (A.U.) 36 1 Ra 3MA+Ra GAPDH

18 Supplementary Figure S11 A Mouse HeLa 36 GAPDH B Pull down 3mg 5mg Ext MOUSE IP DIG MOUSE

19 Supplementary Figure S1 PI(3)P fold change 3 1 HeLa cells IP: PI3P 3 U -S U -S 6 5 p Tubulin

20 Supplementary Figure S13 HEK93T cells PMA IP: -s PMA H7 PD DIG PMA -s PMA H7 PD 4/44 P-ERK1/ 4/44 ERK1/ Tubulin

21 Supplementary Figure S14 MT 1 MGEAEKFHYI YSDLDINVQ LKIGSLEGKR EQKS 34 YKAVLE DPMLKFSGLY QETSDLYVT 61 QVFAEGKPL ALPVRT 76 S 77 YKA FSTRWNWNEW LKLPVKYPDL PRNAQVALTI WDVYGPGKAV 11 PVGGTTVSLF GKYGMFRQGM HDLKVWPNVE ADGSEPTKTP GRT 163 S 164 S 165 T 166 LSED QMSRLAKLTK 181 AHRQGHMVKV DWLDRLT 197 FRE IEMINESEKR SS 1 NFMYLMVE FRVKDDKE YGIVYYEKDG 41 DESSPILTSF ELVKVPDPQM SMENLVESKH HKLARS 76 LRS 79 G PSDHDLKPNA ATRDQLNIIV 31 SYPPTKQLTY EEQDLVWKFR YYLTNQEKAL TKFLKVNWD LPQEAKQALE LLGKWKPMDV 361 EDSLELLSSH YTNPTVRRYA VARLRQADDE DLLMYLLQLV QALKYENFDD IKNGLEPTKK 41 DSQSSVSENV SNSGINSAEI DSSQIITSPL PSVSSPPPAS KTKEVPDGEN LEQDLTFLI 481 SRAKNSTLA NYLYWYVIVE EDQDTQQRD PKTHEMYLNV MRRFSQALLK GDKSVRVMRS 541 LLAAQQTFVD RLVHLMKAVQ RESGNRKKKN ERLQALLGDN EKMNLSDVEL IPLPLEPQVK 61 IRGIIPETAT LFKSALMPAQ LFFKTEDGGK YPVIFKHGDD LRQDQLILQI ISLMDKLLRK 661 ENLDLKLTPY KVLATSTKHG FMQFIQSVPV AEVLDTEGSI QNFFRKYAPS ENGPNGISAE 71 VMDTYVKSA GYVITYILG VGDRHLDNLL LTKTGKLFHI DFGYILGRDP KPLPPPMKLN 781 KEMVEGMGGT QSEQYQEFRK QYTAFLHLR RYSNLILNLF SLMVDANIPD IALEPDKTVK 841 KVQDKFRLDL SDEEAVHYMQ SLIDESVHAL FAAVVEQIHK FAQYWRK MT1