SUPPLEMENTARY INFORMATION

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Eric Logan
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Figure S1 The effect of T198A mutation on p27 stability. a, Hoechst 33342 staining for nuclei (see Fig 1d). Scale bar, 100 μm.

1 Figure S1 The effect of T198A mutation on p27 stability. a, Hoechst staining for nuclei (see Fig 1d). Scale bar, 100 μm. b, Densitometric analysis of wild type and mutant p27 protein levels represented as ratios to 0-h (See Fig 1f). c and d, Western blots of protein extracts from MCF-7 with (+) or without (-) MG132 for 12-h following transfection with wild type and mutant p27 (GFP was co-transfected as a transfection control) (c), and from U2OS cells transfected with RFP-p27WT, the indicated mutant alleles, or an empty RFP vector (C1), or mock transfected (-) for 48-h prior to treatment with (+) or without (-) MG132. e, Densitometric analysis of Western blots of wild type and mutant p27 protein levels from MCF-7 or Hela cells 48-h posttransfection. 1

vacuoles with monodansylcadaverine (MDC) (b). c, U2OS-GFPLC3 cells cultured in the presence or absence of 10% fetal bovine serum (-FBS) for 12-h.

2 Figure S2 The role of p27 and cell cycle in autophagy and cell viability. a and b, MCF-7 cells transfected with YFP-p27WT and mutant allele selected with G418 for 3 weeks were stained for lysosomes with LysoTracker (red) and nuclei with Hoechst (blue) (a) and for acidic vacuoles with monodansylcadaverine (MDC) (b). c, U2OS-GFPLC3 cells cultured in the presence or absence of 10% fetal bovine serum (-FBS) for 12-h. d, Western blots of U2OS-GFPLC3 cell lysates (see also Fig 3a). e, U2OS-GFPLC3 cells cultured for 48-h with or without FBS 24-h post-rnai. f, Cell cycle profiles of MCF-7 transfected with non-targeting (NT) sirna or a p27sirna pool for 48-h with or without rapamycin (100 nm) added 20-h before analysis. g and h, MCF-7 cells incubated with either mimosine (mimo, 300 μm) or nocodazole (noco, 40 ng ml -1 ) for 22-h, followed by treatment with (+) or without (-) rapamycin (Rap, 100 nm) 24-h post-rnai. Arrows indicate apoptotic cells (g). Densitometric analysis of GFP-LC3-II/I (Western blots) of U2OS-GFP-LC3 cells asynchronously grown (Ctl) or synchronized with nocodazole, hydroxylurea (Hu, 100 μm), mimosine, or aphidicolin (Aphi, 5 μg ml -1 ) for 48-h prior to rapamycin treatment for 24-h. Flow cytometric analysis (h). Scale bar, 50 μm. 2

Figure S3 Kinases potentially mediating p27t198 phosphorylation a, Alignment of p27t198 peptide sequence with consensus phosphorylation motifs of a panel of basophilic kinases.

3 Figure S3 Kinases potentially mediating p27t198 phosphorylation a, Alignment of p27t198 peptide sequence with consensus phosphorylation motifs of a panel of basophilic kinases. Box, compatible residues; dashed box, non-essential residues; *, phosphorylation sites; β, basic residue; φ, hydrophobic residue. b, c, and d, Kinase assays using purified AMPK and either recombinant full-length His-p27 (b) or a wild type p27t198 peptide (WT, SGSG-KPGLRRRQT) versus a T198A mutant peptide (c and d) as substrates in the presence or absence of AMP (172.5 μm). Kinase activity was determined by either 32 p-atp incorporation (b and c) or by Western dot blot and densitometric scanning (representative of 3 independent assays) using the pt198 antibody and densitometry analysis (d). CMP, counts per minute. e, Western blots of protein lysates from MCF-7 cells treated with (+) or without (-) LY (LY, 10 μm) for 24-h. f and g, Western blots of protein extracts from MEFs cultured in the absence or presence of UO126 (5 μm) or AICAR (2mM) or both for 2-h or 2-h after UV irradiation (5 μj cm -2 ). 3

Figure S4 The role of cyclin-cdks in autophagy and cell death. a and b, Western blots of protein extracts (a) or immunoprecipitates (b) from 3T3L1 cells treated with (+) or without (-) AICAR (1.

c, d, and e, MCF-7 with stable expression of GFP-LC3 transfected with control (CTL) or indicated sirna pools, each consisting of 3 individual sirnas, for 24-h and cultured for 48-h with or without

The LC3-II bands were quantified by densitometric scanning and the data represent mean +/- SD (e). *, p=0.016; **, p=0.001 (T test).

4 Figure S4 The role of cyclin-cdks in autophagy and cell death. a and b, Western blots of protein extracts (a) or immunoprecipitates (b) from 3T3L1 cells treated with (+) or without (-) AICAR (1.5 mm) for 2-h. #, rainbow marker; NR IgG, normal rabbit IgG; 1X loading, 500 μg; Cdk4:p27 (right panel), densitometric ratio of p27-associated Cdk4 to p27. c, d, and e, MCF-7 with stable expression of GFP-LC3 transfected with control (CTL) or indicated sirna pools, each consisting of 3 individual sirnas, for 24-h and cultured for 48-h with or without 5%FBS (c). Scale bar, 50 μm. Protein extracts were subject to Western blotting using GFP antibody for GFP-LC3 or indicated antibodies (d). The LC3-II bands were quantified by densitometric scanning and the data represent mean +/- SD (e). *, p=0.016; **, p=0.001 (T test). f, Western blots of protein extracts from U2OS-GFPLC3 cells transfected with non-targeting (-) or individual Cdk2 or Cdk4 sirnas, showing the efficacy and specificity of gene silencing. Note p27 levels were increased by Cdk2 but not Cdk4 depletion, indicating sufficient suppression of Cdk2 activity despite a slightly lesser degree of Cdk2 knockdown compared to Cdk4 knockdown. D Scan, densitometric scanning of Western blots (ratio to control); 1X sirna, 5 nm; Cdk2 sirna duplex 12, 5 -GAA ACA AGU UGA CGG GAG AAU U::5 -PUC UCC CGU CAA CUU GUU UCU U; 13, 5 -GGA GUU ACU UCU AUG CCU GUU::5 -PCA GGC AUA GAA GUA ACU CCU U; 14, 5 -GGG CCU AGC UUU CUG CCA UUU::5 -PAU GGC AGA AAG CUA GGC CCU U; Cdk4 sirna duplex 13, 5 -GAG CUC UGC AGC ACU CUU AUU::5 -PUA AGA GUG CUG CAG AGC UCU U; 14, 5 -CAG CAC AGU UCG UGA GGU GUU::5 -PCA CCU CAC GAA CUG UGC UGU U; 15, 5 -GCA CUU ACA CCC GUG GUU GUU::5 -PCA ACC ACG GGU GUA AGU GCU U. 4

5 Figure S5 Extended blots from figures in text. See related Figure legends. 5

Level 2 Biology, 2017

91159 911590 2SUPERVISOR S Level 2 Biology, 2017 91159 Demonstrate understanding of gene expression 2.00 p.m. Wednesday 22 November 2017 Credits: Four Achievement Achievement with Merit Achievement with