HEAHKFLKVEFPARVRSSQATYEIQFGHLQRPTHYNTSWDWARFEVWAHRWMDLSEHGFG Sbjct: 805 HEAHKFLKVEFPARVRSSQATYEIQFGHLQRPTHYNTSWDWARFEVWAHRWMDLSEHGFG 864

Transcription

1 Supplementary Fig S1 a gi sp Q9NTJ4 MAN2C1HUMAN Alpha-mannosidase 2C1 (Alpha-D-mannoside mannohydrolase) (Mannosidase alpha class 2C member 1) (Alpha mannosidase 6A8B) Query: 1 HEAHKFLKVEFPARVRSSQATYEIQFGHLQRPTHYNTSWDWARFEVWAHRWMDLSEHGFG 60 HEAHKFLKVEFPARVRSSQATYEIQFGHLQRPTHYNTSWDWARFEVWAHRWMDLSEHGFG Sbjct: 805 HEAHKFLKVEFPARVRSSQATYEIQFGHLQRPTHYNTSWDWARFEVWAHRWMDLSEHGFG 864 Query: 61 LALLNDCKYGASVRGSILSLSLLRAPKAPDATADTGRHEFTYALMPHKGSFQDAGVIQAA 120 LALLNDCKYGASVRGSILSLSLLRAPKAPDATADTGRHEFTYALMPHKGSFQDAGVIQAA Sbjct: 865 LALLNDCKYGASVRGSILSLSLLRAPKAPDATADTGRHEFTYALMPHKGSFQDAGVIQAA 924 Query: 121 YSLNFPLLALPAPSPAPATSWSAFSVSSPAVVLETVKQAESSPQRRSLVLRLYEAHGSHV 180 YSLNFPLLALPAPSPAPATSWSAFSVSSPAVVLETVKQAESSPQRRSLVLRLYEAHGSHV Sbjct: 925 YSLNFPLLALPAPSPAPATSWSAFSVSSPAVVLETVKQAESSPQRRSLVLRLYEAHGSHV 984 Query: 181 DCWLHLSLPVQEAILCDLLERPDPAGHLTLRDNRLKLT 218 DCWLHLSLPVQEAILCDLLERPDPAGHLTLRDNRLKLT Sbjct: 985 DCWLHLSLPVQEAILCDLLERPDPAGHLTLRDNRLKLT 1022 b AbGST 1:100 1:200 1:500 1:1000 1:2000 1:5000 MAN2C1 EV a MAN IgG EV MAN2C1 Lysate C-MAN236 IgG a MAN AbGST-C-MAN236 MAN2C1 83 kda EV C-MAN kda C-MAN236 1:5000 Ab conc. AbGST AbGST-C-MAN236 -DAPI 1:2000 DAPI 1:1000 -DAPI 1:500 DAPI d EV MAN2C1 83 kda c Ab dillutions Supplementary Figure S1. Identification of MAN2C1 as a -NR. (a) A cdna was recovered from surviving LNCaP cells co-transfected with and a human prostate cdna library (see reference 32 for details). GeneBank BLAST search identified this cdna being á-mannosidase 2C1 (MAN2C1). The C-terminal 236 residues of MAN2C1 were identified, of which 218 were used for a Blast search. (b) Characterization of anti-man2c1 antibody. AntiMAN2C1 antibody was raised in rabbits using recombinant C-MAN236 and affinity purified. The antibody detected FLAG-tagged MAN2C1 transiently expressed in 293T cells by western blot at the indicated dilutions (left panel). Competition of GST or GST-C-MAN236 with anti-man2c1 antibody used at 1:1000 in western blot (right panel). (c) Anti-MAN2C1 antibody immunoprecipitates C-MAN T cells were transiently transfected with an empty vector (EV) or C-MAN236 (FLAG tagged), followed by immunoprecipitation with anti-man2c1 antibody (áman) or a control IgG and western blot analysis with anti-flag antibody (M2). (d) Anti-MAN2C1 antibody recognizes CMAN236 by immunofluorescent (IF) staining. NIH3T3 cells were stably infected with C-MAN236 retrovirus, followed by IF staining with anti-man2c1 antibody at the indicated dilutions (left 4 panels). The specific C-MAN236 signal recognized by the antibody could be competed out by GST-C-MAN236 (right two panels). Images were taken using a fluorescent microscope (Zeiss). The faint nuclear signal of C-MAN236 may be due to the non-confocal nature of the images. Scale bar represents 10 ìm.

2 Supplementary Fig S2 a 62 kda Ctrl shrna shrna MAN2C1 shrna shrna MAN2C1 shrna AKT-P 62 kda AKT 83 kda MAN2C kda 47.5 kda Actin b MAN2C1 Merge Supplementary Figure S2. MAN2C1 reduces function and co-localizes with. (a) MCF7 cells were infected with retrovirus expressing the indicated shrnas. The cells were selected in hygromycin (for shrna) or puromycin (for MAN2C1 shrna) for 2 days to achieve 100% infection, followed by western blot examination for the indicated proteins. (b) Typical z-stack images of MCF7 cells immunofluoroscently stained for (red) and MAN2C1 (green). Merge image is also included. Scale bar represents 10 m.

3 Supplementary Fig S3 MAN2C1(FLAG) Merge Merge with DAPI LNCaP 293T Supplementary Figure S3. Co-localization of with MAN2C1. 293T and LNCaP cells were co-infected with and MAN2C1. Double immunofluorescent staining was performed for (red) and MAN2C1 (green) using an anti-flag antibody. Nuclei were counterstained with DAPI (blue). Scale bar represents 10 m. Images were captured using MP Leica TCS SP5 confocal microscope. Typical z-stack images are shown.

4 Supplementary Fig S4 a 1 1 Catalytic domain Binding to MAN2C1 Cat-MAN C-MAN C-MAN-N C-MAN236 C-MAN236-N C-MAN236-C - - b EV C-MAN-N 10% Cell lysate IgG IP Ab 47.5 kda 16.5 kda C-MAN-N c EV C-MAN236-N 47.5 kda 6.5 kda 10% Cell lysate IgG * IP Ab IgG C-MAN236-N EV C-MAN236-C 47.5 kda 16.5 kda 10% Cell lysate IgG * IP Ab IgG C-MAN236-C Supplementary Figure S4. Mapping the -binding motifs in MAN2C1. (a) MAN2C1 deletion mutants were generated and examined for their interaction with by transient expression of individual MAN2C1 mutants with in 293T cells, followed by IP-western blot analysis. (b) Binding of C-MAN-N to. (c) Binding of C-MAN236-N (left panel) and C-MAN236-C (right panel) to. The asterisks indicate the bands.

5 Supplementary Fig S5 a Empty Vector MAN2C1 b Empty Vector MAN2C1 c 500 M2 3 Tumor volume (mm ) Ctrl MAN2C1 MAN2C1 * sirna AKT-P * Supplementary Figure S5. MAN2C1 promotes DU145 cells-derived xenograft tumor formation in nude mice. (a) DU145 empty vector and MAN2C1 overexpressing cells were implanted into nude mice. Typical images of tumours formed at the termination day are shown. (b) Xenograft tumours from each group were IHC stained with anti-flag (M2) for MAN2C1, anti-, and anti-phospho-akt (AKT- P) antibodies. Images for empty vector or MAN2C1 tumors were from the same individual tumors, respectively. Scale bar represents 10 µm. (c) DU145 cells were first treated with the indicated sirna for 5 2 days and then (2 x 10 ) implanted into NOD/SCID mice. Five mice were used for individual treatments. Animals were terminated at 42 days. The average tumour volumes were graphed and analyzed by 2- tailed student test. *: p < 0.05 (2-tailed student t-test) for MAN2C1 sirna versus Ctrl sirna and MAN2C1/ sirnas versus MAN2C1 sirna.

6 Supplementary Fig S6 a Antibodies C-MAN236 FLAG with DAPI C-MAN µm KDEL C-MAN236 FLAGKDEL with DAPI 10 µm b C-MAN236 C-MAN236/ Cyt Mic Cyt Mic Cyt Mic 47.5 kda 25 kda Distribution of between Cyt and Mic (%) C-MAN kda Calnexin Supplementary Figure S6. associates with C-MAN236 at the ER. (a) DU145 cells were stably transfectesd with C-MAN236. Co-localization of endogenous with C-MAN236 (anti-flag) and co-localization of KDEL with C-MAN236 (anti-flag) were examined. Typical z-stack confocal images are shown. (b) 293T cells were transiently transfected with the indicated transgenes (top). Cells were then separated into cytosolic (Cyt) and ER microsome (Mic) fractions and were examined for the expression of C-MAN236,, and Calnexin (an ER membrane protein) by western blot using anti-ha (), anti-flag (C-MAN236), and anti-calnexin antibodies. The portion of ER-bound in the total (the sum of in Cyt and Mic) was presented under the panel. Star symbol indicates a background band.

7 Supplementary Fig S7 H&E MAN2C1GST MAN2C1 GST-C-MAN236 Supplementary Figure S7. Anti-MAN2C1 antibody specifically recognizes MAN2C1 by IHC. PIN was H&E or IHC stained with the anti-man2c1 antibody ( MAN2C1) in the presence of GST (a non-specific protein) or GST-C-MAN236 (the competitive antigen). Scale bar represents 50 m.

8 Supplementary Fig S8 MAN2C1 Positive Cells (%) Normal PIN Gleason Supplementary Figure S8. Increases of MAN2C1 in -positive PINs and carcinomas of all Gleason scores. The percentage of MAN2C1-positive cells in normal prostate glands, as well as in -positive and negative PINs and carcinomas with different Gleason scores, was determined based on the IHC staining of TMA2TMA5.

9 Supplementary Fig S9 a 100% Percentage Recurrence-Free Survival 80% 60% 40% 20% MAN2C1-Negative MAN2C1-Positive Median survival: 57 months Log-rank test statistic = 4.28 p= % Months Following Radical Prostatectomy b Percentage Recurrence-Free Survival 100% 80% 60% 40% 20% 0% MAN2C1 Expression MAN2C1-Negative MAN2C1-Positive Negative-censored Positive-censored Median s urvival: MAN2C 1 E stimate S td Negative Undefined Undefined P os itive O verall L og-rank test statistic = p = Months After Radical Prostatectomy Supplementary Figure S9. MAN2C1 expression associates with prostate cancer recurrence. Kaplan-Meier analysis of biochemical recurrence-free survival for a subset of patients in our cohort (a, n = 20) and TMA2 (b, n=88) is shown.

10 Supplementary Table S1. Primers and sirna target sequences MAN2C1 Primer 1 CGGAATTCGCCACCATGGCGGCTGCGCCGGCCTT Primer 2 GCTCTAGAGTGTGGCGGAGGCTGAAG Cat-MAN Primer 1 CGGAATTCGCCACCATGGCGGCTGCGCCGGCCTT Primer 2 GCTCTAGACAGGGCCATCACTTCGAT C-MAN Primer 1 CGGAATTCGCCACCATGGCCCTGCCCAAACCG Primer 2 GCTCTAGAGTGTGGCGGAGGCTGAAG C-MAN-N Primer 1 CGGAATTCGCCACCATGGCCCTGCCCAAACCG Primer 2 GCTCTAGACCAGTGTACCTCGGTGTG C-MAN236 Primer 1 CGGAATTCGCCACCATGCATGAGGCCCACAAGTTC Primer 2 GCTCTAGAGTGTGGCGGAGGCTGAAG C-MAN236-N Primer 1 CGGAATTCGCCACCATGCATGAGGCCCACAAGTTC Primer 2 GCTCTAGACAGTGCATAGGTGAACTC C-MAN236-C Primer 1 CGGAATTCGCCACCATGCCGCACAAGGGCTCT Primer 2 GCTCTAGAGTGTGGCGGAGGCTGAAG -N Primer 1 CGGGATCCATGACAGCCATCATCAAA Primer 2 GCTCTAGATCAGTGATTTTTTAGCAGGTA -C Primer 1 CGGGATCCATGCTGGATTACAGACCCGTG Primer 2 GCTCTAGATCAGACTTTTGTAATTTGTGAATGCTG cdna inserts Primer 1 CGGGATCCAGCCCTCACTCCTTCTCTAG Primer 2 CGGAATTCCTACAGGTGGGGTCTTTCATT MAN2C1 RT-PCR GTTCCCAGGCCACCTATGAGA CAGCATCCTGGAAAGAGCCCTT RT-PCR CATAACCCACCACAGCTAGAACT ATCACCACACACAGGCAATGG ACTIN RT-PCR TCCATCATGAAGTGTGACGT AGTACTTGCGCTCAGGAGGA MAN2C1 oligo sirna target ATAGCTGGTCTTCTCACCCTCTTTG MAN2C1 shrna target Santa Cruz # sc sh oligo sirna target TGTCTCTGGTCCTTACTTCTT shrna target GTATAGAGCGTGCAGATAA Primers used to generate MAN2C1 truncation mutants (see Supplementary Fig S4a) Primers used to generate truncation mutants (See Supplementary Fig S4a) Primers used to amplified cdna inserts in the Lib/ surviving LNCaP cells Primers used to perform RT-PCR for the indicated cdnas Targeting sequences used to knock-down MAN2C1 and, respectively. MAN2C1 shran from Santa Cruz was a pool of MAN2C1 shrnas.

11 Supplementary Table S2. Clinical information of the local patient cohort Ethnic Family Primary Secondary Total Capsule Positive Perineural Patient # Age Background History Gleason Gleason Gleason Involvement Nodes Invasion 1 62 Caucasian Yes No 0 Unknown 2 67 Caucasian No No 0 Unknown 3 61 Caucasian Unknown No 0 Unknown 4 57 Caucasian Unknown No 0 Unknown 5 56 Caucasian No No 0 Unknown 6 66 Caucasian Unknown No 0 Unknown 8 57 Caucasian Yes Yes 0 Unknown 8 69 Caucasian No No 0 Unknown 9 61 Caucasian No Yes 0 Unknown Caucasian Unknown No 0 Unknown Caucasian No Yes 0 Unknown Caucasian Unknown No 0 Unknown Caucasian Unknown Yes 0 Unknown Caucasian Yes No 0 Unknown Caucasian No Yes 1 Unknown caucasian No Yes 0 Unknown Caucasian Yes Yes 0 Unknown Caucasian Yes Yes 0 Unknown Unknown Unknown No 0 Unknown Caucasian Yes Yes 0 Unknown Unknown Unknown Yes 0 Unknown Unknown Unknown No 0 Unknown Asian Yes No 0 Unknown Unknown Unknown No 0 Unknown Caucasian Unknown No 0 Unknown Caucasian Unknown No 0 Unknown 27 unknown Caucasian No Yes 0 Unknown Caucasian No No 0 Unknown African American Unknown No 0 Unknown Caucasian Yes No 0 Unknown Unknown Unknown No 0 Unknown Caucasian No No 0 Unknown African American No No 0 Unknown Native Yes No Caucasian Unknown Yes Native No No 0 Unknown African American Yes No Caucasian No No Caucasian No No African American No No 0 0

12 41 68 Unknown Unknown Yes Caucasian Yes Unknown Unknown Unknown Unknown Unknown No unknown Caucasian No No Unknown Unknown No Caucasian No Yes Caucasian Unknown No Caucasian No Yes Unknown Unknown No Caucasian No No Caucasian No Yes 0 0

13 Supplementary Table S3. MAN2C1 expression correlates with a reduction in function in primary prostate cancer tissues derived from TMA2 Normal Akt-P - Akt-P Total Pearson's φ p - MAN2C MAN2C and MAN2C Total and AKT-P MAN2C MAN2C1 and AKT-P MAN2C Total PIN Akt-P - Akt-P Total Pearson's φ p - MAN2C MAN2C and MAN2C < Total and AKT-P < MAN2C MAN2C1 and AKT-P MAN2C Total Carcinoma Akt-P - Akt-P Total Pearson's φ p - MAN2C MAN2C and MAN2C < Total and AKT-P MAN2C MAN2C1 and AKT-P MAN2C Total Note: The indicated correlations (Pearson's φ) and their p values are shown.

14 Supplementary Table S4. MAN2C1 expression correlates with a reduction in function in primary prostate carcinomas derived from TMA5 Carcinoma Akt-P - Akt-P Total Pearson's φ p - MAN2C MAN2C and MAN2C < Total and AKT-P 0.22 < MAN2C MAN2C1 and AKT-P MAN2C Total

15 Supplementary Table S5. MAN2C1 expression correlates with a reduction in function in primary prostate cancer tissues derived from our patient cohort (51 patients) Normal Akt-P - Akt-P Total Pearson's φ p - MAN2C MAN2C and MAN2C Total and AKT-P MAN2C MAN2C1and AKT-P MAN2C Total PIN Akt-P - Akt-P Total Pearson's φ p - MAN2C MAN2C and MAN2C Total and AKT-P MAN2C MAN2C1and AKT-P MAN2C Total Carcinoma Akt-P - Akt-P Total Pearson's φ p - MAN2C MAN2C and MAN2C <0.001 Total and AKT-P MAN2C MAN2C1and AKT-P MAN2C Total

16 Supplementary Table S6. MAN2C1 expression and AKT-P (AKT activation) levels in negative and -positive PINs and carcinomas N Mean Std p MAN2C1 Normal PIN Carcinoma Total 314 AKT-P Normal PIN Carcinoma <0.001 Total 314 MAN2C1 Normal PIN <0.001 Carcinoma <0.001 Total 470 AKT-P Normal PIN <0.001 Carcinoma <0.001 Total 470 Note: p values are in comparison to Normal. Std: standard derivation. Normal prostate glands included patients from our cohort. PINs and carcinomas were from TMA2 and TMA5.

17 Supplementary Table S7. MAN2C1 expression and AKT-P (AKT activation) levels in negative and -positive PINs and carcinomas of different Gleason Scores N Mean Std p MAN2C1 Normal PIN Gleason Gleason Gleason Gleason Gleason Total 313 AKT-P Normal PIN Gleason Gleason Gleason Gleason Gleason <0.001 Total 313 MAN2C1 Normal PIN Gleason Gleason <0.001 Gleason <0.001 Gleason Gleason Total 468 AKT-P Normal PIN <0.001 Gleason Gleason <0.001 Gleason <0.001 Gleason <0.001 Gleason <0.001 Total 468

18 Supplementary Methods Identification of candidate cdnas. Genomic DNA was purified from single surviving cell colonies. Exogenous was confirmed by PCR using a pair of primers (forward primer 5 - CAT AAC CCA CCA CAG CTA GAA CT-3 ; reverse primer 5 -ATC ACC ACA CAC AGG CAA TGG-3 ) for 35 cycles at 94 C for 45 seconds, 55 C for 45 seconds, and 72 C for 45 seconds. The genomic DNA from -positive surviving cells was subjected to PCR amplification using a pair of primers specific for plib (forward primer 5 -CGG GAT CCA GCC CTC ACT CCT TCT CTA G-3 ; reverse primer 5 -CGG AAT TCC TAC AGG TGG GGT CTT TCA TT-3 ). The PCR conditions were 95 C for 10 minutes, 5 cycles of 95 C (45 seconds)- 60 C (45 seconds)-72 C (1 minute), 35 cycles of 95 C (45 seconds)-65 C (45 seconds)-72 C (1 minute), and 72 C for 15 minutes. We were able to PCR-amplify ectopic cdnas from 22 surviving cell clones derived from Lib/ co-infection. The PCR products were subcloned into a TA-PCR cloning vector, which was used to sequence cdnas. One of candidates sequenced was a MAN2C1 fragment containing C-terminal 236 amino acid residues. Detailed analysis of C-MAN236 in plib revealed that the upstream linker, used to construct the cdna library, provided an initiation codon ATG and six additional residues (MAAAAST). Generation of anti-man2c1 antibody. Recombinant GST-C-MAN236 and 6xHis tagged C- MAN236 were isolated from E. coli BL-21. GST-C-MAN236 was injected into 2 rabbits (8-10 weeks old). Rabbits were boosted with 6xHis tagged C-MAN236. The specific antibody was purified from rabbit serum by ammonium sulfate [(NH4) 2 SO 4 ] precipitation, followed by passing through a GST-coupling CNBR activated sepharose 4B (Sigma) column. The flow-through was

19 then applied to a GST-C-MAN236-coupling sepharose 4B column to purify the anti-man2c1 antibody. The antibody specifically recognizes ectopic MAN2C1 and C-MAN236 expressed in 239T and NIH3T3 cells by western blot, IP (Supplementary Figure S1), and immunofluorescence (IF) staining (Supplementary Figure S1).