Supplementary Materials The proteasomal deubiquitinating enzyme PSMD14 regulates macroautophagy by controlling Golgi-to-ER retrograde transport Bustamante HA., et al. Figure S1. siRNA sequences directed against human PSMD14 used for Validation Stage. Figure S2. Primer pairs sequences used for RT-qPCR. Figure S3. The PSMD14 DUB inhibitor CZM increases the Golgi apparatus area. Immunofluorescence microscopy analysis of the Golgi area in parental H4 cells treated for 4 h either with the vehicle (DMSO; Control) or CZM. The Golgi marker GM130 was used to determine the region of interest in each condition. Statistical significance was determined by Student's t-test. Bars represent the mean ± SEM (n =43 cells). ***P <0.001. Figure S4. CZM causes the accumulation of KDELR1-GFP at the Golgi apparatus. HeLa cells expressing KDELR1-GFP were either left untreated or treated with CZM for 30, 60 or 90 min. Cells were fixed and representative confocal images were acquired. Figure S5. Effect of CZM on proteasome activity. Parental H4 cells were treated either with the vehicle (DMSO; Control), CZM or MG132, for 90 min. Protein extracts were used to measure in vitro the Chymotrypsin-like peptidase activity of the proteasome. The enzymatic activity was quantified according to the cleavage of the fluorogenic substrate Suc-LLVY-AMC to AMC, and normalized to that of control cells. The statistical significance was determined by One-Way ANOVA, followed by Tukey’s test. Bars represent the mean ± SD of biological replicates (n=3). **P <0.01; n.s., not significant. Figure S6. Effect of CZM and MG132 on basal macroautophagy. (A) Immunofluorescence microscopy analysis of the subcellular localization of LC3 in parental H4 cells treated with either with the vehicle (DMSO; Control), CZM for 4 h or MG132 for 6 h. Cells were fixed, permeabilized and stained with a rabbit polyclonal antibody to LC3B followed by Alexa-594-conjugated donkey anti-Rabbit IgG. Scale bar 10 μm. (B) Parental H4 cells were treated as in (A) and the protein extracts were analyzed by western blot with a polyclonal antibody to LC3B. Densitometric quantification of the protein levels of LC3B were depicted as the Ratio LC3B-II/LC3B-I. The statistical significance was determined by One-Way ANOVA, followed by Tukey’s test. Bars represent the mean ± SD of biological replicates (n=3). ***P <0.001; n.s., not significant. Figure S7. Distribution of RAB1A upon CZM treatment. Immunofluorescence analysis of endogenous RAB1A in H4 parental cells treated either with vehicle (DMSO; Control) (A-C) or CZM for 4 h (D-F). Cells were fixed, permeabilized, and double stained with a rabbit monoclonal antibody to RAB1A (clone D3X9S) (A and D) and a mouse monoclonal antibody to GM130 (clone35/GM130) (B and E), followed by Alexa-594-conjugated donkey anti-Rabbit IgG and Alexa-488- conjugated donkey anti-Mouse IgG. Merging of the images generated the third picture (C and F). Scale bar, 10 compared to control cells. The statistical significance was determined by Student's t-test. Bars represent the mean ± SEM of the fluorescent signal per cell area (n=173 cells). *P< 0.05. Figure S8. ATG9A is distributed in the swollen Golgi apparatus upon CZM treatment. Immunofluorescence analysis of endogenous ATG9A in H4 parental cells treated either with the vehicle (DMSO; Control) (A-C) or CZM for 4 h (D-F). Cells were fixed, permeabilized, and double stained with a rabbit monoclonal antibody to ATG9A (clone EPR2450(2)) (A and D) and a mouse monoclonal antibody to GM130 (clone35/GM130) (B and E), followed by Alexa-594-conjugated donkey anti-Rabbit IgG and Alexa-488- conjugated donkey anti-Mouse IgG. Merging of the images generated the third picture (C and F). Scale bar, 10 compared to control cells. The statistical significance was determined by Student's t-test. Bars represent the mean ± SEM of the fluorescent signal per cell area (n=93 cells). **P <0.01. Sequence siRNA #1 (5´-GAACAAGUCUAUAUCUCUU-3´) siRNA #2 (5´-GGCAUUAAUUCAUGGACUA-3´) siRNA #3 (5´-AGAGUUGGAUGGAAGGUUU-3´) siRNA #4 (5´-GAUGGUUGUUGGUUGGUAU-3´) Figure S1 Target S Sequence hTBP1 f(5´-TAGTCCAATGATGCCTTACG-3´) r(5´-TGGTCAGAGTTTGAGAATGG-3´) hPSMD14 f(5´-ACCTTAAGAGTTGTAGTTACTGACC-3´) r(5´-TTTAACAGTGCCAGGGAAGAG-3´) hAPP f(5´-CCTAAAGCATTTCGAGCATG-3´) r(5´-GTTTCCGTAACTGATCCTTG-3´) Figure S2 1.5 2.53 *** ) 2 m 1.0 µ 0.5 Golgi Area ( 0.0 Control µM CZM 10 Figure S3 15.24 150 n.s.** 125 100 75 50 normalized (%) 25 Chymotrypsin-like peptidase activity 0 Control 10 µM CZM 10 µM MG132 90 min Figure S5 A Control 10 µM CZM 10 µM MG132 LC3B B 4.03 5 *** B-I 3 4 C L / I I 3 B- 3 n.s. C 2 L o i t a 1 R 0 Control 10 µM CZM 10 µM MG132 Figure S6 RAB1A GM130 Merge A B C Control D E F M CZM µ 10 G 60 1.08 * ) 40 otal (% T 20 Fraction RAB1A Ratio GM130/ 0 Control 10 µM CZM Figure S7 ATG9A GM130 Merge A B C Control D E F M CZM µ 10 G 1.19 50 ** ) 40 otal (% 30 T 20 Fraction ATG9A Ratio GM130/ 10 0 Control 10 µM CZM Figure S8 Target List High-Content siRNA Screening “Ubiquitinome” Gene Symbol NCBI Reference Sequence PCGF2 NM_007144 PSMD14 NM_005805 RNF34 NM_025126 PSMD7 NM_002811 FBXO4 NM_012176 KCTD3 NM_016121 RNF141 NM_016422 UBE2E2 NM_152653 BRPF3 NM_015695 NACA NM_005594 CAND2 XM_944849 EPN1 NM_013333 UBXD4 NM_181713 SIAH2 NM_005067 TRIM23 NM_033228 UBA52 NM_003333 FAU NM_001997 EIF3S5 NM_003754 RSPRY1 NM_133368 MARK4 NM_031417 TRIM42 NM_152616 UBC NM_021009 FBXW10 NM_031456 UBE2E3 NM_182678 MDM2 NM_006879 RBM6 NM_005777 TOM1 NM_005488 ZBTB12 NM_181842 SOCS3 NM_003955 PHF12 NM_020889 FBXO44 NM_183413 DNAJB2 NM_001039550 RNF130 NM_018434 BIRC7 NM_022161 CREBL1 NM_004381 KBTBD2 NM_015483 RNF39 NM_170770 RYBP NM_012234 HRC NM_002152 NEURL NM_004210 VPRBP NM_014703 HECTD3 NM_024602 FAF1 NM_131917 ZNF592 NM_014630 PSMD4 NM_002810 FLJ25076 XM_940609 RNF187 XM_047499 TNK2 NM_001010938 SHFM3 NM_022039 PRPF8 NM_006445 EIF2AK4 NM_001013703 TRIM31 NM_052816 SUMO2 NM_001005849 UCHL3 NM_006002 FLJ20280 NM_017741 UBE2D4 NM_015983 TCEB1 NM_005648 MDM4 NM_002393 RAMP NM_016448 TRIM63 NM_032588 PHF23 NM_024297 UBE2J1 NM_016021 KUA-UEV NM_003349 DDI2 NM_032341 NICE-4 NM_014847 RNF146 NM_030963 UBB NM_018955 CHD5 NM_015557 ZBTB16 NM_001018011 CISH NM_145071 OTUD5 NM_017602 SENP8 NM_145204 ASPSCR1 NM_024083 FLJ43374 NM_198582 LOC652557 XM_942059 HIP2 NM_005339 SF3B3 NM_012426 UBE2V1 NM_001032288 LGALS3BP NM_005567 ZNF216 NM_006007 TRIM56 NM_030961 KIAA1536 NM_020898 SAE1 NM_005500 WDR11 NM_018117 KLHL1 NM_020866 SH3RF2 NM_152550 MNAT1 NM_002431 RAI17 NM_020338 KCTD17 NM_024681 PCF11 NM_015885 USP6 NM_004505 USP42 NM_032172 KCMF1 NM_020122 PHF21A NM_016621 VPS11 NM_021729 TRIM74 NM_198853 KRTAP5-9 NM_005553 KLHL7 NM_001031710 USP26 NM_031907 RNF32 NM_030936 USP16 NM_001001992 TRIM60 NM_152620 USP8 NM_005154 UBE2D2 NM_003339 KCTD12 NM_138444 UFC1 NM_016406 USP10 NM_005153 TA-KRP NM_032505 RPS27A NM_002954 UBE2J2 NM_194457 UBE2M NM_003969 RANBP9 NM_005493 LOC51035 NM_015853 BMI1 NM_005180 RKHD1 NM_203304 PC326 NM_001017977 HACE1 NM_020771 EIF3S3 NM_003756 FANCD2 NM_001018115 CDC34 NM_004359 YAF2 NM_005748 UBXD3 NM_152376 SIP NM_001007214 KCND1 NM_004979 RNF44 NM_014901 PEX10 NM_002617 ZNF151 NM_003443 USP52 NM_014871 ZRANB1 NM_017580 PLAA NM_001031689 TCEB2 NM_207013 BMI1 NM_005180 TCEB3 NM_003198 PDZRN4 NM_013377 RNF186 NM_019062 FLJ25555 NM_152345 LOC441061 XM_941081 BIRC2 NM_001166 BTBD14A NM_144653 STAMBPL1 NM_020799 LOC646862 XM_929820 MIB2 NM_080875 LMO7 NM_005358 PIAS1 NM_016166 FLJ10719 NM_018193 TRIM7 NM_203294 AB026190 NM_014458 LL0XNC01- UBE2NL NM_001012989 237H1.1 NM_001031834 FBXO5 NM_012177 USP9Y NM_004654 HECTD1 NM_015382 TRIM33 NM_033020 DKFZP547C195 NM_207343 LOC646862 XM_929820 TRIM32 NM_012210 TSG101 NM_006292 HECW1 NM_015052 TRIM6-TRIM34 NM_001003819 AIRE NM_000659 FBXO2 NM_012168 KCNC1 NM_004976 WDR26 NM_025160 UNK NM_001080419 SKP1A NM_170679 TRIM8 NM_030912 CUL5 NM_003478 SBBI54 NM_138334 C13ORF22 NM_005800 C20ORF18 NM_031227 TRAF7 NM_206835 RNF167 NM_015528 RBM10 NM_152856 KCNA1 NM_000217 FBXL20 NM_032875 OTUB2 NM_023112 ASB18 NM_212556 SPSB2 NM_032641 LONRF2 NM_198461 GGA3 NM_014001 USP45 XM_371838 SPSB3 NM_080861 PHF5A NM_032758 SHARPIN NM_030974 UBE4B NM_006048 HGS NM_004712 KLHDC5 NM_020782 TIP120A NM_018448 KCNC3 NM_004977 UEVLD NM_018314 RNF121 NM_194452 ATG10 NM_031482 PARK7 NM_007262 CHD4 NM_001273 UBE2N NM_003348 FBXO34 NM_017943 CUEDC1 NM_017949 UBQLN2 NM_013444 BARD1 NM_000465 ZNF185 NM_007150 TAB3 NM_198312 FBXO17 NM_148169 FBXL13 NM_145032 ANKFY1 NM_020740 MID2 NM_052817 USP43 XM_371015 FBXW5 NM_178226 PARC NM_015089 PRPF19 NM_014502 HR NM_018411 WDR40A NM_015397 ZNF297B NM_014007 RAB40C NM_021168 UBL5 NM_024292 FBXL12 NM_017703 PHF19 NM_001009936 BTBD9 NM_152733 KCTD5 NM_018992 PHF6 NM_032335 MARCH4 NM_020814 LRRC29 NM_001004055 ASB14 NM_130387 USP54 NM_152586 FLJ34154 NM_173813 HSHIN6 NM_207320 HUWE1 NM_031407 MYO6 NM_004999 ZBTB1 NM_014950 FBXL2 NM_012157 FBXL22 NM_203373 FBXO39 NM_153230 RNF207 NM_207396 UBE2H NM_182697 RFWD3 NM_018124 ZNF131 NM_003432 UBE2L6 NM_004223 LATS1 NM_004690 PHF16 NM_014735 EEA1 NM_003566 MARCH8 NM_001002266 ITCH NM_031483 FBXL14 NM_152441 BAZ1B NM_032408 BACH2 NM_021813 RFP NM_030950 DDB1 NM_001923 TOM1L1 NM_005486 TRIM2 NM_015271 BIRC3 NM_182962 RNF38 NM_194331 MSL2L1 NM_018133 DTX3 NM_178502 BRE NM_199191 SH3MD4 XM_293090 WDR71 NM_025155 NSMCE1 NM_145080 NOSIP NM_015953 PML NM_033247 RUFY1 NM_025158 C9ORF60 NM_006336 COPS4 NM_016129 SENP1 NM_014554 LOC392188 XM_373238 SIAH1 NM_003031 TRIM29 NM_058193 CCL20 NM_004591 VPS41 NM_080631 COPS7A NM_016319 ATG16L1 NM_198890 FEM1C NM_020177 MGC3123 NM_024107 ATG7 NM_006395 FBXO11 NM_012167