Supplementary Figure S A) The blots shown in Figure B were qualified by using Gel-Pro analyzer software (Rockville, MD, USA). The ratio of LC3II/LC3I to actin was then calculated. The data are represented as mean±sd from three independent experiments. B) The blots in Figure E were qualified and the ratio of LC3II/LC3I to actin was then calculated. The data are represented as mean±sd from three independent experiments. C) HCT cells with stable expression of GFP-LC3 were treated with Embelin as indicated. GFP-LC3 puncta accumulation was observed by a microscope. Quantification of LC3 punctate cells was shown on the right. The data are represented as means±sd of three independent experiments. D-G) The blots in Figures G, H, I and J were qualified and the ratio of LC3II/LC3I to actin was then calculated and shown in D, E, F and G, respectively. The data are represented as mean±sd from three independent experiments. Supplementary Figure S A) The blots in Figure A were qualified and the ratio of LC3II/LC3I to actin was then calculated. The data are represented as mean±sd from three independent experiments. B) MCF7, MCFA, HepG and LO cells were individually treated with specific or control sirnas. 8 h after transfection, cell lysates were analyzed by Western blotting with the indicated antibodies. The data are representative of two biological replicates. C) HCT cells were infected with lentiviruses expressing control shrna or 3 different sets of specific shrnas. 8 h after infection, cell lysates were subjected to Western blot analysis with the indicated antibodies. The data are representative of three biological replicates. D-F) The blots in Figures B, D and E were qualified and the ratio of LC3II/LC3I to actin was then calculated and shown in D, E and F, respectively. The data are represented as mean±sd from three independent experiments.
G) HCT KO cells were transfected with Flag--WT (wild type) or the indicated mutants. h after transfection, cell lysates were analyzed by Western blotting with the indicated antibodies. The data are representative of two biological replicates. H) The blots in Figure F were qualified and the ratio of LC3II/LC3I to actin was then calculated. The data are represented as mean±sd from three independent experiments. Supplementary Figure S3 A) Lysates from HCT cells were immunoprecipitated with anti- antibody or an isotope-matched control antibody. Immunoprecipitates and cell lysates were analyzed by Western blotting. The data are representative of two biological replicates. B) The blots in Figure 3C were qualified and the ratio of to actin was then calculated. The data are represented as mean±sd from three independent experiments. C and D) HCT WT and KO cells were treated with 5μg ml - cycloheximide for the indicated periods of time. The half-life of was measured by Western blot analysis (C). We should mention that amounts of cell lysates were adjusted to achieve similar expression levels of at time. The blots were qualified and the ratio of to actin was then calculated. The data are represented as mean±sd from three independent experiments (D). E) HCT KO cells were transfected with and constructs as indicated. h after transfection, cells were treated with 5μg ml - cycloheximide for the indicated periods of time followed by western blot analysis with the indicated antibodies. The data are representative of three biological replicates. Supplementary Figure S A) Lysates from HEK 93T cells expressing HA- alone or HA- plus Flag- were immunoprecipitated with anti-flag antibody. Immunoprecipitates were then analyzed by Western blotting. The data are representative of two biological
replicates. B) HCT KO cells were transfected with HA- alone or HA- plus Flag- D8A/W3A. h after transfection, cells were treated with CHX for the indicated periods of time. Cell lysates were then analyzed by Western blotting. The blots were qualified and the ratio of HA- to was then calculated. The data are represented as mean±sd from three independent experiments C) Lysates from HEK 93T cells expressing HA- alone or HA- plus Flag- H7A were immunoprecipitated with anti-flag antibody followed by Western blot analysis. The data are representative of two biological replicates. Supplementary Figure S5 A) Purified Flag- CA was incubated with increasing amounts of His- recombinant protein in a total of µl in vitro ubiquitination reaction buffer at 37 C for h. The reaction mixtures were analyzed by Western blotting with anti- antibody. The data are representative of three biological replicates. B) HCT cells expressing specific or control sirnas were treated with µm MG-3 for h. Cell lysates were denatured before immunoprecipitation with anti- and anti- antibodies. Immunoprecipitates were analyzed by Western blotting with anti-ubiquitin antibody. The data are representative of three biological replicates. C) -/- -/- MEF cells were transfected with the indicated plasmids. h after transfection, cell lysates were immunoprecipitated with anti-ha antibody. The input and immunoprecipitates were analyzed by Western blotting. The data are representative of two biological replicates. Supplementary Figure S A) HCT WT and KO cells were treated with EBSS for the indicated periods of time. Cell lysates were subjected to Western blot analysis with the indicated antibodies. The data are representative of three biological replicates. B-D) The blots in Figures B, C and F were qualified and the ratio of LC3II/LC3I 3
to actin was then calculated and shown in B, C and D, respectively. The data are represented as mean±sd from three independent experiments. Supplementary Figure S7 Expression levels of,, and LC3 conversion in the tumors excised from indicated different groups of mice (Figure 5A) were evaluated by Western blot analysis. Left and Right indicate tumors grown in the left and right flanks of mice, respectively. The data are representative of two biological replicates. Supplementary Figure S8 Histological sections of the indicated xenograft tumors were stained with the indicated antibodies. Supplementary Figure S9 A and B) The blots in Figures 5G and 5H were qualified and the ratio of LC3II/LC3I to actin was then calculated and shown in A and B, respectively. The data are represented as mean±sd from three independent experiments. Supplementary Figure S The tumor tissues (T) from human Breast, Lung and Stomach and their adjacent normal tissues (N) were homogenized for protein extraction. Protein extracts were analyzed by Western blotting with the indicated antibodies. As shown in the table, 5 of 3 esophagus cancers (3%), 3 of 5 intestine cancers (%), of 3 breast cancers (33%), of 8 lung cancers (5%), and 3 of stomach cancers (3%) exhibited the great autophagy inhibition along with the elevated expression of p-,, and and the decreased expression of compared to those from their matched adjacent normal tissues. The data are representative of three biological replicates. Supplementary Figure S Histological sections of the tumor tissues from human Breast, Colon, Esophagus,
Stomach, and Lung and their adjacent normal tissues were stained with the indicated antibodies. 5
A C LC3II / LC3I / 8 LC3II / LC3I / HCT GFP-hLC3 + Embelin ng/ml 5ng/ml 3ng/ml ng/ml B 5 3 3 5 7 8 Figure S h 3ng/ml h h h h Cells with GFP-LC3 punctation (%) 3 (ng/ml) (h) h 3ng/ml 5 3 D E LC3II / LC3I / 5 3 3 LC3II / LC3I / 8 3 F G. LC3II / LC3I / 5 5 3 LC3II / LC3I /..8...
A LC3II / LC3I / C HCT Sh-Ctrl + Sh- + Sh- + Sh-3 + E F H 8 LC3II / LC3I / LC3II / LC3I / LC3II / LC3I / 3 8 5 5 8 3 LC3 I LC3 II 3 3 3 B MCF7 MCFA HepG LO Si-Ctrl + + + + Si- + + + + D LC3II / LC3I / 8 3 5 7 8 3 5 7 8 G 5 Flag R75H KO R73H G79E K38R WT 3 5 LC3 I LC3 II PARP p5/7 Figure S LC3 I LC3 II Procaspase3
A Input C HCT WT KO CHX 3 9 3 9 (min) IP: anti-igg IP: anti- 3 5 7 8 B D / /...8....5.5 3 5 7 8 3 5 7 8 Figure S3 E KO + HA- WT Flag Flag- WT Flag- H7A CHX 3 9 3 9 3 9 (min) HA- Flag- 5 3 5 7 8 9 GFP KO + HA- CA Flag Flag- WT Flag- H7A CHX 3 9 3 9 3 9 (min) HA- Flag- 5 3 5 7 8 9 GFP
A HA- Flag- D8A/W3A Input + + + + + + HA- Flag- Ip: anti-flag Figure S B HCT KO HA- + + + + + + + + Flag- D8A/W3A + + + + CHX 3 9 3 9 (min) HA- Flag- /..8... 3 5 7 8 C HA- Flag- H7A Input + + + + + + HA- Flag- Ip: anti-flag
Figure S5 A B HCT E+E+Ub His- Flag- CA + + + + + + 3+ + + + + Si-Ctrl Si- C HA- GFP- Flag- -(Ub)n His- Input IP: anti-ha + + + + + + + + + + + + HA- GFP- +MG3 Input IP IP: anti- IP: anti- 5 7 5 7 5 -(Ub)n -(Ub)n Flag-
Figure S A HCT WT KO EBSS 8 8 (h) LC3 I LC3 II C LC3II / LC3I / 8 3 5 7 8 B LC3II / LC3I / LC3II / LC3I / 8..8... 3 3 3 D
Figure S7 Left: WT Right: KO LC3I LC3II 3 Left: KO-Ctrl Right: KO- SA 3 5 7 8 9 3 5 7 8 9 LC3I LC3II Left: KO-Ctrl Right: KO- Left: KO-Ctrl Right: KO- SD LC3I LC3II 3 5 7 8 9 3 5 7 8 9 LC3I LC3II
Figure S8 WT KO KO- KO- SA KO- SD LC3
Figure S9 A LC3II / LC3I /..8... 3 5 7 8 9 3 5 B LC3II / LC3I /..8...
Figure S Breast Lung Stomach N T N T N T N T N T N3 T3 p- LC3I LC3II 3 p- LC3I LC3II 3 5 p- LC3I LC3II Esophagus Intestine Breast Lung Stomach 3% ( 5 / ) % ( 3 / 5 ) 33% ( / 3 ) 5% ( / 8 ) 3% ( 3 / )
Cancer Lung Normal Cancer Normal Stomach Cancer Normal Esophagus Cancer Colon Normal Cancer Normal Breast LC3 Figure S