EMBO/SourceData huggingface dataset:
Figure 2Nickel-affinity-purified Mpc1/Mpc3 hetero-complex used for SEC-MALLS analysis, showing a 1:1 stoichiometry of the protomers.SEC-MALLS analysis of the hetero-complex. The light scattering trace for Mpc1/Mpc3 is shown as a black line. The masses of the protein-detergent-lipid complex (PDL, green), the detergent-lipid micelle (DL, blue) and the protein (P, red) are indicated. Protein fractions across the peak were assessed by SDS-PAGE and visualised by Coomassie Blue staining (B, inset).Nickel-affinity-purified Mpc3 protein used for SEC-MALLS analysis.SEC-MALLS analysis of Mpc3, colour designation as in (B). The protein fractions across the peak (D, inset) were assessed as in (B).
Figure 3Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in liposomes (n=8) in comparison to empty liposomes (n=6) at a ΔpH of 1.6.Kinetic analysis of pyruvate homo-exchange at ΔpH of 1.6 (n=3). The hetero-complex was assayed for initial rates of uptake in the concentration range of 25-600 μM. The calculated KΜ was 299 μΜ in this experiment and 318 and 409 μΜ in two additional biological repeats.Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in physiological pH (n=4) compared to empty liposomes (n=4).In the absence of a ΔpH, the time course of pyruvate homo-exchange was similar for the Mpc1/Mpc3 proteoliposomes and the empty liposomes (n=4).Inhibition of [14C]-pyruvate homo-exchange by UK5099 (1-100 μM), Zaprinast (1-1000 μM), lonidamine (10-10000 μM) and 7ACC2 (5-500 μM). Data points represent the mean of three technical replicates of a typical experiment. The IC50 measurements have also been independently replicated, three times for UK5099 Zaprinast and 7ACC2 (average IC50 9 ± 7 μM, 18 ± 8 μM and 27 ± 13 μM, respectively) and two times for lonidamine (average IC50, 118 ± 24 μM).[14C]-pyruvate homo-exchange inhibition by the TZDs, pioglitazone and rosiglitazone (n=6).
Figure 4Time course of pyruvate homo-exchange in proteoliposomes at a ΔpH of 1.6 was compared for Mpc3 (n=4), the Mpc1/Mpc3 hetero-complex (n=6) and empty liposomes (n=4).Time course of pyruvate homo-exchange at a ΔpH of 1.6 was compared for Mpc1 (n=4), Mpc1/Mpc3 hetero-complex (n=4) and empty liposomes (n=4).Thermostability analysis via cpm. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc3 (blue lines) (n=3).Thermostability analysis via nanoDSF. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc1 (orange lines) (n=3).
Figure 1Fluorescence was observed in the transformed N. benthamiana leaf epidermal cells, which results from complementation of the N-terminal part of the YFP fused with ATG8a (ATG8a-N-YFP) by the C-terminal part of the YFP fused with NBR1 (NBR1-C-YFP). No fluorescence was observed when ATG8a-N-YFP was coexpressed with unfused C-YFP or with mNBR1-C-YFP or when unfused N-YFP was coexpressed with NBR1-C-YFP. YFP epifluorescence images, bright-field images and overlay images of the same cells are shown.
Figure 2Five weeks-old Arabidopsis wild-type Col-0 plants were placed in the 22°C and 45°C growth chambers and total RNA was isolated from leaf samples collected at indicated times. Transcript levels were determined using real-time qRT-PCR. Error bars indicate SE (n = 3).
Figure 3(A) Four-weeks old transgenic wild-type Col-0 (WT) and atg7-2 mutant plants expressing GFP-ATG8a were treated with (45°C) or without (22°C) heat shock for 3 h and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (B) Numbers of punctate GFP-ATG8a spots representing autophagosomes per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.
Figure 4(Top panel) Four-weeks old transgenic wild-type Col-0 (WT), atg7-2 and nbr1-1 mutant plants expressing GFP-NBR1 were treated with (45°C) or without (22°C) heat shock for 3 h and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (Bottom panel) Numbers of punctate GFP-NBR1 spots per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.
Figure 5(A) Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed in 22°C and 45°C growth for 10 hours and then moved to room temperature for 3-day recovery or (B) sprayed with 20 µM methyl viologen (MV) and kept under light for two days before the picture of representative plants was taken. The experiments were repeated three times with similar results.
Figure 6(A) Electrolyte leakage.(B) Fv/Fm images. (C) Average values for the Fv/Fm images. Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed in 22°C and 45°C growth chambers. Fully expanded leaves were sampled after 10 hours in the indicated temperatures and immediately measured for electrolyte leakage and Fv/Fm. Error bars in (A) and (C) indicate SE (n = 5). According to Duncan's multiple range test (P = 0.05), means of EL or Fv/Fm do not differ significantly if they are indicated with the same letter. The experiments were repeated twice with similar results.
Figure 7(A) Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed into a growth chamber with approximately 50% humidity. The photograph of representative plants was taken 10 days after withholding watering. The experiment was repeated twice with similar results.(B) Seven days-old seedlings Col-0 WT, atg5, atg7 and nbr1 grown on solid MS medium were transferred to the same medium (control) or the same medium containing 0.16 M NaCl and photographed 5 days later. The survived seedlings were scored 5 days after the transfer and the average values and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter.
Figure 8(A) Eight weeks old wild type (WT), atg5, atg7 and nbr1 plants grown under normal growth conditions in a growth chamber. (B) Four weeks old WT, atg5, atg7 and nbr1 plants after being kept in the dark for 6 days. (C) Percentage of plants that survived the various lengths in dark as determined by resumption of growth. Each point represents the average of 10 plants. The experiments were repeated twice with similar results.
Figure 9(A) Responses of Col-0 wild type (WT), atg5, atg7 and nbr1 plants to Botrytis. Wild-type and mutant plants were inoculated by spraying with spore suspension at a density of 2.5×105 spores ml−1, and kept at high humidity. Pictures of representative plants were taken at 4 and 5 days post inoculation (dpi). (B) Quantitative real-time PCR analysis of the B. cinerea ActA (BcActA) transcript levels in infected Arabidopsis plants at 5 dpi. The experiments were repeated twice with similar results.
Figure 10(A) Diagrams of wild-type and mutant NBR1 proteins. The conserved W and I residues in the LIR motif between the two UBA domains were changed to A residues in the NBR1W661A/I664A (mNBR1) mutant protein. (B) Five weeks-old Arabidopsis Col-0 wild type (WT), nbr1 and transgenic nbr1 plants expressing the wild-type NBR1 (lines 3 and 6; see Figure S2) or the NBR1W661A/I664A (mNBR1) (lines 2 and 6; see Figure S2) mutant gene were placed in a 45°C growth chambers for 10 hours and then moved to room temperature for 3-day recovery before the picture as taken. The experiment was repeated three times with similar results.
Figure 11Leaf tissues from wild type (WT) and atg7 expressing NBR1-TAP were collected at indicated hours (h) under 45°C and prepared for soluble and insoluble proteins as described in Materials and Methods. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and probed with a peroxidase-conjugated anti-peroxidase antibody for detection of NBR1-TAP. Protein samples prepared from non-transgenic Col-0 wild-type plants were included as controls.
Figure 12(A) Accumulation of insoluble proteins. Leaf tissues from wild-type (WT), atg7 and nbr1 mutants collected at indicated hours (h) under 45°C for preparation of total, soluble and insoluble proteins as described in Materials and Methods. Total proteins in the starting homogenates and insoluble proteins in the last pellets were determined the percentages of insoluble proteins to total proteins were calculated. (B) Profiles of soluble and insoluble proteins. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and stained with Coomassie brilliant blue. Major proteins accumulated in the pellets from heat-stressed atg7 mutant plants were indicated by arrows.
Figure 13Leaf tissues from wild type (WT), atg7 and nbr1 mutants were collected at indicated hours (h) under 45°C and prepared for soluble and insoluble proteins as described in Materials and Methods. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and probed with anti-ubiquitin monoclonal antibody. The experiment was repeated three times with similar results.
Figure 1   Bovine heart mitochondria were incubated at the indicated pH in absence or presence of 200 μM DPC, treated with 1% (w/v) digitonin and F-ATP synthase was immunoprecipitated with an anti-F-ATP synthase Ab followed by 15% SDS-PAGE. β subunit and CyPD content were detected by Western blotting. The ratio between CyPD and the corresponding β subunit is reported in the lower part of the panel, referring to pH 7.4 values as 100%. Data are an average ± s.e. of 3 independent experiments.   B,C EDTA-SMP were solubilized at the indicated pH values, extracted with 1% (w/v) digitonin and subjected to BN-PAGE in order to separate dimers (Vd) and monomers (Vm), which were identified by Coomassie blue (panel B) or in-gel activity staining (panel C) and analyzed by densitometry, which is reported in the bottom part of each panel. Values report the dimer/monomer ratio, where the ratio at pH 7.4 was taken as 100%. Data are an average ± s.e. of 3 independent experiments. The p-values calculated with the Student t test are shown * p≤0.05 *** p≤0.001.D Sequence of peptides 95-113 of OSCP before (left panel) and after reaction with DPC (right panel) obtained from tryptic digests of the SDS-PAGE band corresponding to the expected molecular mass of OSCP in immunoprecipitated F-ATP synthase. Fragments of the series b and y identified in the LC-MS/MS analysis are indicated on the sequence of the peptides. H112 and H112CeT are indicated in red boldface. Ions y4-y17 show a mass shift of +72 Da in the modified peptide.
Figure 2Permeabilized HEK293T cells expressing wild type (WT) or edited (H112Q or H112Y) OSCP subunit were suspended in KSCN-based media at pH 7.4 (left traces) or pH 6.5 (right traces) and PTP opening was measured as the decrease in absorbance at 540 nm. Where indicated (arrows) 0.1 mM Ca2+ in the absence of further additions (black traces) or in the presence of 0.5 mM DPC (red traces) or 2 µM CsA (blue traces) was added. The bottom panel reports the difference between the absorbance readings before the addition of Ca2+ and at the end of the swelling phase. Data are mean ± s.e. of 5 experiments (triplicate samples) for Ca2+ alone (black bars) and Ca2+ + DPC (red bars) and of 3 experiments (duplicate samples) for Ca2+ + CsA (blue bars). Solid bars, pH 7.4, hatched bars, pH 6.5Mitochondria were prepared from cells, suspended in sucrose buffer at pH 7.4 (solid bars) or 6.5 (hatched bars) in the absence (black bars) or presence (blue bars) of 1.6 µM CsA, extracted with 1% (w/v) digitonin and immunoprecipitated with an F-ATP synthase antibody. The immunoprecipitates were subjected to 12% SDS-PAGE followed by Western blotting for CyPD and α subunit of F-ATP synthase. Each immunodetected band was analyzed by densitometry, the ratio between CyPD and α subunit was measured and expressed relative to the ratio obtained in absence of CsA at pH 7.4, which was taken as 100%. Data are average ± s.e. of 6 independent experiments.HEK293T cells were incubated in a balanced salt solution containing 2-deoxyglucose at pH 7.4 (solid bars) or 6.5 (hatched bars) and anoxia induced by incubation in a BD GasPak EZ Anaerobe Gas-generating Pouch System (BD Biosciences). Cell death was measured based on LDH release (average ± s.e. from 8 independent experiments). In all panels the p values calculated with the Student t test are shown (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).
Figure 3Representative current traces recorded at V(pipette) +10 mV from mitoplasts derived from wild type (WT) or H112Q OSCP mutation-harboring cells.Channel activity (top) and corresponding amplitude histograms (bottom) recorded in wild type mitochondria before (WT pH 7.3) and after acidification to pH 6.5 (WT pH 6.5). Amplitude histograms were obtained from 120 second-long current traces for the corresponding pH values and the panels are from the same experiment.Recordings as in panel B from mitoplasts of cells harboring the OSCP H112Q replacement. Left panel, pH 7.3; middle panel, 440 seconds after acidification to pH 6,5; right panel, 30 seconds after the addition of 5 mM Ba2+ at pH 6.5. Amplitude histograms were obtained from 100 second-long segments and the panels are from the same experiment. Note that both in panels B and C well-visible subconductance states can be detected in the amplitude histograms (peaks between open levels o2 and o3 on left panels). Gaussian fitting has been obtained using the Origin Program set. In all panels c, closed; o1, o2, o3 open states with discrete subconductance.
Figure 1(A) Relative expression of the BBSome subunits in the indicated organs and T cells measured by qPCR. CT values of the BBS genes were normalized to the geometric mean of the CT values of reference genes Gapdh, Tubb2a, and Eef1a1. The expression levels are normalized to those of brain (=1). Mean+SD. Three biological replicates (the result of each biological replicate is a median of three technical replicates).(B) Immunoblot analysis of BBS4 expression in T and B lymphocytes isolated from the lymph nodes and spleen of C57Bl/6 mouse. β-actin staining served as a loading control.(D) Immunoblot analysis of BBS4 expression in testes and thymi lysates of Bbs4+/+ (WT), Bbs4GT/+, and Bbs4GT/GT mice. β-actin staining serves as a loading control. A representative experiment out of three biological replicates in total is shown.(E) Immunoblot analysis of BBS4 expression in the brain lysates of Bbs4+/+, Bbs4GT/GT, and Bbs4KO/KO mice. β-actin staining served as a loading control. A representative experiment out of two biological replicates is shown.(F) Hematoxylin and eosin staining of sections of seminiferous tubules from ~4 weeks old Bbs4+/+ (n=2 mice) and Bbs4GT/GT (n=4) males. Scale bar, 100 µm. Representative images are shown.(G) Genotypic ratio of Bbs4+/+, Bbs4GT/+ or Bbs4KO/+ (Het), and Bbs4GT/GT (GT) or Bbs4KO/KO (KO) offspring at weaning from mating of Bbs4GT/+ (n=145 pups) or Bbs4+/KO (n=168 pups) parents. Binomial test was used for statistical comparison of the observed distribution to the expected Mendelian ratio.(H) Bbs4+/+ and Bbs4GT/GT female littermates at 20 weeks of age. Representative litter out of seven in total. (I) Bbs4+/+ and Bbs4KO/KO female littermates at 20 weeks of age. Representative litter out of five in total. (J) Growth curves of Bbs4-deficient mice, mean ± SD is shown. Females: Bbs4+/+ (n=12 mice), Bbs4GT/GT (n=12), Bbs4KO/KO (n=6). Males: Bbs4+/+ (n=6 mice), Bbs4GT/GT (n=7), Bbs4KO/KO (n=5).(K) Leptin concentration in blood plasma taken from young adult (7-8 weeks) or mid-age (14-20 weeks) mice. Young adult mice: Bbs4+/+ (n=4 mice), Bbs4KO/KO (n=3), analyzed in two independent experiments. Mean+SEM. Mid-age adult mice: Bbs4+/+ (n=7 mice), Bbs4KO/KO (n=5), Bbs4GT/GT (n=3), analyzed in 4 independent experiments. Kruskal-Wallis tests was used for the statistical analysis. Mean+SEM.
Figure 3(A,B) Splenocytes isolated from Bbs4+/+ (n=4 mice) and Bbs4GT/GT (n=3) B-cell transgenic B1-8 littermates were activated with 4-hydroxy-3-nitrophenylacetic acid succinimide ester-loaded T2-Kb cells in three independent experiments. Percentage of activated B cells (gated as CD69+ B220+ IgLλ+ viable cells) in the samples without T2-Kb (negative control), and in the samples with T2-Kb added in ratios 1:10 or 1:3 was determined by flow cytometry. (A) Representative mice are shown. (B) Mean±SEM. Statistical significance was calculated using two-tailed Mann-Whitney test.(C) B cells from 18-20 weeks old Bbs4KO/KO (red circle, n=3 mice), and control mice (Bbs4+/+, dark grey square, n=4, or Bbs4+/KO, light grey square, n=2) were activated with F(ab')2-goat anti-mouse IgM (Mu chain). Percentage of activated B cells (gated as CD69+ CD19+ viable cells) was determined by flow cytometry. Representative experiment out of three biological replicates is shown. Mean±SEM. Statistical significance was calculated using two-tailed Mann-Whitney test.(D) CFSE-loaded T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were incubated with DDAO-labeled WT splenocytes loaded with OVA peptide or with the indicated altered peptide ligands for 20 min. Percentage of T cells conjugated with the APCs was determined by flow cytometry. Four biological replicates were performed. Mean+SEM. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all peptides.500 or 1000 T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were adoptively transferred into RIP.OVA mice followed by infection with Listeria monocytogenes expressing ovalbumin (LM-OVA) on the next day. (E) Glucose level in the urine of mice was monitored on a daily basis. The mouse was considered diabetic when it had urine glucose level ≥ 1000 mg/dL for two consecutive days. Statistical significance was calculated by Log-rank (Mantel-Cox) test.500 or 1000 T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were adoptively transferred into RIP.OVA mice followed by infection with Listeria monocytogenes expressing ovalbumin (LM-OVA) on the next day. (F) Glucose concentration in blood on day 7 post-infection. 500 OT-I ctrl (n=13 mice), 500 OT-I Bbs4 cKO (n=13), 1000 OT-I ctrl (n=9), 1000 OT-I Bbs4 cKO (n=11), analyzed in four independent experiments, mean is shown. Statistical significance was calculated using two-tailed Mann-Whitney test.
Figure 4Cells isolated from bone marrow of Bbs4FL/FL (control) and Vav-iCre Bbs4FL/FL (cKO) mice were analyzed by flow cytometry. (A) Percentage of B220low in bone marrow. Representative experiment out of four in total is shown. Bbs4FL/FL (n=6 mice), Vav-iCre Bbs4FL/FL (n=9). Representative experiment out of six in total is shown. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all tests. Median is shown.Cells isolated from bone marrow of Bbs4FL/FL (control) and Vav-iCre Bbs4FL/FL (cKO) mice were analyzed by flow cytometry. (B) Percentage of B-cell precursors (B220+, IgM- IgD-) in the bone marrow. Representative experiment out of four in total is shown. Bbs4FL/FL (n=6 mice), Vav-iCre Bbs4FL/FL (n=9). Representative experiment out of six in total is shown. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all tests. Median is shown.Cells isolated from , lymph nodes and spleen of Bbs4FL/FL (control) and Vav-iCre Bbs4FL/FL (cKO) mice were analyzed by flow cytometry. (C) Percentage of CD19+ B cells in spleens and lymph nodes of Vav-iCre Bbs4FL/FL (n=12 mice) and Bbs4FL/FL (n=9) mice, six independent experiments. Representative experiment out of six in total is shown. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all tests. Median is shown.Cells isolated from spleen of Bbs4FL/FL (control) and Vav-iCre Bbs4FL/FL (cKO) mice were analyzed by flow cytometry. D) Percentage of late mature B cells (CD19+, IgM-, IgD+) in spleens of Vav-iCre Bbs4FL/FL (n=12 mice), and Bbs4FL/FL mice (n=9). Representative experiment out of six in total is shown. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all tests. Median is shown.(E) Expression of Cxcl12 and Il-7 in the bone marrow from Bbs4+/+ Bbs18+/+ (WT) mice (black circles, n=7 mice), Bbs4+/KO (Het, green circle, n=3), Bbs18+/KO (Het, blue circle, n=3), Bbs4KO/KO (green square, n=5), and Bbs18KO/KO (blue square, n=2) was analyzed by RT-qPCR in five independent experiments. The expression was normalized to Gapdh. The statistical significance of the difference between the BBSome-deficient mice (Bbs4KO/KO and Bbs18KO/KO) and controls were calculated using two-tailed Mann-Whitney test. Median is shown.(F) MEF cell lines were derived from a single Bbs4+/+ (WT) or a single Bbs4KO/KO embryos. The expression of Cxcl12 in these cell lines was analyzed by RT-qPCR. The expression was normalized to Gapdh and to Bbs4+/+ MEFs (=1) for each experiment. Three biological replicates. One sample t test was used for the statistical analysis.(G) Expression of Cxcl12 and canonical-WNT responsive gene Alpl in untreated (NT) ST2 cells, or ST2 cells treated with WNT3A or WNT5A was analyzed by RT-qPCR. The expression was normalized to Gapdh and untreated ST2 sample (=1) for each experiment. Four biological replicates were performed. One sample t test was used for the statistical analysis.
figf1(a) MCF7 cells stably expressing Myc-TEV-Flag-Beclin 1 (MEF-Beclin 1) and parental controls were lysed and subjected to MEF-tag-based purification. Proteins bound to MEF-Beclin 1 were isolated and detected by SDS-PAGE and silver staining. Seven bands specific for MEF-Beclin 1 are numbered. (b) Six bands were identified by nanoflow LC-MS/MS and MASCOT software.(c) MCF7 cells were lysed and subjected to immunoprecipitation using the indicated antibodies.(d) Differential subcellular fractionation. Post-nuclear supernatant of A549 cell lysates were spun at 20,000g for 10 min and the supernatant was further spun at 100,000g for 60 min. Equivalent quantities of each supernatant and pellets from the same number of cells were subjected to western blotting with the indicated antibodies, including anti-Sec61β (ER), cathepsin D (lysosome), EEA1 (early endosome), transferrin receptor (plasma membrane and recycling endosome) and GAPDH (cytosol).(e) Gel filtration analysis. The supernatant fraction of A549 cell (or Atg14L knockdown) lysates were subjected to a Superose 6 column and each fraction was immunoblotted with the indicated antibody (see Supplementary Information, Fig. S2e). Relative amounts of each fraction determined by densitometry were plotted. Vo, void fraction. The elution pattern of each protein was reproduced in several experiments. Full scans of the gel and blots in a, c and d are available in Supplementary Information, Fig. S6.
figf2(a) A549 cells were infected with adenovirus harbouring shRNA targeting Atg14L, Rubicon, UVRAG or a control (luciferase). Cell lysates were subjected to immunoblotting with the antibodies indicated. The asterisk in the Rubicon blot represents a non-specific cross-reacting band.(b) Quantification of GFP-positive dots per cell. Images were taken from cells shown in c and the number of GFP-positive dots was determined. At least 140 cells were counted per experiment and the values are mean ± s.d. of three independent experiments. (c) A monoclonal A549 cell line stably expressing GFP-LC3 was infected with adenovirus harbouring shRNA targeting Atg14L, Rubicon or control (luciferase). The cells were cultured under nutrient-rich or starvation conditions. GFP was observed by confocal laser microscopy. Scale bar, 10 μm. The full scan of the blot in a is available in Supplementary Information, Fig. S6.