EMBO/SourceData huggingface dataset:
Figure 4. ACE2 expression is upregulated in the lungs from SARS-CoV MA15 infected C57Bl/6 mouse.Intranasal instillation of 102, 103 PFU of SARS-CoV MA15 in 50 µl of PBS or mock-infected with PBS alone, lungs were harvested 24 hrs later and total RNA was isolated and subjected to microarray analysis. ACE2 expression data was retrieved from Gene Expression Omnibus (GEO) microarray database (NCBI Accession: PRJNA149057; GEO: GSE33266).
Figure 5. ACE2 expression is upregulated in primary human airway epithelial cells in response to wild type MERS-CoV (icMERS-CoV EMC2012).Human airway epithelial cells were infected with a multiplicity of infection of 5 PFU per cell. Infected samples were collected as indicated above. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA391962; GEO: GSE100504).
Figure 6. ACE2 expression is upregulated in human airway epithelial cells in response to rhinovirus infection.Primary epithelial cells from 4 donors were exposed to rhinovirus or medium only (used as control). 24 hrs post-infection, gene expression was assessed using Affymetrix U133 plus 2.0 human GeneChips. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA137767; GEO: GSE27973).
Figure 7. ACE2 expression is upregulated in human bronchial epithelial cells (HBECs) after IFNβ and H1N1 influenza stimulation.HBECs were treated with IFNβ (1000U/ml) or wild type H1N1 influenza (A/PR/8/34). Control samples were incubated with fresh medium under the same conditions. HBECs were harvested at indicated time points (0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 18 hours post-treatment). Total RNA was extracted and analyzed using the Affymetrix High Throughput Array. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA121751; GEO: GSE19392).
Figure 8. ACE2 expression is upregulated in human primary keratinocytes exposed to IFNα and IFNγ.Primary keratinocytes from 3 donors were either untreated (control) or exposed to cytokines IFNα, IFNγ. 24 hrs after treatment, total RNA was extracted for subsequent microarray analysis. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA153023; GEO: GSE36287).
Figure 1(A) NIH-3T3 cells were transduced with retroviral vectors expressing M45 or GFP. Two days after transduction cells were stimulated with the TLR4 agonist LPS (10 µg/ml), the TLR2 agonist LTA-SA (10 µg/ml), or IL-1β (20 ng/ml). IκBα levels were determined by immunoblotting.(B) NIH-3T3 cells were transduced with an M45-expressing or an empty retroviral vector. 30 min after stimulation with IL-1β (10 ng/ml) cells were fixed, and subcellular localization of the NF-κB p65 subunit was analyzed by immunofluorescence.(C) NIH-3T3 cells expressing an NF-κB-dependent secreted alkaline phosphatase (SEAP) reporter were transduced with retroviral vectors expressing M45 or GFP and stimulated 78 h later with the TLR2 agonist Pam3CSK4 (Pam.,1 µg/ml), the TLR4 agonist LPS (0.1 µg/ml), or IL-1β (5 ng/ml). SEAP activity in the supernatant was quantified 15 h after stimulation and is shown as fold induction of SEAP activity of stimulated cells compared to non-stimulated cells (mean ± SD)(D) NIH-3T3 cells transduced with retroviral vectors expressing M45 or GFP were stimulated with IL-1β (20 ng/ml, 15 min) or TNFα (10 ng/ml, 5 min). Phosphorylated and total p38 in cell lysates was detected by immunoblotting.
Figure 2(A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam3CSK4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting.(B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD).(C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam3CSK4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), R837 (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).
Figure 3(A) 293A cells were transfected with plasmids expressing HA-tagged M45 or an unrelated MCMV control protein (m143) and Flag-tagged IKK subunits. Lysates were subjected to immunoprecipitation (IP) using an anti-HA antibody. Immunoprecipitates were analyzed by immunoblotting (IB) using anti-Flag antibody. Whole cell lysates (WCL) were immunoblotted using Flag-, HA-, and actin-specific antibodies, respectively.(B) Nemo−/− MEFs were transfected with HA-tagged IKK subunits and HA-tagged M45 or an unrelated MCMV control protein ( m142), respectively. IP and IB were performed using anti-HA and anti-Flag antibodies, respectively. WCL were immunoblotted with anti-NEMO antibody and as described for panel A.(C) rip1−/− MEFs were transfected with HA-tagged M45 or an unrelated MCMV control protein (m142) and Flag-tagged NEMO. IP was done with an anti-HA antibody. Immunoprecipitates and WCL were analyzed by immunoblotting using the indicated antibodies.(D) 293A cells were transfected with plasmids expressing HA-tagged M45 or m143 (as a control). Lysates were subjected to anti-HA immunoprecipitation. Immunoblotting of precipitates and the WCL was done with antibodies as indicated.(E) NIH-3T3 cells were infected for 4 hours with RM45 (MCMV expressing HA-tagged M45), ΔM45, or Rm142 (MCMV expressing HA-tagged m142) at an MOI of 5. Cell lysates were subjected to IP and IB with antibodies as indicated.
Figure 4(B) NIH-3T3 cells were transduced with retroviruses expressing full length M45, truncated M45 proteins, or GFP. After stimulation with IL-1β (20 ng/ml, 15 min), IκBα levels were determined by immunoblotting.(C) 293A cells were transfected with plasmids encoding Flag-tagged NEMO and HA-tagged full-length M45, truncated M45, or an unrelated MCMV control protein (m142), respectively. Lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody. Immunoprecipitates and the whole cell lysates (WCL) were analyzed by immunoblotting (IB) with the indicated antibodies.(D) Primary BMDMs were mock infected or infected with GFP-expressing wt MCMV (wt), ΔM45 mutant (ΔM45), M45 revertant (RM45), or MCMVs expressing Ct or Nt3 (RCt and RNt3) at an MOI of 1. 17 h postinfection cells were stimulated for 4 hours with TLR9 agonist CpG (0.5 µM) or TLR7 agonist R838 (0.1 µM) in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SEM).
Figure 5(A) NIH-3T3 cells were infected with MCMV ΔM45 or the revertant virus, RM45, at an MOI of 3. Cells were harvested at indicated time points postinfection, and the levels of IKK subunits, M45, IE1, and actin in lysates were analyzed by immunoblotting.(B) Murine fibroblasts (NIH-3T3, 10.1) or human embryonic kidney 293A cells were transduced with retroviral vectors expressing M45 or GFP, harvested 72 hours later, and analyzed by immunoblotting.(C) ts20 cells harboring a immunoblotting-sensitive E1 enzyme were incubated for 14 h at a permissive (35°C) or a restrictive (40.5°C) temperature. Cells were then mock-infected or infected with RM45, and analyzed 10 h postinfection by immunoblotting(D) NIH-3T3 cells were mock infected or infected with RM45 (MOI of 5) and treated 2 hpi with 10 mM ammonium chloride (NH4Cl) or a mix of lysosomal protease inhibitors (PI-mix). 9 hpi, levels of IKK subunits, M45, IE1, and actin were determined by immunoblotting. ((E) NIH-3T3 cells were stably transduced with retroviral vectors expressing M45 or GFP, and treated for the indicated times with PI-mix, 10 mM NH4Cl, or 5 µM lactacystin, respectively. Levels of the indicated proteins were analyzed by immunoblotting.
Figure 6(A) NIH-3T3 cells stably expressing Flag-tagged NEMO were mock infected or infected with wt MCMV-GFP or MCMV-GFP-ΔM45 at an MOI of 6. Cells were fixed 6 hpi, and NEMO distribution was analyzed by immunofluorescence. The arrow indicates the region which is shown in higher magnification in the right corner of the picture.(B) NIH-3T3 cells were transfected with Flag-NEMO and M45-HA expressing plasmids as indicated. 24 h later, cells were fixed and double immunofluorescence staining was performed using antibodies against the HA and Flag tags. Regions shown in higher magnification in the left corner of the pictures are indicated by arrows.(C) NIH-3T3 cells stably expressing Flag-NEMO were mock infected or infected with MCMV-M45-HA or MCMV-ΔM45 at an MOI of 7. Seven hpi, cells were fixed and analyzed as described for panel B.(D) NIH-3T3 cells were transfected with expression plasmids for Flag-NEMO and M45-HA or empty vector as indicated. Plasmids encoding GFP-tagged LC3, Rab5, or Rab7 were cotransfected. 24 hours posttransfection cells were fixed and used for anti-Flag immunofluorescence staining.
Figure 7(A) atg5−/− and atg5+/+ MEFs were mock infected or infected with MCMVΔM45 or the revertant virus, RM45, at an MOI of 10. Eight hpi cells were harvested and levels of the indicated proteins were analyzed by immunoblotting.(B) 10.1 fibroblasts were mock infected or infected with wt MCMV or ΔM45 at an MOI of 5. Cells were left untreated or treated 30 min after infection with 10 mM NH4Cl to block lysosomal degradation of LC3-II. Cells were harvested at the indicated time points, and LC3-II levels in cell lysates were analyzed by immunoblotting using an LC3-II-specific antibody. LC3-II levels were quantified by densitometric analysis and normalized using the corresponding actin levels. Fold increases are shown relative to mock-infected cells.
Figure 8(A) NIH-3T3 cells stably expressing GFP-LC3 were transduced with retroviral vectors expressing M45 or Ct3. Three days later, cells were analyzed by confocal laser scanning microscopy. GFP-LC3 dots were counted in 50 cells per sample. Results are shown as box and whisker plots. The bottom and top of the box represent the first and third quartile, respectively. The horizontal line within the box represents the median. The minima and maxima within 1.5-fold interquartile range are shown as whiskers. Dots represent outliers. Significance was determined using ANOVA (***, p<0.001). M45 and Ct3 were detected by immunoblot(B) Fluorescence images of cells described above. Large GFP-LC3 aggregates are indicated by arrow heads.(C) Electron micrograph of a double-membrane autophagosome in an M45-expressing cell.(D) Autophagosomes were counted in 10 randomly selected M45- and Ct3-expressing cells (2-3 TEM sections per cell). Results are shown in box and whisker plots as described for panel A. Significance was determined using the student's t-test (***, p<0.001).(E) Epifluorescence image of an M45-expressing cell with large GFP-LC3 structures(F) TEM image of the same cell reveals large aggregates marked with 'A'. (G, H) Successive enlargement of the region marked in panel F shows an example of an aggregate-associated autophagosome.
Figure 1(A) Clone P15 induces HA-LC3 lipidation. 293T cells were transfected with P15 plus plasmids expressing HA-LC3A and/or the apoptotic inhibitor p35 (as shown), and lysed for western blotting against the indicated molecules (anti‐HA for HA-LC3A). The figure shows that expression of P15 induces HA-LC3A conversion to a lower molecular weight form indicative of protein lipidation. This activity remains unchanged by p35.(B) P15 induces GFP-LC3 translocation to a vesiculated pattern. 293T cells were transfected with the indicated plasmids mixed with vectors expressing GFP-LC3A and p35. The known autophagic inducer bNIP3L constituted a positive control. Representative confocal microscopy pictures are shown.(C) TMEM59 induces HA-LC3 lipidation in different cell lines lacking the SV40 large T−antigen plasmid amplification system. Cells were transfected with the shown plasmids, vectors expressing HA-LC3A (left) or HA-LC3B (right) and GST (as transfection control), and lysed for western blotting against the indicated molecules.(D) TMEM59 induces GFP-LC3 activation. 293 cells were transfected with the indicated plasmids and GFP-LC3A (top) or GFP-LC3B (bottom). Representative confocal microscopy pictures are shown.(E) Quantification of the phenotype in D. The number of GFP-LC3‐positive vesicles per transfected cell was scored for at least fifty cells. Data are expressed as means ±s.d. of one representative experiment of three repetitions.Source data for this figure is available on the online supplementary information page.
Figure 2(A) TMEM59 ID is required for HA-LC3 lipidation. 293 cells were transfected with full‐length TMEM59 (FL) or a deleted version lacking the ID (ΔID, Δ263-323), HA-LC3A (left) or HA-LC3B (right) and GST. Cells were lysed for western blotting against the shown molecules.(B) The ID is necessary for GFP-LC3 activation. 293 cells were transfected with the indicated TMEM59 constructs and GFP-LC3A or GFP-LC3B (as indicated). Representative confocal pictures are shown.(C) Quantification of the phenotype in B. Scoring and data expression were as in Figure 1E.(D) TMEM59 ID (amino acids 263-323) suffices for HA-LC3A lipidation. 293 cells were transfected with the indicated CD16:7 chimera (Control: empty chimera) and vectors encoding HA-LC3A and GST, subjected to aggregation with the shown amounts of anti‐CD16 antibody and lysed for western blotting. The right panel shows control WBs demonstrating equal loading (ACTIN), transfection (GST) and chimera expression (CD16) in unaggregated samples, as all experimental points per chimera derive from a single transfection.(E) Comparable surface levels of CD16:7 constructs. Transfected 293 cells were processed for anti‐CD16 flow cytometry. The graph displays percentages of positive cells (left axis) and means of fluorescence of positive cells (MF, right axis) obtained from triplicates. Data are expressed as means ±s.d. of the triplicates.(F) The ID suffices for HA-LC3B lipidation.(G) The ID suffices for GFP-LC3 activation. 293 cells were transfected with the indicated chimeras and GFP-LC3A or GFP-LC3B, aggregated and mounted. Representative confocal microscopy pictures are shown. Activated GFP-LC3 appears as a collapsed mass of indiscernible vacuoles.(H) Quantification of the phenotype in G. As individual vesicles could not be counted, quantification was done as the percentage of transfected cells showing redistributed GFP-LC3. At least 10 different fields were counted (about 400 cells). The experiment was repeated three times. Data are expressed as means ±s.d. of the triplicates.Source data for this figure is available on the online supplementary information page.
Figure 3(A, B) Ability of serial C‐terminal deletions of TMEM59 to activate LC3. 293 cells were transfected with the indicated TMEM59 C‐terminal deletions, HA-LC3A (A) or HA-LC3B (B) and GST, and lysed for western blotting against the indicated molecules.(C) Amino acids 263-281 retain the full potential of TMEM59 ID to promote HA-LC3 conversion. 293 cells were transfected with the shown CD16:7 chimeras, HA-LC3A or HA-LC3B (as indicated) and GST, and subjected to anti‐CD16 aggregation before lysing them for western blotting. The lower panels show control WBs (unaggregated samples).D Amino acids 263-281 suffice for GFP-LC3 activation and retain the full potential of TMEM59 ID to promote GFP-LC3 activation. 293 cells were transfected with the indicated CD16:7 chimeras and GFP-LC3A or GFP-LC3B (as indicated), and subjected to anti‐CD16 aggregation before fixing them for microscopy. The graph shows percentages of transfected cells exhibiting redistributed GFP-LC3. Quantification and data expression were done as in Figure 2H.(E) Amino acids 282-323 of TMEM59 lack LC3 activation potential. 293 cells were transfected with the shown chimeras, HA-LC3A or HA-LC3B and GST, and processed for western blotting against the indicated molecules. Control WBs of unaggregated samples are shown in the lower panels.(F) Surface expression levels of CD16:7 chimeras. Procedures and data expression were as in Figure 2E. The figure shows that the functional differences observed between CD16:7 chimeras (C-E) are not caused by differential surface expression.
Figure 4(A) Endocytic vesicles containing aggregated CD16:7-263-281 become labelled with GFP-LC3. JAR cells were transfected with vectors expressing the indicated CD16:7 chimeras and GFP-LC3A, and subjected to anti‐CD16 aggregation for 8 h before staining for the endocytosed chimera (red). Representative confocal pictures are shown. The inset highlights a vesicle where the peripheral GFP-LC3 staining is particularly distinguishable. The right panel displays control WBs showing that chimera aggregation for 8 h activates HA-LC3A conversion in JAR cells.(B) Colocalization of endocytosed CD16:7-263-281 with EEA1 and CD63. JAR cells were transfected with CD16:7-263-281 and subjected to aggregation before staining them for the endocytosed chimera (red) and EEA1 or CD63 (green; FITC‐coupled primary antibodies), as indicated. Representative confocal microscopy pictures are shown.(C) Immunoelectron microscopy assay showing that endocytosed CD16:7-263-281 localizes to LC3−labelled, single−membrane vesicles. JAR cells were transfected with the CD16:7-263-281 chimera and a construct expressing human IgG1 fused to LC3A. Cells were subjected to anti−CD16 aggregation for 8 h and processed for immunoelectron microscopy. Thick gold signal (18 nm): aggregated, endocytosed chimera; thin gold signal (12 nm): IgG1-LC3. Arrows indicate single membrane (black) or IgG1-LC3A (white). Scale bar: 400 nm.(D) Lysosomal inhibition increases HA-LC3II levels promoted by CD16:7-263-281. 293 cells were transfected with the indicated chimera and HA-LC3A, and subjected to anti−CD16 aggregation in the absence or presence of bafilomycin (200 nM, added 4 h post aggregation) before lysing them for western blotting against the indicated molecules.(E) Aggregation of CD16:7-263-281 promotes its own degradation. 293 cells were transfected with the indicated constructs, aggregated and lysed for western blotting. Shown are overexposed anti‐CD16 WBs.(F) Degradation of CD16:7-263-281 is inhibited by ATG5 depletion. 293 cells were transfected with the indicated siRNAs and subsequently with the shown CD16:7 constructs, aggregated and lysed for anti‐CD16 western blotting (left panel). The right panel displays control WBs showing ATG5 depletion.Source data for this figure is available on the online supplementary information page.
Figure 5(A) Identification of critical amino acids for HA-LC3 conversion. 293 cells were transfected with the indicated CD16:7-263-281 mutants and HA-LC3A, subjected to anti‐CD16 aggregation and lysed for western blotting against the indicated molecules. Asterisks mark mutations with reduced activity. Shown is one representative experiment of six repetitions.(B) Identification of critical amino acids for GFP-LC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7-263-281 mutants and GFP-LC3A, aggregated and stained for the endocytosed chimera (red). Preparations were scored by blindly counting the number of red vesicles (chimera) and green vesicles (GFP-LC3A) per cell, as well as the number of red vesicles colocalizing with green ones. The percentage of green vesicles colocalizing with red ones was close to 100% for all mutants, that is, virtually no GFP-LC3A vesicles were unrelated to endocytosed chimera (not shown). At least 50 cells were scored per experimental point. The experiment was repeated three times. The graph shows the number of GFP-LC3A vesicles per cell expressed as the percentage of the value obtained for the wild−type chimera (left axis), and the percentage of chimera vesicles labelled with GFP-LC3A (right axis). Data are expressed as means ±s.d. of the triplicates. Asterisks indicate significant differences with respect to wild−type values (paired Student's t−test; P<0.01).(C) Representative confocal microscopy pictures of the phenotype produced by the indicated mutations. Procedures were as in B.(D) Simultaneous mutation of the four essential amino acids to alanine blocks HA-LC3 conversion induced by CD16:7-263-281. 293 cells were transfected with the indicated chimeras (4M, quadruple mutant) and HA-LC3A, aggregated and lysed for western blotting.(E) Simultaneous mutation of the four essential amino acids blocks GFP-LC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7-263-281 constructs and GFP-LC3A, aggregated and stained for the endocytosed chimera (red). Representative confocal microscopy images are shown.(F) Quantification of the phenotype in E. Data gathering and expression were as in B.Source data for this figure is available on the online supplementary information page.
Figure 6(A) Apposition events between GFP-ATG16L1 and endocytosed CD16:7-263-281. JAR cells were transfected with CD16:7-263-281 and GFP-ATG16L1 or GFP-BECLIN (as indicated), aggregated for 4 h and stained for the endocytosed chimera (red). Representative confocal microscopy pictures are shown. Two different examples are provided for GFP-ATG16L1.(B-D) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation using agarose beads coupled to glutathione (IP, immunoprecipitation; TL, total lysate). Shown are WBs against the indicated molecules. (B) AU-ATG16L1 co‐precipitates with wild‐type (WT) TMEM59-GST but not with a mutated version where the four essential amino acids were mutated to alanine (TMEM59-4M-GST). (C) Full‐length TMEM59 and TMEM59-Δ282 co‐precipitate with GST-ATG16L1, whereas the respective 4M versions do not. (D) AU-ATG16L1 co‐precipitates with a fusion protein between GST and the minimal active peptide of TMEM59 (GST-263-281), but not with a 4M version (GST-263-281-4M) or a GST fusion protein with the inactive portion of TMEM59-ID (GST-282-323).(E) HA-ATG16L1 expressed in bacteria co−precipitates with a GST-263-281 recombinant protein purified from bacterial cultures, but not with a 4M version of the same construct or GST-282-323. The indicated GST partners were expressed in bacteria and purified using agarose beads coupled to glutathione. The loaded beads were then used for HA-ATG16L1 pull−down from crude bacterial lysates. Shown are WBs against the indicated molecules (PD, pull−down). A Coomasie staining of a protein gel with the purified GST fusion proteins is shown. The right panel compares the amount of HA-ATG16L1 pulled down by GST-263-281 with the signal provided by direct anti−HA immunoprecipitation. This result shows that about 20-25% of the available HA-ATG16L1 protein is precipitated by GST-263-281. Asterisks indicate irrelevant bands in B and D.Source data for this figure is available on the online supplementary information page.
Figure 7(A-F) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione. Shown are WBs against the indicated molecules. (A) A deleted version of ATG16L1 lacking the WD domain (HA-ATG16L1-ΔWD) does not co‐precipitate with TMEM59-GST. (B) TMEM59 does not co‐precipitate with ATG16L1-ΔWD fused to GST. (C) The WD domain of ATG16L1 (HA-ATG16L1-WD) suffices to co‐precipitate with TMEM59-GST. (D) TMEM59 co‐precipitates with ATG16L1-WD fused to GST. (E) HA-ATG16L1-WD does not bind a 4M version of TMEM59-GST. (F) TMEM59-4M does not co‐precipitate with the ATG16L1-WD fused to GST.Source data for this figure is available on the online supplementary information page.