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<article doi="10.1038/emboj.2010.74" abstract="Autophagy, a critical process for bulk degradation of proteins and organelles, requires conjugation of Atg8 proteins to phosphatidylethanolamine on the autophagic membrane. At least eight different Atg8 orthologs belonging to two subfamilies (LC3 and GATE-16/GABARAP) occur in mammalian cells, but their individual roles and modes of action are largely unknown. In this study, we dissect the activity of each subfamily and show that both are indispensable for the autophagic process in mammalian cells. We further show that both subfamilies act differently at early stages of autophagosome biogenesis. Accordingly, our results indicate that LC3s are involved in elongation of the phagophore membrane whereas the GABARAP/GATE-16 subfamily is essential for a later stage in autophagosome maturation."><fig id="1924"><title></title><label>Figure 1</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1924"/><sd-panel panel_id="5015">(A) <sd-tag id="sdTag11" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing <sd-tag id="sdTag2" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag3" source="sdapi" category="entity" entity_type="gene" role="intervention" text="LC3B" ext_ids="81631" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="MAP1LC3B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LC3B</sd-tag> (left panel) or <sd-tag id="sdTag12" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag4" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of LC3 siRNAs (A, B, C), using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in <sd-tag id="sdTag8" source="sdapi" category="entity" entity_type="undefined" role="component" text="EBSS" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">EBSS</sd-tag> medium in the absence or presence of 0.1 μM <sd-tag id="sdTag9" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="Baf A" ext_ids="CHEBI:22689" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="bafilomycin A1" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">Baf A</sd-tag> and subjected to <sd-tag id="sdTag264" source="sdapi" category="assay" entity_type="" role="" text="western blot" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">western blot</sd-tag> analysis after lysis with RIPA extraction buffer.<graphic href="https://api.sourcedata.io/file.php?panel_id=5015"/></sd-panel><sd-panel panel_id="5016">(B) Quantification of relative <sd-tag id="sdTag17" source="sdapi" category="entity" entity_type="protein" role="assayed" text="p62" ext_ids="Q13501" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="SQSTM1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">p62</sd-tag> and <sd-tag id="sdTag18" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag19" source="sdapi" category="entity" entity_type="protein" role="assayed" text="GATE‐16" ext_ids="P60520" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">GATE‐16</sd-tag> level (upper panel) and degradation (lower panel) was performed as described in 'Materials and methods'. Mean±s.d. of three independent experiments is presented.<graphic href="https://api.sourcedata.io/file.php?panel_id=5016"/></sd-panel><sd-panel panel_id="5017">(C) <sd-tag id="sdTag25" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing <sd-tag id="sdTag26" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag27" source="sdapi" category="entity" entity_type="gene" role="component" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> were transfected with non‐targeting siRNA or a pool of LC3 siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐<sd-tag id="sdTag28" source="sdapi" category="entity" entity_type="protein" role="assayed" text="p62" ext_ids="Q13501" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="SQSTM1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">p62</sd-tag> after fixation, and analysed by <sd-tag id="sdTag24" source="sdapi" category="assay" entity_type="" role="" text="confocal microscopy" ext_ids="BAO_0000453" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="confocal microscopy" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">confocal microscopy</sd-tag>. The <sd-tag id="sdTag29" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag> intensity of three independent experiments was measured as described in 'Materials and methods' (right panel). Scale bar: 20 μm.<graphic href="https://api.sourcedata.io/file.php?panel_id=5017"/></sd-panel></fig><fig id="1925"><title></title><label>Figure 2</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1925"/><sd-panel panel_id="5018">(A) <sd-tag id="sdTag36" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing <sd-tag id="sdTag37" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag38" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> (left panel) or <sd-tag id="sdTag39" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag33" source="sdapi" category="entity" entity_type="gene" role="intervention" text="LC3B" ext_ids="81631" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="MAP1LC3B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LC3B</sd-tag> (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of <sd-tag id="sdTag41" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GABARAP" ext_ids="11337" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAP" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GABARAP</sd-tag> siRNAs (<sd-tag id="sdTag34" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GABARAP" ext_ids="11337" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAP" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GABARAP</sd-tag>, <sd-tag id="sdTag35" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GABARAPL1" ext_ids="11337" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAP" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GABARAPL1</sd-tag>, and <sd-tag id="sdTag42" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag>) using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in <sd-tag id="sdTag45" source="sdapi" category="entity" entity_type="undefined" role="component" text="EBSS" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">EBSS</sd-tag> medium in the absence or presence of 0.1 μM <sd-tag id="sdTag46" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="Baf A" ext_ids="CHEBI:22689" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="bafilomycin A1" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">Baf A</sd-tag> and subjected to <sd-tag id="sdTag265" source="sdapi" category="assay" entity_type="" role="" text="western blot" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">western blot</sd-tag> analysis after lysis with RIPA extraction buffer.<graphic href="https://api.sourcedata.io/file.php?panel_id=5018"/></sd-panel><sd-panel panel_id="5019">(B) Quantification of relative <sd-tag id="sdTag52" source="sdapi" category="entity" entity_type="protein" role="assayed" text="p62" ext_ids="Q13501" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="SQSTM1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">p62</sd-tag> and <sd-tag id="sdTag53" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag51" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LC3B" ext_ids="Q9GZQ8" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="MAP1LC3B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">LC3B</sd-tag> level (upper panel) and degradation (lower panel) were performed as described in 'Materials and methods'. Mean±s.d. of three independent experiments is presented.<graphic href="https://api.sourcedata.io/file.php?panel_id=5019"/></sd-panel><sd-panel panel_id="5020">(C) <sd-tag id="sdTag62" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing <sd-tag id="sdTag63" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag61" source="sdapi" category="entity" entity_type="gene" role="component" text="LC3B" ext_ids="81631" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="MAP1LC3B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LC3B</sd-tag> were transfected with non‐targeting siRNA or a pool of <sd-tag id="sdTag64" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GABARAP" ext_ids="11337" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAP" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GABARAP</sd-tag> siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐<sd-tag id="sdTag66" source="sdapi" category="entity" entity_type="protein" role="assayed" text="p62" ext_ids="Q13501" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="SQSTM1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">p62</sd-tag> after fixation, and analysed by <sd-tag id="sdTag67" source="sdapi" category="assay" entity_type="" role="" text="confocal microscopy" ext_ids="BAO_0000453" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="confocal microscopy" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">confocal microscopy</sd-tag>. The <sd-tag id="sdTag68" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag> intensity of three independent experiments was measured as described in 'Materials and methods' (right panel). Scale bar: 20 μm.<graphic href="https://api.sourcedata.io/file.php?panel_id=5020"/></sd-panel></fig><fig id="1926"><title></title><label>Figure 3</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1926"/><sd-panel panel_id="5021">(A) Bulk protein degradation was tested in <sd-tag id="sdTag73" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells that were transfected with non‐targeting siRNA (control siRNA), a pool of <sd-tag id="sdTag75" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GABARAP" ext_ids="11337" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAP" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GABARAP</sd-tag>/<sd-tag id="sdTag76" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> siRNAs, or a pool of LC3 siRNAs. The rate of long‐lived protein degradation was measured in cells incubated in either <sd-tag id="sdTag79" source="sdapi" category="entity" entity_type="undefined" role="component" text="αMEM" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">αMEM</sd-tag> (control) or <sd-tag id="sdTag77" source="sdapi" category="entity" entity_type="undefined" role="intervention" text="EBSS" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">EBSS</sd-tag> (starvation) medium, in the presence or absence of 10 mM of <sd-tag id="sdTag80" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="3‐MA" ext_ids="CHEBI:38635" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="3-methyladenine" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">3‐MA</sd-tag>, 72 h after siRNA transfection. Values expressing the protein degradation percentage are represented as the mean±s.d. of three determinations.<graphic href="https://api.sourcedata.io/file.php?panel_id=5021"/></sd-panel><sd-panel panel_id="5022">(B) <sd-tag id="sdTag87" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing <sd-tag id="sdTag88" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag84" source="sdapi" category="entity" entity_type="gene" role="intervention" text="LC3B" ext_ids="81631" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="MAP1LC3B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LC3B</sd-tag> (left panel) or <sd-tag id="sdTag89" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag90" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> (right panel) were transfected with a pool of <sd-tag id="sdTag91" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GABARAP" ext_ids="11337" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAP" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GABARAP</sd-tag>/<sd-tag id="sdTag92" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> siRNAs or a pool of LC3, respectively, and with non‐targeting siRNA. Seventy‐two hours after transfection, the cells were collected after 5 h of starvation and the relative level of <sd-tag id="sdTag93" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag86" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LC3B" ext_ids="Q9GZQ8" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="MAP1LC3B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">LC3B</sd-tag> or <sd-tag id="sdTag94" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag95" source="sdapi" category="entity" entity_type="protein" role="assayed" text="GATE‐16" ext_ids="P60520" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">GATE‐16</sd-tag> was measured using <sd-tag id="sdTag96" source="sdapi" category="assay" entity_type="" role="" text="flow cytometry" ext_ids="BAO_0000005" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="flow cytometry" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">flow cytometry</sd-tag>. Values represent the mean±s.d. of three experiments.<graphic href="https://api.sourcedata.io/file.php?panel_id=5022"/></sd-panel></fig><fig id="1927"><title></title><label>Figure 4</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1927"/><sd-panel panel_id="5023">(A) <sd-tag id="sdTag102" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing <sd-tag id="sdTag98" source="sdapi" category="entity" entity_type="protein" role="reporter" text="YFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">YFP</sd-tag>‐<sd-tag id="sdTag99" source="sdapi" category="entity" entity_type="gene" role="component" text="Atg5" ext_ids="9474" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG5" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">Atg5</sd-tag> were transfected with non‐targeting siRNA (control siRNA), a pool of LC3 siRNAs, or a pool of <sd-tag id="sdTag104" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GABARAP" ext_ids="11337" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAP" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GABARAP</sd-tag>/<sd-tag id="sdTag105" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> siRNAs using DharmaFect reagent. Seventy‐two hours after transfection, the cells were incubated for 2 h in <sd-tag id="sdTag107" source="sdapi" category="entity" entity_type="undefined" role="component" text="EBSS" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">EBSS</sd-tag> medium, fixed, and immunostained with anti‐<sd-tag id="sdTag100" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag> antibodies. Quantification of <sd-tag id="sdTag101" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg5" ext_ids="Q9H1Y0" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG5" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg5</sd-tag> and <sd-tag id="sdTag108" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag>‐labelled puncta structures from three independent experiments is presented on the right panel. Scale bar: 20 μm.<graphic href="https://api.sourcedata.io/file.php?panel_id=5023"/></sd-panel><sd-panel panel_id="5024">(B) <sd-tag id="sdTag113" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing <sd-tag id="sdTag114" source="sdapi" category="entity" entity_type="protein" role="reporter" text="YFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">YFP</sd-tag>‐<sd-tag id="sdTag115" source="sdapi" category="entity" entity_type="gene" role="component" text="Atg5" ext_ids="9474" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG5" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">Atg5</sd-tag> were treated as in (A) and monitored by <sd-tag id="sdTag112" source="sdapi" category="assay" entity_type="" role="" text="live microscopy" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">live microscopy</sd-tag>. Quantification of the lifespan of <sd-tag id="sdTag116" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg5" ext_ids="Q9H1Y0" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG5" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg5</sd-tag>‐labelled puncta structures is presented. Mean±s.d. of three independent experiments is presented. *P0.05, **P0.001.<graphic href="https://api.sourcedata.io/file.php?panel_id=5024"/></sd-panel><sd-panel panel_id="5025">(C) <sd-tag id="sdTag133" source="sdapi" category="entity" entity_type="protein" role="reporter" text="YFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">YFP</sd-tag>‐<sd-tag id="sdTag134" source="sdapi" category="entity" entity_type="gene" role="component" text="Atg5" ext_ids="9474" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG5" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">Atg5</sd-tag> cells were transfected with siRNA pool and starved as in (A). Cryo sections of fixed cells were immunolabelled using anti‐<sd-tag id="sdTag135" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag> antibodies and analysed by <sd-tag id="sdTag137" source="sdapi" category="assay" entity_type="" role="" text="TEM" ext_ids="BAO_0000455" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="transmission electron microscopy" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">TEM</sd-tag> as described in 'Materials and methods'. Quantification of the <sd-tag id="sdTag136" source="sdapi" category="entity" entity_type="gene" role="component" text="Atg5" ext_ids="9474" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG5" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">Atg5</sd-tag>‐labelled structures size is presented at the right panel. *P0.05, **P0.001.<graphic href="https://api.sourcedata.io/file.php?panel_id=5025"/></sd-panel></fig><fig id="1928"><title></title><label>Figure 5</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1928"/><sd-panel panel_id="5026">(A) Control <sd-tag id="sdTag140" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells and <sd-tag id="sdTag141" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing <sd-tag id="sdTag142" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag139" source="sdapi" category="entity" entity_type="gene" role="intervention" text="LC3B" ext_ids="81631" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="MAP1LC3B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LC3B</sd-tag> or <sd-tag id="sdTag143" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag144" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> were starved for 2 h in <sd-tag id="sdTag146" source="sdapi" category="entity" entity_type="undefined" role="component" text="EBSS" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">EBSS</sd-tag> medium fixed and immunostained with anti‐<sd-tag id="sdTag148" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag> antibodies. Quantification of <sd-tag id="sdTag149" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag>‐labelled puncta structures from three independent experiments is presented at the right panel. Arrowheads represent <sd-tag id="sdTag150" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg‐16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg‐16</sd-tag>‐labelled puncta. Scale bar: 20 μm.<graphic href="https://api.sourcedata.io/file.php?panel_id=5026"/></sd-panel><sd-panel panel_id="5027">(B) <sd-tag id="sdTag155" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing <sd-tag id="sdTag156" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag153" source="sdapi" category="entity" entity_type="gene" role="intervention" text="LC3B" ext_ids="81631" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="MAP1LC3B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LC3B</sd-tag> (left panel) or <sd-tag id="sdTag157" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag158" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> (middle panel) were starved for 2 h in <sd-tag id="sdTag160" source="sdapi" category="entity" entity_type="undefined" role="component" text="EBSS" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">EBSS</sd-tag> medium. The cells were fixed and their cryo sections were immunolabelled as in ( Figure 4B). Quantification of the size of <sd-tag id="sdTag161" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐labelled structures (right panel) from three independent experiments is presented. *P0.05, **P0.001.<graphic href="https://api.sourcedata.io/file.php?panel_id=5027"/></sd-panel></fig><fig id="1929"><title></title><label>Figure 6</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1929"/><sd-panel panel_id="5028">(A, B) Control <sd-tag id="sdTag164" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells and <sd-tag id="sdTag165" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing <sd-tag id="sdTag166" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag177" source="sdapi" category="entity" entity_type="gene" role="intervention" text="LC3B" ext_ids="81631" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="MAP1LC3B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LC3B</sd-tag> (A) or <sd-tag id="sdTag167" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag168" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> (B) were transfected with the reciprocal siRNA pools using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in <sd-tag id="sdTag171" source="sdapi" category="entity" entity_type="undefined" role="component" text="EBSS" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">EBSS</sd-tag>, subjected to immunostaining with anti‐<sd-tag id="sdTag173" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag> after fixation, and analysed by <sd-tag id="sdTag174" source="sdapi" category="assay" entity_type="" role="" text="confocal microscopy" ext_ids="BAO_0000453" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="confocal microscopy" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">confocal microscopy</sd-tag>. Quantification of cells containing <sd-tag id="sdTag175" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag> structures larger than 2 μm (A) or <sd-tag id="sdTag176" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag>‐labelled puncta structures (B) from three independent experiments in comparison to control and to G to A mutants (D) is presented at the lower panel. Scale bar: 20 μm.<graphic href="https://api.sourcedata.io/file.php?panel_id=5028"/></sd-panel><sd-panel panel_id="5029">(C) <sd-tag id="sdTag183" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing silent <sd-tag id="sdTag184" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag182" source="sdapi" category="entity" entity_type="gene" role="intervention" text="LC3B" ext_ids="81631" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="MAP1LC3B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LC3B</sd-tag> or <sd-tag id="sdTag185" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag186" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> were transfected with their siRNA pools using DharmaFect reagent. After 72‐h interval, the cells were incubated for 2 h in <sd-tag id="sdTag188" source="sdapi" category="entity" entity_type="undefined" role="component" text="EBSS" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">EBSS</sd-tag> medium and subjected to immunostaining with anti‐<sd-tag id="sdTag190" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag> and anti‐<sd-tag id="sdTag191" source="sdapi" category="entity" entity_type="protein" role="assayed" text="p62" ext_ids="Q13501" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="SQSTM1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">p62</sd-tag> antibodies followed by fixation. The cells were analysed by <sd-tag id="sdTag192" source="sdapi" category="assay" entity_type="" role="" text="confocal microscopy" ext_ids="BAO_0000453" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="confocal microscopy" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">confocal microscopy</sd-tag>. Arrows represent cells, which do not express <sd-tag id="sdTag193" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag> proteins whereas arrowheads represent cells, which express <sd-tag id="sdTag194" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐proteins. Quantification of <sd-tag id="sdTag195" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag>‐labelled puncta structures from three independent experiments is presented at the right panel. Scale bar: 20 μm.<graphic href="https://api.sourcedata.io/file.php?panel_id=5029"/></sd-panel><sd-panel panel_id="5030">(D) <sd-tag id="sdTag198" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing <sd-tag id="sdTag199" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag267" source="sdapi" category="entity" entity_type="gene" role="intervention" text="LC3B" ext_ids="81631" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="MAP1LC3B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LC3B</sd-tag>G120A or <sd-tag id="sdTag200" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag268" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag>G116Awere transfected with either pool of <sd-tag id="sdTag201" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GABARAP" ext_ids="11337" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAP" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GABARAP</sd-tag>/<sd-tag id="sdTag202" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> siRNAs or LC3 siRNAs, respectively, and a non‐targeting siRNA. The cells were starved for 2 h, fixed, and immunostained with anti‐<sd-tag id="sdTag205" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag> and anti‐<sd-tag id="sdTag206" source="sdapi" category="entity" entity_type="protein" role="assayed" text="p62" ext_ids="Q13501" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="SQSTM1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">p62</sd-tag> antibodies. Quantification is presented in (A, B) at the lower panels. Scale bar: 20 μm.<graphic href="https://api.sourcedata.io/file.php?panel_id=5030"/></sd-panel><sd-panel panel_id="5031">(E) <sd-tag id="sdTag209" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing <sd-tag id="sdTag210" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag208" source="sdapi" category="entity" entity_type="gene" role="intervention" text="LC3B" ext_ids="81631" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="MAP1LC3B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LC3B</sd-tag> (left panel) or <sd-tag id="sdTag211" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag212" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> (right panel) were transfected with the reciprocal siRNA pool or with non‐targeting siRNA and starved for 2 h in <sd-tag id="sdTag214" source="sdapi" category="entity" entity_type="undefined" role="component" text="EBSS" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">EBSS</sd-tag> medium. The cells were fixed and their cryo sections were immuno‐labelled using anti‐<sd-tag id="sdTag215" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag> antibodies and analysed by <sd-tag id="sdTag216" source="sdapi" category="assay" entity_type="" role="" text="TEM" ext_ids="BAO_0000455" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="transmission electron microscopy" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">TEM</sd-tag> as described in 'Materials and methods'. Quantification of cells containing <sd-tag id="sdTag217" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag> structures larger than 2 μm (left panel) or the size of <sd-tag id="sdTag218" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag>‐labelled structures (right panel) is presented.<graphic href="https://api.sourcedata.io/file.php?panel_id=5031"/></sd-panel></fig><fig id="1930"><title></title><label>Figure 7</label><graphic href="https://api.sourcedata.io/file.php?figure_id=1930"/><sd-panel panel_id="5032">(A) Control <sd-tag id="sdTag223" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells and <sd-tag id="sdTag224" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells stably expressing <sd-tag id="sdTag225" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag234" source="sdapi" category="entity" entity_type="gene" role="intervention" text="LC3B" ext_ids="81631" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="MAP1LC3B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LC3B</sd-tag> or <sd-tag id="sdTag226" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag227" source="sdapi" category="entity" entity_type="gene" role="intervention" text="GATE‐16" ext_ids="11345" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="GABARAPL2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">GATE‐16</sd-tag> were transfected with <sd-tag id="sdTag260" source="sdapi" category="entity" entity_type="protein" role="reporter" text="Hs" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">Hs</sd-tag><sd-tag id="sdTag262" source="sdapi" category="entity" entity_type="gene" role="intervention" text="Atg4A" ext_ids="115201" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG4A" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">Atg4A</sd-tag>C77A‐<sd-tag id="sdTag235" source="sdapi" category="entity" entity_type="protein" role="reporter" text="Myc" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">Myc</sd-tag>‐<sd-tag id="sdTag236" source="sdapi" category="entity" entity_type="protein" role="reporter" text="His6" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">His6</sd-tag>. After 48 h interval, the cells were incubated for 2 h in <sd-tag id="sdTag229" source="sdapi" category="entity" entity_type="undefined" role="component" text="EBSS" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">EBSS</sd-tag>, and subjected to immunostaining with anti‐<sd-tag id="sdTag231" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag> and anti‐myc followed by <sd-tag id="sdTag232" source="sdapi" category="assay" entity_type="" role="" text="confocal microscopy" ext_ids="BAO_0000453" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="confocal microscopy" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">confocal microscopy</sd-tag> analysis. Quantification of cells containing <sd-tag id="sdTag233" source="sdapi" category="entity" entity_type="protein" role="assayed" text="Atg16" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">Atg16</sd-tag> structures larger than 2 μm from three independent experiments is presented at the right panel. Scale bar: 20 μm.<graphic href="https://api.sourcedata.io/file.php?panel_id=5032"/></sd-panel><sd-panel panel_id="5033">(B) <sd-tag id="sdTag240" source="sdapi" category="entity" entity_type="cell_line" role="component" text="HeLa" ext_ids="CVCL_0030" norm_text="" ext_dbs="CVCL" in_caption="True" ext_names="HeLa" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://identifiers.org/cellosaurus:">HeLa</sd-tag> cells were transfected with <sd-tag id="sdTag261" source="sdapi" category="entity" entity_type="protein" role="reporter" text="Hs" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">Hs</sd-tag><sd-tag id="sdTag263" source="sdapi" category="entity" entity_type="gene" role="intervention" text="Atg4A" ext_ids="115201" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG4A" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">Atg4A</sd-tag>C77A‐<sd-tag id="sdTag241" source="sdapi" category="entity" entity_type="protein" role="reporter" text="Myc" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">Myc</sd-tag>‐<sd-tag id="sdTag242" source="sdapi" category="entity" entity_type="protein" role="reporter" text="His6" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">His6</sd-tag> or empty vector (mock). After a 48 h interval, the cells were incubated for 2 h in <sd-tag id="sdTag245" source="sdapi" category="entity" entity_type="undefined" role="component" text="EBSS" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">EBSS</sd-tag>, fixed, and their ultrathin sections were analysed by <sd-tag id="sdTag246" source="sdapi" category="assay" entity_type="" role="" text="TEM" ext_ids="BAO_0000455" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="transmission electron microscopy" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">TEM</sd-tag> as described in 'Materials and methods'. *P0.05.<graphic href="https://api.sourcedata.io/file.php?panel_id=5033"/></sd-panel></fig></article>