experimental models hek293a cells  (ATCC)

Journal: bioRxiv

Article Title: ATG16L2 creates a VAIL-dedicated heterodimer with ATG16L1

doi: 10.64898/2026.06.01.729256

Figure Lengend Snippet: A . Schematic overview of ATG16 family proteins. ATG16L1α, ATG16L1β and ATG16L2 share an N-terminal ATG5-interacting motif (AFIM), a central coiled-coil domain (CCD) and a C-terminal WD40 β-propeller. The β isoform-specific insertion in ATG16L1β and the WIPI2-interaction region present in ATG16L1 but absent from ATG16L2 are indicated. Percent amino-acid identity between ATG16L1β and ATG16L2 is shown for the N-terminal region, CCD, middle/disordered region and WD40 β-propeller, highlighting strong conservation of the N-terminal AFIM-containing region and WD40 β-propeller, but limited conservation within the CCD and intervening region. B . Immunoblot analysis of LC3B and GABARAP lipidation in wild-type (WT) and ATG16L1/TECPR1-DKO HEK293A cells with or without ATG16L2 reconstitution, left untreated (1) or treated with 50 nM Torin1 and 100 nM bafilomycin A1 (2) or 5 µM monensin (3) for 2 h. Stain-Free is a loading control. C, D . Quantification of LC3B-II ( C ) and GABARAP-II ( D ) from immunoblots in ( B ), normalized to Stain-Free. E . Strep-TactinXT pull-down of TwinStrep-V5-ATG16L2 from ATG16L1/TECPR1-DKO HEK293A cells co-expressing HaloTag-ATG16L1β or HaloTag-ATG16L2. Input and pull-down fractions were immunoblotted for the indicated proteins. F . Schematic of the ATG16L1 AFIM mutant (L21W/R24D; LWRD) used to disrupt ATG5 binding. G . Strep-TactinXT pull-down of TwinStrep-V5-ATG16L1β WT or LWRD mutant from ATG16L1/TECPR1-DKO HEK293A cells. Input and pull-down fractions were immunoblotted for the indicated proteins. H . Schematic of the ATG16L compositions analyzed and corresponding immunoblot of LC3B and GABARAP lipidation in HEK293A cells expressing the indicated ATG16L combinations, left untreated or treated with 50 nM Torin1 plus 100 nM bafilomycin A1 or 5 µM monensin for 2 h. ATG16L composition #1, WT cells expressing endogenous ATG16L1; ATG16L composition #2, ATG16L1/TECPR1-DKO cells expressing ATG16L1β L21W/R24D; ATG16L composition #3, ATG16L1/TECPR1-DKO cells co-expressing ATG16L1β L21W/R24D and ATG16L2. Stain-Free is a loading control. I, J . Quantification of LC3B-II ( I ) and GABARAP-II ( J ) from immunoblots in ( H ), normalized to Stain-Free. Data information: Quantifications are normalized as indicated in the graphs. Bars show mean ± SD; symbols denote independent experiments. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple-comparisons test ( C,D,I,J ). Adjusted p-values are indicated in the graphs.

Article Snippet: Consistent with Human Protein Atlas data ( Jin et al , 2023 ), we were unable to detect ATG16L2 in HEK293A cells by MS and observed detectable ATG16L2 peptides in only one of five U2OS samples ( ).

Techniques: Western Blot, Staining, Control, Expressing, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: ATG16L2 creates a VAIL-dedicated heterodimer with ATG16L1

doi: 10.64898/2026.06.01.729256

Figure Lengend Snippet: A . MS-based abundance of ATG16L2 in WT HEK293A and U2OS cells. Raw LFQ intensities are shown relative to the limit of detection (LOD). B . Strep-TactinXT pull-down of TwinStrep-V5-ATG16L1β WT or LWRD mutant from ATG16L1/TECPR1-DKO HEK293A cells, showing ATG5 binding in the input and pull-down fractions. For the WT sample, a 10-fold diluted lysate was used, resulting in 10-fold less protein loaded in the input and 10-fold less material incubated with the Strep-TactinXT beads, as indicated. C . Strep-TactinXT pull-down of TwinStrep-V5-ATG16L2 from ATG16L1/TECPR1-DKO HEK293A cells co-expressing HaloTag-ATG16L1β or HaloTag-ATG16L2, left untreated or treated with 5 µM monensin for 30 min. D . Schematic of the ATG16L compositions analyzed and corresponding immunoblot of LC3B and GABARAP lipidation in HEK293A cells expressing the indicated ATG16L combinations, left untreated or treated with 50 nM Torin1 plus 100 nM bafilomycin A1 or 5 µM monensin for 2 h. ATG16L composition #1, WT cells expressing endogenous ATG16L1; ATG16L composition #2, ATG16L1/TECPR1-DKO cells expressing ATG16L1β aa70-607; ATG16L composition #3, ATG16L1/TECPR1-DKO cells co-expressing ATG16L1β aa70-607 and ATG16L2. Stain-Free is a loading control. E . Quantification of LC3B-II from immunoblots in ( D ), normalized to Stain-Free. F–M . Immunoblot analysis of LC3B and GABARAP lipidation in WT and ATG16L1/TECPR1-DKO HEK293A cells xpressing ATG16L1β L21W/R24D with or without ATG16L2, treated as indicated. Stain-Free is a loading control.

Article Snippet: Consistent with Human Protein Atlas data ( Jin et al , 2023 ), we were unable to detect ATG16L2 in HEK293A cells by MS and observed detectable ATG16L2 peptides in only one of five U2OS samples ( ).

Techniques: Mutagenesis, Binding Assay, Incubation, Expressing, Western Blot, Staining, Control

Journal: bioRxiv

Article Title: ATG16L2 creates a VAIL-dedicated heterodimer with ATG16L1

doi: 10.64898/2026.06.01.729256

Figure Lengend Snippet: A . Schematic representation of ATG16L1β, ATG16L2 and the domain-swap chimeras 1-2-1 and 2-1-2. Numbers indicate the origin of the N-terminal/CCD/C-terminal regions, with 1 denoting ATG16L1β-derived sequence and 2 denoting ATG16L2-derived sequence. B . Immunoblot analysis of LC3B and GABARAP lipidation in WT and ATG16L1/TECPR1-DKO HEK293A cells expressing the indicated chimeras, left untreated (1), treated for 2 h with 50 nM Torin1 plus 100 nM bafilomycin A1 (2), or treated with 5 µM monensin together with 5 µM MRT68921 and 10 µM SAR405 (3). Stain-Free is a loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. C, D . Quantification of LC3B-II ( C ) and GABARAP-II ( D ) from immunoblots in ( B ), normalized to Stain-Free. E . Confocal images of ATG16L1/TECPR1-DKO U2OS cells expressing HaloTag-1-2-1 or HaloTag-2-1-2, stained for WIPI2, left untreated or treated with 50 nM Torin1 plus 100 nM bafilomycin A1 for 30 min. Scale bars, 10 μm. F . Quantification of WIPI2-positive structures containing the indicated HaloTag chimera from images in ( E ). Total cells per condition/images per condition: 252/25 (1-2-1 untreated), 259/25 (1-2-1 Torin1+BafA1), 241/25 (2-1-2 untreated), 284/25 (2-1-2 Torin1+BafA1). G . Confocal images of ATG16L1/TECPR1-DKO U2OS cells expressing HaloTag-1-2-1 or HaloTag-2-1-2, stained for LAMP1, left untreated or treated with 5 µM monensin for 15 min. Scale bars, 10 μm. H . Quantification of LAMP1-positive structures containing the indicated HaloTag chimera from images in ( G ). Total cells per condition/images per condition: 244/25 (1-2-1 untreated), 248/25 (1-2-1 monensin), 236/25 (2-1-2 untreated), 222/25 (2-1-2 monensin). Data information: Quantifications are normalized as indicated in the graphs. Bars show mean ± SD; symbols denote independent experiments. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple-comparisons test ( C,D ). Adjusted p-values are indicated in the graphs. For image quantifications; Dot colors indicate independent experiments with each doth representing one image; large open symbols indicate the mean of each independent replicate; black horizontal lines indicate the overall mean. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple-comparisons test ( F,H ). Adjusted p-values are indicated in the graphs.

Article Snippet: Consistent with Human Protein Atlas data ( Jin et al , 2023 ), we were unable to detect ATG16L2 in HEK293A cells by MS and observed detectable ATG16L2 peptides in only one of five U2OS samples ( ).

Techniques: Derivative Assay, Sequencing, Western Blot, Expressing, Staining, Control

Journal: bioRxiv

Article Title: ATG16L2 creates a VAIL-dedicated heterodimer with ATG16L1

doi: 10.64898/2026.06.01.729256

Figure Lengend Snippet: A . Immunoblot analysis of LC3B and GABARAP lipidation in WT and ATG16L1/TECPR1-DKO HEK293A cells expressing the indicated chimeras, left untreated (1), treated for 2 h with 50 nM Torin1 plus 100 nM bafilomycin A1 (2), or treated with 5 µM monensin together with 5 µM MRT68921 and 10 µM SAR405 (3). Stain-Free is a loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. B, C . Quantification of LC3B-II ( B ) and GABARAP-II ( C ) from immunoblots in ( A ), normalized to Stain-Free. Data information: Quantifications are normalized as indicated in the graphs. Bars show mean ± SD; symbols denote independent experiments. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple-comparisons test ( B,C ). Adjusted p-values are indicated in the graphs.

Article Snippet: Consistent with Human Protein Atlas data ( Jin et al , 2023 ), we were unable to detect ATG16L2 in HEK293A cells by MS and observed detectable ATG16L2 peptides in only one of five U2OS samples ( ).

Techniques: Western Blot, Expressing, Staining, Control

Journal: bioRxiv

Article Title: ATG16L2 creates a VAIL-dedicated heterodimer with ATG16L1

doi: 10.64898/2026.06.01.729256

Figure Lengend Snippet: A . Confocal images of ATG16L1/TECPR1-DKO U2OS cells expressing mNeonGreen-ATG16L2 WT or an ATG16L2 Δβ-propeller mutant, stained for LAMP1, left untreated or treated with 5 µM monensin for 15 min. Scale bars, 10 μm. B . Quantification of LAMP1-positive structures containing mNeonGreen-ATG16L2 from images in ( A ). Total cells per condition/images per condition: 284/30 (ATG16L2 untreated), 258/30 (ATG16L2 monensin), 245/30 (ATG16L2 Δβ-propeller untreated), 231/30 (ATG16L2 Δβ-propeller monensin). C . Structural comparison of the ATG16L1 and ATG16L2 β-propeller domains. The ATG16L1 β-propeller structure was obtained from the AlphaFold Protein Structure Database model AF-Q676U5-F1-v6 and visualized for residues aa306–607. The ATG16L2 β-propeller structure was obtained from the AlphaFold Protein Structure Database model AF-Q8NAA4-F1-v6 and visualized for residues aa322–619. Residues selected for alanine substitution in ATG16L2, and corresponding CASM-relevant residues in ATG16L1, are highlighted. D . Immunoblot analysis of LC3B lipidation in ATG16L1/TECPR1-DKO HEK293A cells expressing ATG16L1β L21W/R24D F467A together with WT or mutant ATG16L2 proteins, treated with 5 µM monensin for 2 h. E . Quantification of LC3B-II from immunoblots in ( D ), normalized to Stain-Free. All ATG16L2 mutants were compared with WT ATG16L2. F . Immunoblot analysis of LC3B lipidation in ATG16L1/TECPR1-DKO HEK293A cells expressing ATG16L1β L21W/R24D together with WT ATG16L2. Cells were left untreated, treated with 5 µM monensin, treated with 5 µM monensin plus 100 nM bafilomycin A1, or analyzed in the absence or presence of Flag-SopF as indicated. G . Quantification of LC3B-II from immunoblots in ( F ), normalized to Stain-Free. Data information: Quantifications are normalized as indicated in the graphs. Bars show mean ± SD; symbols denote independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple-comparisons test ( E,G ). Adjusted p-values are indicated in the graphs. For image quantifications; Dot colors indicate independent experiments with each doth representing one image; large open symbols indicate the mean of each independent replicate; black horizontal lines indicate the overall mean. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple-comparisons test ( B ). Adjusted p-values are indicated in the graphs.

Article Snippet: Consistent with Human Protein Atlas data ( Jin et al , 2023 ), we were unable to detect ATG16L2 in HEK293A cells by MS and observed detectable ATG16L2 peptides in only one of five U2OS samples ( ).

Techniques: Expressing, Mutagenesis, Staining, Comparison, Western Blot

Journal: bioRxiv

Article Title: ATG16L2 creates a VAIL-dedicated heterodimer with ATG16L1

doi: 10.64898/2026.06.01.729256

Figure Lengend Snippet: A . Confocal images of ATG16L1/TECPR1-DKO U2OS cells expressing HaloTag-ATG16L1α or HaloTag-ATG16L1β in the absence or presence of mNeonGreen-ATG16L2, stained for WIPI2 after treatment with 5 µM monensin for 15 min. Scale bars, 10 μm. B . Quantification of WIPI2-positive structures containing the indicated HaloTag-ATG16L1 isoform from images in ( A ), in the absence or presence of mNeonGreen-ATG16L2. Total cells per condition/images per condition: 227/25 (ATG16L1α monensin), 261/25 (ATG16L1α + ATG16L2 monensin), 242/25 (ATG16L1β monensin), 232/25 (ATG16L1β + ATG16L2 monensin). C . Confocal images of ATG16L1/TECPR1-DKO U2OS cells expressing HaloTag-ATG16L1α or HaloTag-ATG16L1β in the absence or presence of mNeonGreen-ATG16L2, stained for LAMP1 after treatment with 5 µM monensin for 15 min. Scale bars, 10 μm. D . Quantification of LAMP1-positive structures containing the indicated HaloTag-ATG16L1 isoform from images in ( C ), in the absence or presence of mNeonGreen-ATG16L2. Total cells per condition/images per condition: 236/25 (ATG16L1α monensin), 241/25 (ATG16L1α + ATG16L2 monensin), 236/25 (ATG16L1β monensin), 263/25 (ATG16L1β + ATG16L2 monensin). E . Immunoblot analysis of LC3B and GABARAP lipidation in ATG16L1/TECPR1-DKO HEK293A cells expressing ATG16L1α or ATG16L1β in the absence or presence of ATG16L2, treated for 1 h with 5 µM monensin together with 5 µM MRT68921 and 10 µM SAR405 or with 50 nM Torin1 plus 100 nM bafilomycin A1. Stain-Free is a loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. F, G . Quantification of LC3B-II ( F ) and GABARAP-II ( G ) from immunoblots in ( E ), normalized to Stain-Free. Data information: Quantifications are normalized as indicated in the graphs. Bars show mean ± SD; symbols denote independent experiments. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple-comparisons test ( F,G ). Adjusted p-values are indicated in the graphs. For image quantifications; Dot colors indicate independent experiments with each doth representing one image; large open symbols indicate the mean of each independent replicate; black horizontal lines indicate the overall mean. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple-comparisons test ( B,D ). Adjusted p-values are indicated in the graphs.

Article Snippet: Consistent with Human Protein Atlas data ( Jin et al , 2023 ), we were unable to detect ATG16L2 in HEK293A cells by MS and observed detectable ATG16L2 peptides in only one of five U2OS samples ( ).

Techniques: Expressing, Staining, Western Blot, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Human and viral chemokines differentially modulate G protein signaling, β-arrestin recruitment and chemotaxis mediated by the viral G protein-coupled receptor ORF74

doi: 10.1186/s12964-025-02546-9

Figure Lengend Snippet: Agonist and inverse agonist activities on β-arrestin1/2 recruitment to ORF74. The β-arrestin recruitment assays were performed using HEK293A cells co-transfected with pcDNA3.1(+)-ORF74-LgBiT and pEF1α-IRES SmBiT-ARRB1 or pEF1α-IRES SmBiT-ARRB2. Cells were stimulated with increasing concentrations of chemokines, and luminescence signals were measured for ( A ) β-arrestin1 and ( B ) β-arrestin2 recruitment. Data are presented as % of the maximal CXCL1 response set at 100%, with mean ± SD of three independent experiments, each performed in duplicate. Curves were fitted to a four-parameter logistic model

Article Snippet: HEK293A.ORF74 and HEK293A cells were detached with 0.25% Trypsin-EDTA (Thermo Fisher Scientific, #25200056), resuspended in growth medium and incubated at room temperature (RT) for 2 h. Then, cells were washed twice and resuspended in cold assay buffer [Hank’s Balanced Salt Solution (HBSS; Thermo Fisher Scientific), 20 mM HEPES buffer (Thermo Fischer Scientific), 0.5% FBS, pH 7.4].

Techniques: Transfection