Journal: bioRxiv

Article Title: Mono-ADP-ribosylation by ARTD10 restricts Chikungunya virus replication by interfering with the proteolytic activity of nsP2

doi: 10.1101/2020.01.07.896977

Figure Lengend Snippet: ( a,b ) HEK293 cells were transfected with in vitro transcribed 3 EGFP wt and mutant replicons as indicated. Twenty-four hpt, the cells were treated with either vehicle, MG132 or Bafilomycin A1 (Baf.A1) for 6 h to inhibit proteasomal or lysosomal protein degradation, respectively. ( a ) Gaussia luciferase was measured in duplicates at 30 hpt. ( b ) Whole cell lysates were analyzed for processed nsP2 and nsP3 (GFP) by immunoblotting (n = 1). ( c-h ) HEK293 cells were (co-)transfected with RNA of the 3 EGFP replicons and the replicon CASA mutant as indicated (n = 3). ( c ) Representative measurement of Gaussia luciferase activity of the supernatants of the transfected cells in counts per second (CPS) at the indicated times (mean of two technical replicates). ( d ) Gaussia luciferase activity normalized to the mean of the 3 EGFP replicon wt for each experiment 30 hpt. Error bars indicate SD (n = 3; 2 technical replicates measured per n; two-tailed Mann-Whitney test). ( e ) Whole cell lysates were analyzed for processed nsP3 (GFP) by immunoblotting. ( f,g ) Flow cytometry was used to determine GFP positive cells ( f ) as well as their mean GFP fluorescence intensity (MFI) 30 hpt ( g ) (n = 3). ( h ) Representative visualization of the MFI of the GFP positive cells from panels f and g with the “Modal” option scaling all channels to a percentage of the maximum count. ( i-n ) HEK293 cells were transfected with plasmids encoding anti-GFP-nanobody-nsP3-macro wt or V33E mutant. Twenty-four h later cells were transfected with in vitro transcribed 3 EGFP wt and V33E mutant replicons as indicated. ( i,j ) Flow cytometry was used to determine GFP positive cells as well as their MFI 30 hpt (n = 3). ( k ) Representative visualization of the GFP intensity of the GFP positive cells. ( l ) Gaussia luciferase activity normalized to the mean of the 3 EGFP replicon V33E for each experiment 30 hpt. Error bars indicate SD (n = 3; 2 technical replicates measured per n). ( m,n ) Whole cell lysates were analyzed for processed nsP3 (GFP) by immunoblotting and the amount of nsP3- 3 EGFP was quantified by densitometry in relation to the loading control α-tubulin (n = 3). ( P-values for panel d : wt vs. CASA P = 0.0022; wt vs. V33E P = 0.0022; wt vs. V33E+CASA P = 0.3095; CASA vs. V33E+CASA P = 0.0022; V33E vs. V33E+CASA P = 0.0022; panel l : V33E vs. V33E + anti-GFP-nanobody-nsP3-macro P = 0.0022; V33E vs. V33E + anti-GFP-nanobody-nsP3-macro-V33E P = 0.0022; anti-GFP-nanobody-nsP3-macro vs. anti-GFP-nanobody-nsP3-macro-V33E P = 0.0022) .

Article Snippet: Twenty-four hpt with in vitro transcribed replicon RNA cells were treated with vehicle (DMSO), 25 µM MG132 (Sigma) or 200 nM Bafilomycin A1 (Baf.A1) (Enzo Life Sciences) for 6 h. Subsequently, supernatants were collected and cells were lysed with RIPA buffer and subjected to SDS-PAGE and immunoblotting for analysis.

Techniques: Transfection, In Vitro, Mutagenesis, Luciferase, Western Blot, Activity Assay, Two Tailed Test, MANN-WHITNEY, Flow Cytometry, Fluorescence

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of LC3 family protein expression and lipidation. ( A ) Total cell lysates from primary human fibroblasts treated with BafA1, Ex527 or TSA for 48h were analyzed by Western blot as indicated. ( B – E ) Densitometric quantifications of the immunoreactive bands from n = 3 independent experiments. Raw numbers were first normalized to tubulin, and the resulting values were related to the similarly tubulin-normalized control (Ctrl). ( B ) Autophagic flux analyses in cells treated as indicated for 48h. Analysis was performed for each ATG8 protein by determining the difference, between BafA1-treated and BafA1-untreated cells, of the intensity of the lipidated bands running below the unlipidated bands [Flux = (LC3II/Tub) with BafA1 —(LC3II/Tub) without BafA1 ]. Significant changes (by one-way ANOVA on ranks) versus the control: * p ≤ 0.05; ** p ≤ 0.01. ( C ) Raw intensities of all lipidated protein bands (tubulin-normalized). Significant changes (by two-way ANOVA) versus BafA1-untreated cells: * p ≤ 0.05; ** p ≤ 0.01. ( D ) Raw intensities of all lipidated and unlipidated protein bands (tubulin-normalized). Statistical analysis was done as in ( C ). ( E ) Quantification of SIRT1 and p62 expression (tubulin-normalized).

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Expressing, Western Blot, Control

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3A and LC3B in response to pharmacological sirtuin inhibition. Primary human fibroblasts treated with BafA1, Ex527 or TSA as indicated were immunostained with LC3A, LC3B and p62 antibodies and analyzed microscopically. ( A ) Cellular localization of LC3A, LC3B and p62 after treatment with BafA1 alone. ( B ) Cellular localization of LC3A, LC3B and p62 after treatment with Ex527 and BafA1. ( C ) Cellular localization of LC3A, LC3B and p62 after treatment with TSA and BafA1. ( D ) Image analytical quantification of the relative nuclear fraction of LC3A and LC3B in cells treated as indicated and counterstained with DAPI. Significant changes (by two-way ANOVA) versus the control: ## p ≤ 0.01; ### p ≤ 0.001; significant changes between the LC3s: *** p ≤ 0.001. ( E ) Relative co-localization of LC3A and LC3B with p62 quantified by image analysis. Statistical analysis was done as in D. Data in ( D ) and ( E ) were derived from three photographed, independent experiments ( n = 3), in which 10–50 cells per image were analyzed. ( F ) Magnified images of cells immunostained for LC3A or LC3B including the chromatin counterstain (DAPI).

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Inhibition, Control, Derivative Assay

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3C and LC3B in response to pharmacological sirtuin inhibition. Primary human fibroblasts were treated with BafA1, Ex527 or TSA and analyzed as in . ( A ) Cellular localization of LC3C, LC3B and p62 after treatment with BafA1 alone. ( B ) Cellular localization of LC3C, LC3B and p62 after treatment with Ex527 and BafA1. ( C ) Cellular localization of LC3C, LC3B and p62 after treatment with TSA and BafA1. ( D ) Quantification of the relative nuclear fraction of LC3C and LC3B in cells treated as indicated. Significant changes (by two-way ANOVA) versus the control: ### p ≤ 0.001; significant changes between the LC3s: ** p ≤ 0.01; *** p ≤ 0.001. ( E ) Relative co-localization of LC3C and LC3B with p62 quantified by image analysis. Statistical analysis was done as in D. Data in ( D ) and ( E ) were derived from three photographed, independent experiments ( n = 3), in which 12–45 cells per image were analyzed. ( F ) Magnified images of cells immunostained for LC3C or LC3B including the chromatin counterstain (DAPI).

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Inhibition, Control, Derivative Assay

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3A, LC3B and LC3C in response to siRNA-mediated Sirtuin1 knock-down. ( A ) Cellular localization of LC3A, LC3B and p62 after transfection with Sirtuin1 siRNA (siSIRT1-RNA) or scrambled RNA (scrSIRT1-RNA) under BafA1 treatment. ( B ) Cellular localization of LC3C, LC3B and p62 after transfection with Sirtuin1 siRNA (siSIRT1-RNA) or scrambled RNA (scrSIRT1-RNA) under BafA1 treatment. ( C ) Quantification of the relative nuclear fraction of LC3A, LC3B and LC3C in cells transfected as indicated. ( D ) Relative co-localization of LC3A, LC3B and LC3C with p62 quantified by image analysis. Significant changes (by two-way ANOVA) versus scrSIRT1-treated cells: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Data in ( C ) and ( D ) were derived from three photographed, independent experiments ( n = 3), in which 12–45 cells per image were analyzed. ( E ) Sirtuin1 protein expression after transfection with scrSIRT1-RNA or siSIRT1-RNA. Tubulin was used as loading control.

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Knockdown, Transfection, Derivative Assay, Expressing, Control

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3A, LC3B and LC3C in response to pharmacological sirtuin inhibition under starvation conditions. Primary human fibroblasts were grown under starvation conditions and treated with BafA1 and Ex527. ( A ) Cellular localization of LC3A, LC3B and p62 after treatment with Ex527 and BafA1. ( B ) Cellular localization of LC3C, LC3B and p62 after treatment with Ex527 and BafA1. ( C ) Quantification of the relative nuclear fraction of LC3A, LC3B and LC3C in cells treated as indicated. ( D ) Relative co-localization of LC3C, LC3B and LC3A with p62 quantified by image analysis. Significant changes (by two-way ANOVA) versus starvation-only treated cells: ** p ≤ 0.01; *** p ≤ 0.001. Data in ( C ) and ( D ) were derived from three photographed, independent experiments ( n = 3), in which 12–45 cells per image were analyzed.

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Inhibition, Derivative Assay

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Western blot analysis of the cellular localization of LC3A, LC3B and LC3C in response to pharmacological sirtuin inhibition under normal and starvation conditions. Primary human fibroblasts were grown under normal or starvation conditions and were treated with BafA1, Ex527 or TSA, as indicated. Nuclear and cytosolic fractions of the cells were prepared and investigated for their protein content of LC3A, LC3B and LC3C. The nuclear marker histone H3 and the cytosolic marker tubulin were analyzed in selected fractions for general separation quality control purposes.

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Western Blot, Inhibition, Marker, Control

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3A, LC3B, LC3C after siRNA-mediated LC3B knock-down. Primary human fibroblasts were treated with BafA1 after transfection with ( A ) scrLC3B-RNA, ( B ) siLC3B-RNA, ( C ) siLC3B-RNA and Ex527 treatment, ( D ) siLC3B-RNA and starvation, and ( E ) siLC3B-RNA and starvation and Ex527 treatment. ( F ) Quantification of the relative nuclear fraction of LC3A and LC3C in cells treated as indicated. ( G ) Relative co-localization of LC3A and LC3C with p62 quantified by image analysis. Significant changes (by two-way ANOVA) versus scrLC3B: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; significant changes versus siLC3B: # p ≤ 0.05; ## p ≤ 0.01. ( H ) LC3B protein expression in cells transfected with scrLC3B-RNA or siLC3B-RNA. Tubulin was used as loading control. ( I ) Control of knock-down and antibody specificity under immunocytochemistry conditions. Whole-cell signal intensities were quantified and evaluated by two-way ANOVA. Data in ( F ), ( G ) and ( I ) were derived from three photographed, independent experiments ( n = 3), in which 12–45 cells per image were analyzed.

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Knockdown, Transfection, Expressing, Control, Immunocytochemistry, Derivative Assay

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3A, LC3B, LC3C after siRNA-mediated LC3A knock-down. Primary human fibroblasts were treated with BafA1 after transfection with ( A ) scrLC3A-RNA and ( B ) siLC3A-RNA, (C) siLC3A-RNA and Ex527 treatment, ( D ) siLC3A-RNA and starvation, and ( E ) siLC3A-RNA and starvation and Ex527 treatment. ( F ) Quantification of the relative nuclear fraction of LC3B and LC3C in cells treated as indicated. ( G ) Relative co-localization of LC3B and LC3C with p62 quantified by image analysis. Significant changes (by two-way ANOVA) versus scrLC3A: ** p ≤ 0.01; *** p ≤ 0.001; significant changes versus siLC3A: # p ≤ 0.05; ## p ≤ 0.01. ( H ) LC3A protein expression in cells transfected with scrLC3A-RNA or siLC3A-RNA. Tubulin was used as loading control. ( I ) Control of knock-down and antibody specificity under immunocytochemistry conditions. Whole-cell signal intensities were quantified and evaluated by two-way ANOVA. Data in ( F ), ( G ) and ( I ) were derived from three photographed, independent experiments ( n = 3), in which 12–45 cells per image were analyzed.

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Knockdown, Transfection, Expressing, Control, Immunocytochemistry, Derivative Assay

Journal: Cells

Article Title: Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

doi: 10.3390/cells9102315

Figure Lengend Snippet: Analysis of the cellular localization of LC3A, LC3B, LC3C after siRNA-mediated LC3C knock-down. Primary human fibroblasts were treated with BafA1 after transfection with ( A ) scrLC3C-RNA, ( B ) siLC3C-RNA, ( C ) siLC3C-RNA and Ex527 treatment, ( D ) siLC3C-RNA and starvation, and ( E ) siLC3C-RNA, starvation and Ex527 treatment. ( F ) Quantification of the relative nuclear fraction of LC3A and LC3B in cells treated as indicated. ( G ) Relative co-localization of LC3A and LC3B with p62 quantified by image analysis. Significant changes (by two-way ANOVA) versus scrLC3C: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; significant changes versus siLC3C: # p ≤ 0.05; ### p ≤ 0.001. ( H ) LC3C protein expression in cells transfected with scrLC3C-RNA or siLC3C-RNA. Tubulin was used as loading control. ( I ) Control of knock-down and antibody specificity under immunocytochemistry conditions. Whole-cell signal intensities were quantified and evaluated by two-way ANOVA. Data in ( F ), ( G ) and ( I ) were derived from three images per treatment from three photographed, independent experiments ( n = 3), in which 12–45 cells per image were analyzed.

Article Snippet: Bafilomycin A1 (BafA1) was from Biozol (Eching, Germany).

Techniques: Knockdown, Transfection, Expressing, Control, Immunocytochemistry, Derivative Assay

Journal: FEBS Open Bio

Article Title: Autophagosome marker, LC 3, is released extracellularly via several distinct pathways

doi: 10.1002/2211-5463.70150

Figure Lengend Snippet: Establishment of HiBiT‐LC3B knock‐in 293T cells. (A) Schematic presentation of the human LC3B genomic locus. The inserted HiBiT tag sequence is shown in red. (B) Detection of HiBiT‐LC3B, ATG5, or FIP200 using western blotting. WT, HiBiT‐LC3B‐KI, HiBiT‐LC3B KI/ATG5 KO, or HiBiT‐LC3B KI/FIP200 KO 293T cells were treated with or without 100 n m of bafilomycin A1 (BafA1), or the medium was replaced with Hank's balanced salt solution (HBSS) for 6 h. The cells were then lysed, and the lysate was used for western blotting to measure the amount of endogenous FIP200 (1st panel), ATG5 (2nd panel), HiBiT fused LC3B (3rd panel), LC3B (4th panel), p62 (5th panel), and alpha‐tubulin (6th panel) using antibodies recognizing each endogenous protein or anti‐HiBiT antibodies. (C) HiBiT‐dependent NanoLuc luciferase activities (HiBiT activity) of the cell lysate from WT or KI cells. HiBiT activities in the cell lysate used in (B) were measured. Data are presented as mean ± SD of three technical replicates ( n = 3). Experiments were independently repeated at least twice with similar results. **** P < 0.0001; n.s., not significant (two‐way ANOVA and Tukey's multiple comparisons test). (D) Effect of MG132, a proteasome inhibitor, on the accumulation of LC3B. HiBiT‐LC3B KI, HiBiT‐LC3B KI ATG5 KO, or HiBiT‐LC3B KI FIP200 KO 293T cells were treated with 100 n m BafA1 and/or 10 μm MG132, or mock for 12 h. The cells were then lysed, and HiBiT activities were measured. Data are presented as mean ± SD of three technical replicates ( n = 3). Experiments were independently repeated at least twice with similar results. **** P < 0.0001; n.s., not significant (two‐way ANOVA and Tukey's multiple comparisons test). (E) Bafilomycin A1 induces lysosomal accumulation of HiBiT‐LC3B. HiBiT‐LC3B KI 293T cells were transiently transfected with LAMP1‐GFP (green; lysosomal marker) and treated for 6 h with 100 n m BafA1 (bottom panels) or without (top panels). Cells were fixed and immunostained with an anti‐HiBiT antibody (magenta; detecting HiBiT‐LC3B) and Hoechst 33342 (cyan; nuclei). Insets show magnified views of the boxed regions. Scale bar = 10 μm.

Article Snippet: Additionally, BafA1 (AdipoGen Life Sciences, Furinsdorf, Switzerland) was added to the medium at a final concentration of 100 n m , and the cells were cultured for 6 h to confirm the inhibition of lysosomal acidification.

Techniques: Knock-In, Sequencing, Western Blot, Luciferase, Activity Assay, Transfection, Marker

Journal: FEBS Open Bio

Article Title: Autophagosome marker, LC 3, is released extracellularly via several distinct pathways

doi: 10.1002/2211-5463.70150

Figure Lengend Snippet: Bafilomycin A1 treatment significantly enhances HiBiT‐LC3B secretion. (A) Effect of bafilomycin A1 treatment on HiBiT‐LC3B secretion. WT or HiBiT‐LC3B‐KI 293T cells were treated with or without 100 n m of BafA1 for 6 h. Then, the cells were lysed and HiBiT activity was measured (left graph). The 10k ppt (middle graph) or 100k ppt (right graph) fractions of the culture supernatants from the HiBiT‐LC3B KI cells were collected, and HiBiT activities were measured in the presence or absence of detergent; the secretion rate was calculated by dividing the HiBiT activity of the 10k ppt fraction by the sum of the HiBiT activity of the cell culture. Data are presented as mean ± SD of three technical replicates ( n = 3). Experiments were independently repeated at least twice with similar results. * P < 0.05 and ** P < 0.01 (Student's t‐test). (B) Distribution of secreted HiBiT‐LC3B from BafA1‐treated cells. After 6 h, BafA1 (100 n m )‐treated HiBiT‐LC3B KI cells were harvested and fractionated via serial centrifugation as shown in Fig. . HiBiT activities of the 10k ppt (2nd graph), 100k ppt (3rd graph), and 100k supernatant (4th graph) fractions were measured with or without 0.1% Triton X‐100. The distribution of HiBiT activities in each fraction is shown in the pie chart on the left. Data are presented as mean ± SD of three technical replicates ( n = 3). Experiments were independently repeated at least twice with similar results.

Article Snippet: Additionally, BafA1 (AdipoGen Life Sciences, Furinsdorf, Switzerland) was added to the medium at a final concentration of 100 n m , and the cells were cultured for 6 h to confirm the inhibition of lysosomal acidification.

Techniques: Activity Assay, Cell Culture, Centrifugation

Journal: FEBS Open Bio

Article Title: Autophagosome marker, LC 3, is released extracellularly via several distinct pathways

doi: 10.1002/2211-5463.70150

Figure Lengend Snippet: Secretion of the LC3 family of proteins. (A) Expression of HiBiT‐tagged LC3 family proteins. Empty or LC3 family protein‐expressing vectors were transfected into the 293T cells. After 24 h of transfection, the expression levels of LC3 family proteins (top panel) and alpha‐tubulin (bottom panel) were measured using western blotting with anti‐HiBiT or anti‐alpha‐tubulin antibodies, respectively. (B) Detection of HiBiT‐tagged LC3 family proteins. The cells were treated with or without 100 n m of BafA1 for 6 h after plasmid transfection. Cells were harvested, and HiBiT activities were measured. Data are presented as mean ± SD of three technical replicates ( n = 3). Experiments were independently repeated at least twice with similar results. (C) Detection of the secreted HiBiT‐tagged LC3 family proteins. Culture supernatants in the experiment of (B) were harvested, and HiBiT activities were measured in the presence or absence of 0.1% Triton X‐100. Data are presented as mean ± SD of three technical replicates ( n = 3). Experiments were independently repeated at least twice with similar results. (D) The secretion rate of LC3 family proteins. The secretion rate was calculated by dividing the HiBiT activity of the 10k supernatant fraction by the sum of the HiBiT activity of the cell culture. Data are presented as mean ± SD of three technical replicates ( n = 3). Experiments were independently repeated at least twice with similar results.

Article Snippet: Additionally, BafA1 (AdipoGen Life Sciences, Furinsdorf, Switzerland) was added to the medium at a final concentration of 100 n m , and the cells were cultured for 6 h to confirm the inhibition of lysosomal acidification.

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, Activity Assay, Cell Culture

Journal: FEBS Open Bio

Article Title: Autophagosome marker, LC 3, is released extracellularly via several distinct pathways

doi: 10.1002/2211-5463.70150

Figure Lengend Snippet: Lipid modification of LC3 and autophagosome formation are partially involved in LC3 secretion. (A) Secretion of HiBiT‐LC3B from HiBiT‐LC3B KI, HiBiT‐LC3B KI/ATG5 KO, or HiBiT‐LC3B KI/FIP200 KO 293T cells. The HiBiT‐LC3B KI cells were treated with or without 100 n m of BafA1, or the medium was replaced with HBSS for 6 h. Cells and culture supernatants were harvested, and HiBiT activities were measured in the presence or absence of 0.1% Triton X‐100. The secretion rate was calculated by dividing the HiBiT activity of the 10k ppt fraction by the sum of the HiBiT activity of the cell culture. Data are presented as mean ± SD of three technical replicates ( n = 3). Experiments were independently repeated at least twice with similar results. **** P < 0.0001; n.s., not significant (two‐way ANOVA and Tukey's multiple comparisons test). (B) Detection of secreted HiBiT‐LC3B using western blotting. After 12 h of cultivation with or without 100 n m of BafA1, the cells and culture supernatants of HiBIT‐LC3B KI (top panel), HiBiT‐LC3B KI/ATG5 KO (middle panel), and HiBiT‐LC3B KI/FIP200 KO (bottom panel) were harvested. Then, the culture supernatants were centrifuged using the protocol shown in Fig. . HiBiT‐LC3B in each fraction was detected via western blotting using an anti‐HiBiT tag antibody. The numbers below the panels represent the relative amounts of LC3 proteins in the 10k or 100k ppt fractions compared to those in the cell fraction, calculated based on band intensity. (C) Expression of HiBiT‐tagged LC3B deltaG mutant. Empty vector, HiBiT‐LC3B WT, and HiBiT‐LC3B deltaG mutant expressing vectors were transiently expressed in 293T cells, and the expression level of HiBiT‐LC3B was measured using western blotting with an anti‐HiBiT antibody. (D) Secretion from the HiBiT‐tagged LC3B deltaG mutant. HiBiT‐LC3B WT or HiBiT‐LC3B deltaG mutant were transiently expressed in 293T cells. After 24 h of cultivation, the cells were treated with or without 100 n m BafA1, or the medium was replaced with HBSS and incubated for 6 h. Cells and culture supernatants were harvested, and HiBiT activities were measured in the presence or absence of 0.1% Triton X‐100. The secretion rate was calculated by dividing the HiBiT activity of the 10k ppt fraction by the sum of the HiBiT activity of the cell culture. Data are presented as mean ± SD of three technical replicates ( n = 3). Experiments were independently repeated at least twice with similar results. **** P < 0.0001 (two‐way ANOVA and Tukey's multiple comparisons test).

Article Snippet: Additionally, BafA1 (AdipoGen Life Sciences, Furinsdorf, Switzerland) was added to the medium at a final concentration of 100 n m , and the cells were cultured for 6 h to confirm the inhibition of lysosomal acidification.

Techniques: Modification, Activity Assay, Cell Culture, Western Blot, Expressing, Mutagenesis, Plasmid Preparation, Incubation

Journal: FEBS Open Bio

Article Title: Autophagosome marker, LC 3, is released extracellularly via several distinct pathways

doi: 10.1002/2211-5463.70150

Figure Lengend Snippet: Receptor binding of LC3 is also involved in LC3 secretion. (A) Schematic presentation of LC3‐receptor interaction‐mediated selective autophagy. (B) Expression levels of HiBiT‐tagged LC3B WT or LC3B mutants. Indicated vectors were transfected into the 293T cells. After 24 h of cultivation, HiBiT‐LC3B proteins (top panel) or alpha‐tubulin (bottom panel) in cells were detected using western blotting. (C) Secretion of receptor‐binding‐deficient LC3B mutants. HiBiT‐LC3B WT or mutants were transiently expressed in 293T cells. After 24 h of cultivation, the cells were treated for 6 h with or without 100 n m of BafA1. Cells and culture supernatants were harvested, and HiBiT activities were measured in the presence or absence of 0.1% Triton X‐100. The secretion rate was calculated. Data are presented as mean ± SD of three technical replicates ( n = 3). Experiments were independently repeated at least twice with similar results. ** P < 0.01, **** P < 0.0001; n.s., not significant (two‐way ANOVA and Tukey's multiple comparisons test). (D) LC3‐II formation of receptor‐binding‐deficient LC3B mutants. After 24 h of transfection, the cells were treated with or without 100 n m BafA1 for 6 h, followed by harvesting. HiBiT‐LC3B proteins (top panel) or alpha‐tubulin (bottom panel) were then detected using western blotting with the same protocol shown in (B). (E) Secretion of receptor‐binding‐deficient and/or delta G LC3B mutants. Each vector was transfected into WT or FIP200 KO cells. After 24 h of transfection, the cells were treated with or without 100 n m BafA1 for 6 h, then harvested, and their HiBiT activities were detected. The secretion rate of HiBiT‐LC3B was calculated using the same protocol as in (C). Data are presented as mean ± SD of three technical replicates ( n = 3). Experiments were independently repeated at least twice with similar results. **** P < 0.0001 (Two‐way ANOVA and Tukey's multiple comparisons test). (F) LC3‐II formation of receptor‐binding‐deficient and or delta G LC3B mutants in WT 293T cells. Western blotting was performed using the same protocol as shown in (B). (G) LC3‐II formation of receptor‐binding‐deficient and or delta G LC3B mutants in FIP200 KO 293T cells. (H) Secretion of autophagy receptors. Each receptor was transiently expressed in 293T cells. After 24 h of cultivation, the cells were treated with or without 100 n m of BafA1 and incubated for 6 h. Cells and culture supernatants were harvested, and HiBiT activities were measured in the presence or absence of 0.1% Triton X‐100. The secretion rate was calculated. Data are presented as mean ± SD of three technical replicates Data are presented as mean ± SD of three technical replicates ( n = 3). Experiments were independently repeated at least twice with similar results. **** P < 0.0001 (Two‐way ANOVA and Tukey's multiple comparisons test). The expression level of HiBiT‐tagged autophagy receptors and alpha‐tubulin in cells was confirmed by western blotting. (I) Schematic presentation showing the possible LC3B secretion pathways. LC3B could be secreted via the following five different pathways: (1) autophagy secretion, (2) autophagy‐independent but lipid modification‐dependent secretion, (3) receptor binding‐dependent secretion, (4) autophagy, lipid modification‐independent secretion, and (5) lysosome secretion.

Article Snippet: Additionally, BafA1 (AdipoGen Life Sciences, Furinsdorf, Switzerland) was added to the medium at a final concentration of 100 n m , and the cells were cultured for 6 h to confirm the inhibition of lysosomal acidification.

Techniques: Binding Assay, Expressing, Transfection, Western Blot, Plasmid Preparation, Incubation, Modification