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