Published Erratum
doi: 10.1073/pnas.2535281122.
Epub 2025 Dec 24.
Correction for Li et al., BCL2 regulates antibacterial autophagy in the intestinal epithelium
- PMID: 41439717
- PMCID: PMC12772163
- DOI: 10.1073/pnas.2535281122
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Published Erratum
Correction for Li et al., BCL2 regulates antibacterial autophagy in the intestinal epithelium
Proc Natl Acad Sci U S A.
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Figures
Bacterial infection promotes phosphorylation of BCL2 and induces dissociation of the BCL2–BECN1 complex in small intestinal epithelial cells. (A) Schematic of the BCL2–BECN1 interaction and its role in activating autophagy. BECN1 initiates autophagosome formation (9). BCL2 complexes with BECN1 to inhibit autophagy, and metabolic signals arising from starvation or exercise trigger dissociation of the BCL2–BECN1 complex as a prerequisite to autophagy (12, 13). The mechanism requires phosphorylation of BCL2, which induces dissociation from BECN1 and promotes autophagy (15). (B) Immunofluorescence microscopy of LC3 in the small intestines of uninfected and S. Typhimurium–infected conventional wild-type mice. Mice were infected intragastrically with 5 × 109 colony-forming units (CFU) of S. Typhimurium (SL1344) and killed 24 h later. Sections of the distal small intestine were stained with rabbit anti-LC3 and Cy3-conjugated anti-rabbit IgG (red) and counterstained with DAPI (blue) to detect nuclei. [Scale bar, 50 μm (Left); 10 μm (Right)]. (C) Immunofluorescence microscopy of LC3 in the small intestinal villus tips of uninfected and S. Typhimurium–infected germ-free wild-type mice. Mice were infected and LC3+ structures were detected as in B. Examples of LC3+ puncta are indicated with arrowheads. (Scale bar, 10 μm.) Results are representative of images from n = 3 mice per group and five independent experiments. Immunofluorescence controls are shown in SI Appendix, Fig. S1. (D) Immunoblot analysis of small intestinal epithelial cells from uninfected or S. Typhimurium–infected germ-free wild-type mice. Mice were infected intragastrically as in B. Epithelial cell lysates were blotted and probed with antibodies against BCL2, p-BCL2, LC3, and Mito70 (loading control). LC3-I and LC3-II denote nonlipidated and lipidated LC3, respectively. Each lane represents one mouse. Results are representative of three independent experiments. (E) p-BCL2 and BCL2 band densities in D were measured by scanning densitometry and the ratio of the densities was calculated. Each data point represents one mouse. (F) Immunofluorescence microscopy of organoids derived from the small intestines of wild-type C57Bl/6 mice. Organoids were infected with 106 CFU of S. Typhimurium (SL1344) for 4 h, then fixed, embedded, and sectioned. Sections were detected with rabbit anti-LC3 and Cy3-conjugated anti-rabbit IgG (red) and counterstained with DAPI (blue) to detect nuclei. Arrowheads indicate examples of LC3+ puncta. (Scale bar, 10 μm.) (G) LC3+ structures in images from E were counted. Each data point is the average number of LC3+ puncta in each cell of organoids from an individual mouse; at least 10 individual organoids were counted from each mouse (n = 3 mice per group). Results are representative of three independent experiments. (H) Immunoblot analysis of wild-type small intestinal organoids with and without S. Typhimurium infection. Organoids cultured from wild-type mice were infected with 106 CFU of S. Typhimurium for 4 h. Organoid lysates were immunoblotted with antibodies against BCL2, p-BCL2, LC3, and Mito70 (loading control). LC3-I and LC3-II denote nonlipidated and lipidated LC3, respectively. Results are representative of three independent experiments. (I) Coimmunoprecipitation of BECN1 with BCL2 in small intestinal epithelial cells from uninfected or S. Typhimurium–infected germ-free mice. Epithelial cell lysates were immunoprecipitated with mouse anti-BECN1, blotted, and probed with mouse anti-BCL2-HRP plus rabbit anti-BECN1 (Novus Biologicals) and goat anti-rabbit IgG-HRP. The presence of two BECN1 isoforms in the immunoprecipitate has been observed in published reports (16–18). Each lane represents one mouse. Results are representative of three independent experiments. (J) Band intensities from the BECN1 immunoprecipitations in I were measured by scanning densitometry and the ratios of the BCL2 and BECN1 densities were calculated. Each data point represents one mouse. uninf., uninfected; S. Tm, Salmonella Typhimurium; sm. int. epith. cells, small intestinal epithelial cells; p-BCL2, phosphorylated BCL2; IP, immunoprecipitation. Means ± SEM are plotted; *P < 0.05; ***P < 0.001; ****P < 0.0001 by Student’s t test.
BCL2 phosphorylation promotes antibacterial autophagy in small intestinal epithelial cells. (A) Schematic depicting how the BCL2AAA mutation impacts the BCL2–BECN1 interaction and autophagy activation. Activating signals promote BCL2 phosphorylation at three conserved amino acid residues: T69, S70, and S87 (15). Bcl2AAA mice harbor a knock-in Bcl2 allele in which these residues are mutated, rendering BCL2 nonphosphorylatable and unable to release BECN1 to initiate autophagy. These mice are thus deficient in stimulus-induced but not basal autophagy (13). (B) Immunoblot of small intestinal organoids from wild-type (WT) and Bcl2AAA mice. Organoids were infected with S. Typhimurium–GFP (106 CFU) for 4 h or were left uninfected. Immunoblots were detected with antibodies against BCL2, p-BCL2, LC3, and Mito70 (loading control). LC3-I and LC3-II denote nonlipidated and lipidated LC3, respectively. Results are representative of three independent experiments. (C) Immunofluorescence microscopy of small intestines from infected and uninfected conventionally raised wild-type and Bcl2AAA littermates. Mice were infected intragastrically with S. Typhimurium–GFP (5 × 109 CFU per mouse) and were killed 24 h later. Sections of the distal small intestine were stained with rabbit anti-LC3 and Cy3-conjugated anti-rabbit IgG (red), and goat anti-GFP and DyLight 488-conjugated anti-goat IgG (green). Intrinsic GFP fluorescence is destroyed by tissue fixation, necessitating antibody detection of GFP. Sections were counterstained with DAPI (blue) to detect nuclei. (Scale bar, 50 μm.) (D) Immunofluorescence microscopy of small intestinal villus tips from uninfected and S. Typhimurium–infected mice. Mice were infected and tissues stained as in C. Examples of LC3+ structures are indicated with arrowheads, and examples of intracellular S. Typhimurium–GFP are indicated with arrows. (Scale bar, 10 μm.) Images are representative n = 6 mice per group and two independent experiments. Immunofluorescence controls are shown in SI Appendix, Fig. S1. (E) LC3+ puncta in images from D were counted. Each data point is the average number of LC3+ puncta per cell from one mouse (n = 6 mice per group). At least 200 crypt-villus units were counted per mouse, and all epithelial cells in each crypt-villus unit were counted (note that enterocytes at the villus tips typically have more LC3+ puncta than enterocytes in the lower regions of the crypt-villus unit) (5). (F) Cell-associated S. Typhimurium–GFP in images from D were counted. Each data point is the average number of S. Typhimurium per intestinal epithelial cell (IEC) from one mouse (n = 6 mice per group); at least 200 crypt-villus units were counted per mouse. (G) Immunofluorescence microscopy of small intestinal organoids grown from wild-type and Bcl2AAA littermates. Organoids were infected with 106 CFU of S. Typhimurium–GFP for 4 h, then fixed, embedded, and sectioned. Sections were detected with rabbit anti-LC3 and Cy3-conjugated anti-rabbit IgG (red), and goat anti-GFP, DyLight 488-conjugated anti-goat IgG (green), and DAPI (blue) to detect nuclei. Arrowheads indicate examples of LC3+ puncta. (Scale bar, 5 μm.) Images are representative of organoids from n = 5 mice per group and two independent experiments. (H) LC3+ puncta in images from G were counted. Each point is the average number of LC3+ puncta in each organoid cell from one mouse (n = 5 mice per group); at least 10 organoids were counted from each mouse. (I) Intracellular S. Typhimurium–GFP in images from G were counted. Each point represents organoids from one mouse (n = 8 mice per group and three independent experiments). (J) Numbers of intracellular S. Typhimurium–GFP in organoids grown from wild-type and Bcl2AAA littermates as determined by dilution plating. Each point represents organoids from one mouse (n = 6 mice per group). (K) Bacterial burdens CFU in the small intestine (sm. int.), mesenteric lymph node (MLN), spleen, and liver of wild-type and Bcl2AAA littermates 24 h after intragastric infection with 5 × 109 CFU S. Typhimurium–GFP. Bacterial counts were determined by dilution plating. Each point represents an individual mouse, and data are from three independent experiments. Geometric means ± SEM are plotted. WT, wild-type; S. Tm, Salmonella Typhimurium; GFP, green fluorescent protein; IEC, intestinal epithelial cell; p-BCL2, phosphorylated BCL2; CFU, colony-forming units. Means ± SEM are plotted except where noted; *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant by Student’s t test.
Erratum for
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BCL2 regulates antibacterial autophagy in the intestinal epithelium.Proc Natl Acad Sci U S A. 2024 Dec 3;121(49):e2410205121. doi: 10.1073/pnas.2410205121. Epub 2024 Nov 27. Proc Natl Acad Sci U S A. 2024. PMID: 39602254 Free PMC article.
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