EMBO/soda-nel huggingface dataset:
ATF3 induces RAB7 to govern autodegradation in paligenosis, a conserved cell plasticity program (2021)
Figure 1

Figure 1. Secretory cell structure is dismantled rapidly and in a choreographed pattern in paligenosis

A. Left - Single-level electron micrograph images of zymogenic chief cells (ZCs) after 24h vehicle or high-dose tamoxifen (HDT) treatment from FIB-SEM 3D tomography. G = representative secretory granules; N = nuclei. Scale bar, 2μm. Right - Stacked EM images rendered into 3D projections using Amira software. Cells outlined by dashed yellow line.

B. Representative immunofluorescence images of stomach unit bases at homeostasis, 6 hour, and 24h HDT stained with GIF (red, granules) and LAMP1 (green, lysosomes) Scale bar, 25μm. Stomach unit base outlined by dashed white line.

C. Representative TEM images of normal and injured ZCs (scale bar, 2μm). Right (insets)- higher magnification images of boxed regions from left show emergence of double-membraned autophagic vesicles engulfing mature granules, mitochondria, and rough ER 24h after HDT injury. Scale bar, 500nm. Arrowheads mark vesicular degradation events in the cell lacking double membranes. Cells outlined by dashed yellow line.

D. Graph of the number of zymogenic chief cell granules per cell in vehicle vs. 24h HDT treatment. Each data point=total granules in a cell in a single TEM section with cells from 2 separate mice plotted together (each cell is a data point; cells from the same mouse are grouped by color shade). Red line indicates mean across all the mice.

E. Graph of the number of degradation events scored as in D. A degradation event was counted if electron dense-cargo or organelles were surrounded by a single membrane (lysosome) or double membrane (autophagosome). Red line indicates mean across all the mice.

F. Representative TEM images of normal and injured acinar cells. Scale bar, 2μm. Right (insets)- higher magnification images of boxed regions from left show emergence of double-membraned autophagic vesicles engulfing mature granules, mitochondria, and rough ER 24h after cerulein (CER) injury. Scale bar, 500nm. Arrowheads mark degradation events without double membranes.

G. Graph of the number of acinar cell granules per cell in vehicle vs. 24h CER treatment. Each data point=total granules in a cell in a single cell counted from one mouse. Red line indicates mean.

H. Graph of the number of degradation events scored in vehicle vs. 24h CER treatment. Each data point=total events in a cell in a single cell counted from one mouse. Red line indicates mean.

I. mRNA expression by qRT-PCR of Rab5, Rab7a, Rab7b, and Rab9. Significance determined by one-way ANOVA, Tukey post hoc. Each data point represents the mean of triplicate technical replicates from a single mouse, n=3-4 mice in independent experiments. Error bars denote standard deviation.
ATF3 induces RAB7 to govern autodegradation in paligenosis, a conserved cell plasticity program (2021)
Figure 2

Figure 2. ATF3 transcriptionally upregulates Rab7b early in paligenosis, and RAB7 protein is associated with large lysosomal aggregates

A. Top - Representative RAB7 expression in base of stomach units (ZCs) in vehicle or 24h HDT by immunohistochemistry. Arrowheads point to RAB7 vesicle aggregates. Scale bar 20µm. Bottom - Representative ATF3 expression in base of stomach units (ZCs) by immunohistochemistry. ATF3 appears in nuclei following 24h high-dose tamoxifen (HDT). Scale bar 20µm. Stomach unit base outlined by dashed black line.

B. Atf3 mRNA expression by qRT-PCR in vehicle, 6 hour, 24 hour, and 72 hour HDT treatment. Each data point represents the mean of triplicate technical replicates from a single mouse, n=3-4 mice in independent experiments. Significance determined by One-way ANOVA, Tukey. Error bars denote standard deviation.

C. Western blot of ATF3 expression over HDT and CER timecourses with β-Actin loading control.

D. Top­ - Representative RAB7 staining in pancreatic acinar cells. Arrowheads indicate RAB7 vesicles. Scale bar 20µm. Bottom - Representative ATF3 expression in pancreatic acinar cells. Nuclei express ATF3 after 24h cerulein (CER) injury. Scale bar 20µm.

E. ChIP-PCR on mouse stomach tissue at vehicle and 6 hour HDT. Chromatin probed with ATF3, Histone H3 antibodies, or a Rabbit IgG control. Also included is the 2% chromatin input and water to control for DNA contamination. Rab7b amplicon is from a conserved, putative ATF3-binding site in the first intron. Amplicon from Atf3 is a positive control from a previously characterized ATF3-binding site; negative control is from a site in Rab9a lacking putative ATF3 binding motifs.
ATF3 induces RAB7 to govern autodegradation in paligenosis, a conserved cell plasticity program (2021)
Figure 3

Figure 3. ATF3 transcriptionally upregulates RAB7B in vitro to govern formation of autodegradative vesicles

A. mRNA expression of ATF3 by qRT-PCR in control, ATF3 knockdown (KD), and ATF3 overexpression (OE) AGS cell lines generated using a PiggybacTM transposon system. Significance by one-way ANOVA, Dunnett post-hoc. Datapoint = mean of technical triplicates in a single mouse. n=3 mice. Error bars denote standard deviation.

B. mRNA expression of RAB7B by qRT-PCR in AGS cell lines from 3A. Statistics and plotting as for panel A.

C. Top - Immunofluorescence for RAB7 (red) and DAPI (blue, nuclei) in control, ATF3 knockdown (KD), and ATF3 overexpression (OE) AGS cells. Arrowhead indicates aggregated RAB7 vesicular formation similar to those observed in tissue. Bottom - Immunofluorescence for ATF3 (purple) and Cathepsin D (CTSD, green, lysosomes) in AGS cells. Scale bar, 20µm.

D. Left - Immunofluorescence for anti-FLAG (RAB7B) (red) and DAPI (blue, nuclei) in ATF3 knockdown; RAB7B overexpression (ATF3-KD RAB7B-OE) AGS cells show large vesicular structures. Right - Immunofluorescence for anti-FLAG (RAB7B) (red) and DAPI (blue, nuclei) in ATF3 overexpression; RAB7B GTPase mutant (ATF3-OE RAB7BT22N) AGS cells fail to make vesicles. Scale bar, 20µm.
ATF3 induces RAB7 to govern autodegradation in paligenosis, a conserved cell plasticity program (2021)
Figure 4

Figure 4. Atf3 coordinates cellular autodegradation after injury in paligenosis stage I

A. Representative immunofluorescence images of pancreatic acinar cells after vehicle or 24h cerulein (CER) treatment stained for RAB7 (red) and Cathepsin D (CTSD, green, lysosomes). Top row is wild-type, bottom row is Atf3−/−. Yellow arrowhead highlights RAB7 and CTSD co-expression. White arrowhead points to an area of RAB7 expression only. Scale bar, 20μm.

B. Representative immunofluorescence images of gastric chief cells after vehicle or 24h high-dose tamoxifen (HDT) treatment stained for RAB7 (red) and Cathepsin D (CTSD, green, lysosomes). Top row is wild-type, bottom row is Atf3−/−. Yellow arrowhead highlights RAB7 and CTSD co-expression. White arrowhead points to an area of RAB7 expression only. Scale bars 20μm. Stomach unit base outlined by dashed white line.

C. Representative immunofluorescence images of gastric chief cells after vehicle or 24h HDT treatment stained for Pepsinogen C (PGC, red, granules) and MAP1LC3B (green, autophagy marker). Top row is wild-type, bottom row is Atf3−/−. Scale bar, 50μm. Stomach unit base outlined by dashed white line.

D. Transmission electron micrograph images of zymogenic chief cells (ZCs) after 24h vehicle or HDT treatment in WT (top) and Atf3−/− mice (bottom). Scale bar, 1μm. Arrowheads indicate cell components being degraded.

E. Quantification of degradation events per cell counted from EM images of ZCs after 24h HDT treatment in WT and Atf3−/−mice as shown in panel 3D. Each data point=total granules in a cell in a single TEM section with cells from 2 separate mice plotted together (data point colors indicate cells from a single mouse). Red line indicates mean across all the mice.
ATF3 induces RAB7 to govern autodegradation in paligenosis, a conserved cell plasticity program (2021)
Figure 5

Figure 5. Atf3 loss increases cell death and reduces proliferation after paligenotic injury

A. Representative immunofluorescence images of gastric units after vehicle or 72h high-dose tamoxifen (HDT) treatment stained for GIF (red, granules), GSII (green, neck cells and SPEM marker), BRDU (white, proliferation) and DAPI (blue, nuclei). Top row is wild-type, bottom row is Atf3−/−. Yellow arrowheads point to areas of base dropout where ZCs have been lost. Scale bar, 50μm. Stomach units outlined by dashed white line.

B. Proliferation as quantified by number of BrdU+ cells per unit in WT and Atf3−/−vehicle±HDT. Each data point = mean of counts from 40-50 gastric glands from an individual mouse, n=3-4 mice per treatment group. Significance by unpaired two-tailed t-test. Error bars denote standard deviation.

C. Plot and statistical analysis as for Panel B but quantifying BrdU+ cells only in the bottom 1/3 of the unit, which are derived mostly from paligenosis and not from isthmal and neck stem and progenitor cells.

D. Death of ZCs after injury plotted by quantifying the base dropout rate, which is the fraction of glands that do not reach the muscularis mucosa from the lumen. Each data point is the mean of 80-100 glands from an individual mouse, n=4 mice per treatment group. Significance by unpaired two-tailed t-test. Error bars denote standard deviation.

E. Representative immunohistochemistry of Cleaved-Caspase 3 in pancreatic acinar cells marking apoptosis in vehicle or 24h cerulein (CER) treatment. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm.

F. Apoptosis quantified following 24h CER injury by the number of Cleaved-Caspase 3+ cells from a 40x field. Each data point is mean of positive cells in 4-5 40x fields in an individual mouse, n=3 mice per treatment group. Significance by unpaired two-tailed t-test. Error bars denote standard deviation.

G. Representative immunohistochemistry of BrdU in pancreatic acinar cells marking proliferation in vehicle or 7 days of repeated cerulein injury. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm.

H. Quantification of proliferation at peak ADM as counted by the number of BrdU+ cells from a 20x field following 7D cerulein treatment. Plotting and statistical analysis as per panel F.
ATF3 induces RAB7 to govern autodegradation in paligenosis, a conserved cell plasticity program (2021)
Figure 6

Figure 6. ATF3 is expressed in human SPEM

Serial sections of human tissue. Representative immunohistochemistry of GSII (top; spasmolytic polypeptide-expressing metaplasia [SPEM] marker and neck cells) counterstained with hematoxylin (blue) and eosin (pink). Representative staining of ATF3 (bottom) counterstained with eosin (pink). Tissue was taken from regions adjacent to gastric adenocarcinoma. Scale bars left - 50μm right - 20μm. Arrowheads indicate strongly expressing ATF3 cells and the corresponding region in the same gland labeled with GSII in a serial tissue section. GSII at the base of gastric glands in the body of the stomach indicate transition to SPEM.
The association of AMPK with ULK1 regulates autophagy. (2010)
Figure 1

AMPK interacts with ULK1 directly.

(A) 293T cells were transfected with Flag-ULK1 or control empty vector and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblot analysis with the indicated antibodies. (B) 293T cells were co-transfected with Flag-AMPKα2 and HA-ULK1. Cell lysates were subjected to immunoprecipitation using anti-Flag antibody followed by SDS-PAGE/immunoblot analysis with anti-HA and anti-Flag antibodies. (C) 293T cells were transiently transfected with Flag-AMPKα2 or Flag-AMPKβ1. After 24 h, immunoprecipitation was performed using anti-Flag or control anti-HA antibodies and analyzed by immunoblotting with anti-ULK1 and anti-Flag polyclonal antibodies. (D) 293T cell lysates were subjected to immunoprecipitation with anti-ULK1 polyclonal antibody or control preimmune rabbit serum (NRS), followed by immunoblot analysis with anti-ULK1 and anti-AMPKα antibodies. (E) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti-AMPKα antibody followed by immunoblotting with anti-ULK1 and anti-AMPKα antibodies. (F) Purified His6-AMPKα1/β1/γ1 fusion proteins (250 ng) were mixed with purified His6-tagged ULK1 (1 µg) or Bcl-XL (200 ng) proteins in 1% Triton X-100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti-ULK1 antibody. The resulting protein complexes and 10% of the input proteins were analyzed by immunoblotting with anti-His-Tag polyclonal antibody.
The association of AMPK with ULK1 regulates autophagy. (2010)
Figure 2

AMPK and mTORC1 interact with ULK1 through the PS domain and the kinase domain of ULK1, respectively.

(A) A schematic of ULK1 illustrating its kinase domain, proline/serine-rich domain, C-terminal (CT) domain, and regions for binding to AMPK and mTORC1. (B, C) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutants of ULK1 and subjected to immunoprecipitation with anti-HA antibody. The resulting immune complexes were analyzed by immunoblotting with antibodies specific for mTOR, raptor, AMPKα or HA-tag.
The association of AMPK with ULK1 regulates autophagy. (2010)
Figure 3

The AMPK-ULK1 interaction is important for ULK1-mediated autophagy.

(A) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutant (Δ654–828) ULK1 and subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies. (B) U-2OS cells stably expressing GFP-LC3 were infected with lentivirus expressing human Ulk1 shRNA (shhUlk1) or scrambled shRNA (shScr) and subjected to selection with 1 µg/ml puromycin for 5 days. The cells were then transfected with wild type mouse Ulk1 (mUlk1), mUlk1 Δ654–828 or control empty vector for 24 h, incubated in serum-free DMEM overnight, and cultured in complete medium with or without 10 mM metformin for 20 h. The images were obtained using a fluorescence microscope. The scale bars represent 10 µm. (C) The number of GFP-LC3 dots per GFP-positive cell in (B) was quantified (mean ± s.d.; n = 81). (D) U-2OS cells stably expressing GFP-LC3 were transfected with 0, 2 or 4 µg of pcDNA3 encoding HA-tagged ULK1 deletion mutant (554–828). The total amount of plasmid DNA used for each transfection was normalized at 4 µg with empty vector. After 24 h transfection, the cells were starved in nutrient-free medium for 1.5 h, fixed in 4% paraformaldehyde, and analyzed by fluorescence microscopy. The number of GFP-LC3 dots per GFP-positive cell was quantified (mean ± s.d.; n = 100). Statistical significance was determined by Student's t-test and the asterisks indicate P<0.0001.
The association of AMPK with ULK1 regulates autophagy. (2010)
Figure 5

Raptor phosphorylation by AMPK is required for AICAR-induced autophagy in TSC2<sup>−/−</sup> MEF cells.

(A) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were transfected with GFP-LC3, treated with 2 mM AICAR or control vehicle for 4 h, and then analyzed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was counted (mean ± s.d.; n = 60). The scale bars represent 10 µm. (B) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were treated with 2 mM AICAR for the indicated times. Cell lysates were prepared in RIPA buffer and analyzed by immunoblotting with the indicated antibodies. The levels of p62 are listed relative to those of untreated WT raptor cells, which were set as 1. (C) Schematic representation of AMPK-mediated suppression of the inhibitory effect of mTORC1 on the ULK1 autophagic complex.
DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. (2002)
Figure 1.

Morphological changes induced by DAPk family proteins. (A) Quantification of cell death in 293 cells 24 h after transfection with luciferase, p55/TNFR1, DRP-1 Δ73 or DAPk ΔCaM, and GFP. Graphs show the percentage of GFP-positive cells with altered cell morphology. (B) Photographs of GFP-positive cells cotransfected with luciferase (1), p55/TNFR1 (2), DRP-1 Δ73 (3), or DAPk ΔCaM (4). (C) Photographs showing the overlay of GFP and phase–contrast images of 293 cells 24 h after transfection with GFP and luciferase (1) or with GFP-DAPk (2 and 3); both a blebbing GFP-DAPk–expressing cell (2, arrows indicate blebs) and spread nontransfected cells are shown (3). (D) Hoechst staining (4–6) of GFP-positive 293 cells (1–3) 60 h after cotransfection with GFP and DAPk ΔCaM (1 and 4), DRP-1 Δ73 (2 and 5), or p55/TNFR1 (3 and 6). Arrows point to condensed nuclei resulting from DRP-1 Δ73 or DAPk ΔCaM transfections (4 and 5).
DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. (2002)
Figure 2.

DAPk family proteins do not induce cell death via the mitochondrial pathway. (A) Scoring loss of mitochondrial membrane potential (ΔΨm). 293 cells expressing luciferase (black), DRP-1 Δ73 (pink), DAPk ΔCaM (purple), and Bax (green) were assayed for loss of mitochondrial membrane potential 24 h after transfection using flow cytometric analysis of TMRM fluorescence. The fraction of TMRM-negative cells calculated from three experiments is shown in the graph below. The small increase in TMRM fluorescence in the kinase-transfected cells is not statistically significant. (B) Scoring the release of cytochrome C from mitochondria. 293 cells cotransfected with luciferase (1 and 2), Bax (3 and 6), DRP-1 Δ73 (7 and 8), or DAPk ΔCaM (9 and 10) and GFP constructs were incubated for 24 h and immunostained using anti–cytochrome C antibodies. Cells were visualized for cytochrome C (left) or GFP (right) fluorescence. Bax-transfected spread (3 and 4) or blebbed (5 and 6) cells are shown. In fields that also contain nontransfected cells, arrows point to GFP-positive cytochrome C–stained cells. (C) Assessing the effects of Bcl-2 and Bcl-XL. 293 cells were cotransfected with luciferase, DRP-1 Δ73, DAPk ΔCaM, or Bax, and either Bcl-2 or Bcl-XL together with GFP. GFP-positive cells were visualized by fluorescent microscopy and scored for the appearance of cell death morphology 24 h after transfection. Total cell death refers to the sum of all morphological changes observed.
DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. (2002)
Figure 3.

High resolution morphology of DAPk family protein-induced cell death. (A) Transmission electron micrographs of 293 cells transfected with luciferase (1) p55/TNFR1 (2a–2c), DRP-1 Δ73 (3a and 3b; part of fig. 3b is magnified on its right), and DAPk ΔCaM (4). The different stages of autophagic development are depicted as follows: immature autophagic vesicles (double arrow), mature autophagic vesicles and autolysosomes (black arrow), and residual bodies (dashed arrow). Empty vacuoles (white arrow) are also shown. Arrowheads show condensed and fragmented chromatin. m, mitochondria; g, Golgi apparatus. Bar, 1 μm. (B) Transmission electron micrographs of MCF-7 cells transfected with luciferase (1), p55/TNFR1 (2a–2c), DRP-1 Δ73 (3a and 3b), and DAPk ΔCaM (4). Immature autophagic vesicles (double arrow), autophagic vesicles (black arrow), and residual bodies (dashed arrow) are shown. Insets in B, 2b and 2c, correspond to empty vacuoles, whereas those in B, 3a and 4, correspond to autophagic vesicles. Arrowheads show condensed and fragmented chromatin. m, mitochondria; dm, darkened mitochondria; n, nucleus; g, Golgi apparatus; v, vacuoles; ly, lysosomes; er, endoplasmic reticulum.
DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. (2002)
Figure 4.

DRP-1 induces autophagy in various cell lines. (A) Quantification of autophagy in 293 and MCF-7 cells. Graphs show the percentage of MDC-positive cells resulting from luciferase or DRP-1 Δ73 transfections (mean ± SD calculated from triplicates of 100 cells each; note that total cell number comprises transfected and nontransfected cells). These experiments were repeated three times with reproducible results. (B) Photographs of MDC-stained MCF-7 cells from the same experiment shown in A. Arrows point to MDC-positive cells. Transfections: luciferase (1a and 1b) and DRP-1 Δ73 (2a and 2b) at low (a) and high (b) magnifications.
DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. (2002)
Figure 5.

DAPk family proteins induce caspase-independent cell death in various cell lines. (A) Quantification of cell death-associated morphologies in 293 cells. Graphs show the percentage of cells harboring all spectrum of morphological changes occurring as a result of luciferase, p55/TNFR1, DRP-1 Δ73, or DAPk ΔCaM transfections in the presence or absence of the caspase inhibitors BD-fmk or z-VAD-fmk (100 μM; mean ± SD calculated from triplicates of 100 cells each). This experiment was repeated three times with reproducible results. (B) Quantification of cell death-associated morphologies in MCF-7 cells. Experimental conditions were performed as in A in the presence or absence of BD-fmk (100 μM). (C) Graphs show the percentage of cells harboring the complete spectrum of death-associated morphological changes occurring as a result of cotransfections of luciferase, p55/TNFR1, DRP-1 Δ73, or DAPk ΔCaM with the caspase inhibitor crmA or control luciferase vector (mean ± SD calculated from triplicates of 100 cells each). Immunoblot shows the expression levels of transfected HA-tagged DAPk and DRP-1 Δ73, confirming that cotransfection with crmA did not alter expression of the death-inducing proteins. (D) Transmission electron micrographs of 293 cells transfected with p55/TNFR1 (1) or DRP-1 Δ73 (2a and 2b; parts of these figures are magnified at the right) and treated with the caspase inhibitor BD-fmk (100 μM). Immature autophagic vesicles (double arrow), autophagic vesicles (black arrow), and autolysosomes (white arrow) are shown. m, mitochondria; g, Golgi apparatus. (E) Western blots of cell lysates prepared from HeLa cells transfected with luciferase, p55/TNFR1, DRP-1 Δ73, or DAPk ΔCaM and GFP and incubated for 24 h. Blots were reacted with anti-PARP antibodies, anti–caspase-8 antibodies, anti–caspase-3 antibodies, anti-HA antibodies for DRP-1 Δ73 and DAPk ΔCaM detection, and anti–β-tubulin antibodies to quantitate protein loading.
DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. (2002)
Figure 6.

DRP-1 and DAPk dominant negative mutants reduce membrane blebbing resulting from p55/TNFR1 transfections in 293 cells. (A) 293 cells were cotransfected with p55/TNFR1 in combination with DAPk death domain fused to GFP or DRP-1 K42A and pEGFP-N1 vector. A control luciferase vector was also used. GFP-positive cells were visualized by fluorescent microscopy and scored for the appearance of membrane blebbing 24 h after transfection. (B) Photographs of the various categories of p55/TNFR1-transfected 293 cells: spread cells (1), blebbed cells (2), condensed cells (3), cells displaying a fragmented cytoplasm (4), and cells carrying fragmented nuclei within a noncondensed cytoplasm (5). (C) Quantification of each death morphology observed 24 h after transfection with GFP, p55/TNFR1, and either luciferase or DRP-1 K42A.