EMBO/soda-nel huggingface dataset:
Paneth cells as a site of origin for intestinal inflammation (2013)
f1

a, b, Immunoblot for LC3 conversion in isolated primary intestinal epithelial cells (a) (n = 5/4) and for autophagy proteins in primary intestinal epithelial cell scrapings (b) (n = 3). WT, wild type. c, Transmission electron microscopy (TEM) of crypts. Note autophagic vacuoles in various stages of evolution in Xbp1Dgr;IEC hypomorphic Paneth cells. d, e, Crypt showing GFP–LC3 punctae (d), quantified in e (n = 10; unpaired Student’s t-test; mean ± s.e.m.). Scale bar, 5 &mgr;m. f, g, Immunoblot of silenced MODE-K cells (f) and primary intestinal epithelial cell scrapings (g) for the PERK–eIF2&agr; branch (n = 3). h, i, Promoter sequence qPCR for Map1lc3b (LC3b) (h) and Atg7 (i) after anti-ATF4 ChIP (unpaired Student’s t-test; mean ± s.e.m.). j, GFP–LC3 punctae per crypt after treatment with tamoxifen, and vehicle or salubrinal (n = 10; one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.). k, Enteritis histology score after salubrinal and tamoxifen co-treatment (n = 12/14/13; median shown; Kruskal–Wallis with post-hoc Holm’s-corrected Mann–Whitney U-test). Results represent three (a, f, g) or two (c, e, h, i) independent experiments. *P  0.05, ***P  0.001.
Paneth cells as a site of origin for intestinal inflammation (2013)
f2

a, TEM of crypts of Lieberkühn (n = 2). Scale bars, 2 &mgr;m. b, Representative haematoxylin and eosin stainings. Note transmural inflammation extending through muscularis propria (white arrow) into serosa (black arrow) in Atg7/Xbp1Dgr;IEC and Atg16l1/Xbp1Dgr;IEC mice scored in c and e. Scale bars, 50 &mgr;m, except for lower two panels (10 &mgr;m). c, Enteritis histology score (n = 26/12/18/27; 10–18 weeks; median shown; Kruskal–Wallis with post-hoc Holm’s-corrected Mann–Whitney U-test). d, Crypt TEM in indicated genotypes (n = 2). Scale bars, 2 &mgr;m. e, Enteritis histology score (n = 11; 18 weeks; median shown; Kruskal–Wallis with post-hoc Holm’s-corrected Mann–Whitney U-test). NS, not significant. f, Xbp1 mRNA splicing (Xbp1u, unspliced; Xbp1s, spliced) of crypts; densitometry in g (n = 7; unpaired Student’s t-test; mean ± s.e.m.). h, GRP78 (green) immunofluorescence, white arrows indicate GRP78+ crypts (DAPI, blue; n = 5). Scale bars, 10 &mgr;m. *P  0.05, **P  0.01, ***P  0.001.
Paneth cells as a site of origin for intestinal inflammation (2013)
f3

a, shCtrl or shXbp1 MODE-K cells were co-silenced for Atg16l1 (siAtg16l1) or with scrambled siRNA (siCtrl), and analysed by flow cytometry for annexin V and propidium iodide (PI; n = 3; one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.). b, Immunoblot of primary intestinal epithelial cell scrapings (n = 3). c, Immunoblot of cytoplasmic extracts from shCtrl or shXbp1 MODE-K cells after TNF stimulation. d, TUNEL+ intestinal epithelial cells (IECs) per 100 crypts after BAY11-7082 or vehicle treatment (n = 3/4/4; one-way ANOVA with post-hoc Holm’s-corrected unpaired Student’s t-test; mean ± s.e.m.). e, Enteritis histology score of mice treated with BAY11-7082 or vehicle (n = 10/10/9; median shown; Kruskal–Wallis with post-hoc Holm’s-corrected Mann–Whitney U-test). f, Immunoblot of intestinal epithelial cell scrapings for (p-)IRE1&agr; after IRE1&agr; immunoprecipitation (IP). &bgr;-Actin, loading control of whole lysates. g, Immunoblot of primary intestinal epithelial cell scrapings (n = 4). h, i, Enteritis histology score of indicated genotypes (h, n = 15/16/14/15; i, n = 5/10/12; median shown; Kruskal–Wallis with post-hoc Holm’s-corrected Mann–Whitney U-test). j, Enteritis histology score of specific pathogen-free (SPF) and germ free (GF) housed mice (n = 10/9/7/7; median shown; Kruskal–Wallis with post-hoc Holm’s-corrected Mann–Whitney U-test). k, Representative images of p-I&kgr;B&agr; immunoreactivity under conditions as in j (n = 4). Scale bars, 20 &mgr;m. Results represent four (f), three (c) or two (a, b) independent experiments. *P  0.05, **P  0.01, ***P  0.001.
Paneth cells as a site of origin for intestinal inflammation (2013)
f4

a, Immunoblot of primary intestinal epithelial cell scrapings from mice treated with or without rapamycin for 14 consecutive days (n = 3). b, Enteritis histology score for experiment as in a (n = 4; median shown; Mann–Whitney U-test). c, Representative images of EYFP-Rosa26/D6-cre+/– reporter mice and EYFP-Rosa26 (controls). Co-localization of Defa6 Cre-driven EYFP expression (yellow) with lysozyme-expressing Paneth cells (red; n = 3). DAPI, blue; scale bars, 50 &mgr;m. d, Immunoblots of crypt intestinal epithelial cells from Xbp1Dgr;PC and wild-type controls (n = 2). e, f, Representative confocal images of lysozyme (green) expressing Paneth cells (e) with quantification of crypts with indicated number of lysozyme+ granulated dots in f (n = 5; unpaired Student’s t-test; mean ± s.e.m.). DAPI, blue; scale bars, 10 &mgr;m. g, Immunohistochemistry for p-eIF2&agr; (n = 3). Scale bars, 20 &mgr;m. h, Representative haematoxylin and eosin images of Xbp1Dgr;PC and wild-type mice scored in i. Scale bars, 50 &mgr;m. i, Enteritis scoring in Xbp1fl/fl (WTfl), Defa6-cre+ (WTcre) and Defa6-cre+;Xbp1fl/fl (Xbp1Dgr;PC) mice (n = 21/26/29; median shown; Kruskal–Wallis with post-hoc Holm’s-corrected Mann–Whitney U-test). Results represent two (b, d) independent experiments. *P  0.05, **P  0.01, ***P  0.001.
Feeding activates FGF15-SHP-TFEB-mediated lipophagy in the gut (2022)
Figure 1

Figure 1. Paradoxical feeding activation of gut autophagy is dependent on both SHP and FGF15.

(A-E) C57BL/6, SHP-KO, and FGF15-KO or littermate mice were fasted overnight and then re-fed for 6 h.

(A-C) Levels of the indicated proteins detected by IB in (A) liver and intestinal extracts from C57BL/6 mice and in intestinal extracts from (B) C57BL/6 or SHP-KO mice or (C) FGF15-KO mice or littermates. Band quantitation was done using Image J, and the LC3 II/I ratio and p62/actin and lamp1/actin ratios are shown at the right.

(D, E) Endogenous LC3 was detected by IHC in intestinal sections from (D) SHP-KO and C57BL/6 mice or (E) FGF15-KO mice and littermates. Scale bar, 20 μm.

(A-C) The mean and standard deviation (SD) are plotted. Statistical significance was determined by (A) two-tailed Student's t-tests or (B, C) two-way ANOVA with the Tukey post-test. P-values are indicated (n=3 mice).
Feeding activates FGF15-SHP-TFEB-mediated lipophagy in the gut (2022)
Figure 4

Figure 4. Feeding-mediated increases in intestinal TG and ApoB48 levels are enhanced in SHP-KO and FGF15-KO mice.

(A-F) C57BL/6, SHP-KO, and FGF15-KO or littermate mice as indicated were fasted for 12 h and then, re-fed for 6 h.

(A-D) Intestinal TG levels in (A) C57BL/6 and SHP-KO mice or (C) FGF15-KO mice and littermates and levels of the indicated proteins in intestinal extracts from (B) C57BL/6 and SHP-KO mice or (D) FGF15-KO mice and littermates with quantitation of the bands at right. (A, C) n=3-5 mice, (B, D) n=3 mice.

(E, F) Endogenous ApoB48 levels were detected by IHC in intestinal sections of SHP-KO and C57BL/6 mice (E) or FGF15-KO mice and littermates (F). Scale bar, 20 μm.

(G) HT29 cells were transfected with GFP-LC3 and with SHP siRNA or SHP expression plasmids and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h as described in Materials and Methods. The control group (Veh) was treated with vehicle for FGF19, control plasmid DNA, and scrambled siRNA. The FGF19 groups were treated with control DNA or SHP expression plasmid or SHP siRNA or control scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20). Scale bar, 10 μm.

(H) HT29 cells were treated with 50 ng/ml FGF19 with or without 100 nM bafilomycin A1 for 12 h. LC3 and actin levels in cell extracts were determined by IB. Quantification of bands is shown at the right (n=3 culture plates).

(I) HT29 cells were transfected with Lamp1 siRNA or scrambled siRNA and 48 h later were treated with 50 ng/ml FGF19 for the indicated times. ApoB48 and actin levels were determined by IB (n=2 culture plates).

(A-D, G, H) The mean and SD are plotted. Statistical significance was determined by (A-D, H) two-way ANOVA with the Tukey post-test or (G) one-way ANOVA with the Tukey post-test. P-values are indicated, (A, C) n=3-5 mice, (B, D) n=3 mice.
Feeding activates FGF15-SHP-TFEB-mediated lipophagy in the gut (2022)
Figure 5

Figure 5. In HT29 cells, FGF19 activates lipophagy in a manner dependent on SHP, and FGF19 reduces TG and ApoB48 levels via lipophagy.

(A) HT29 cells were transfected with GFP-LC3 and with Atg7 siRNA or control scrambled RNA (siCtl) and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h as described in Materials and Methods. The control group (Veh) was treated with vehicle for FGF19 and control siRNA. The FGF19 groups were treated with Atg7 siRNA or scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20). Scale bar, 20 μm.

(B) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA and 48 h later were treated with 50 ng/ml FGF19 for 12 h. LC3 and actin levels in cell extracts determined by IB (n=3 culture plates).

(C, D) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA (siCtl) for 48 h and then treated with oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 12 h.

(C) Levels of TG in cell extracts (n=5 culture plates).

(D) ApoB48 and actin levels in cell extracts determined by IB. Bands were quantified and the LC3-II/LC3-I ratio is shown at the right (n=3 culture plates).

(A-D) The mean and SD are plotted. Statistical significance was determined by one-way ANOVA with the Tukey post-test. P-values are indicated.
Feeding activates FGF15-SHP-TFEB-mediated lipophagy in the gut (2022)
Figure 6

Figure 6. TFEB partners with SHP in the transcriptional induction of Ulk1 and Atgl.

(A) Transcription factor binding motifs were identified de novo with MEME-ChIP analysis. Selected unique motifs within SHP binding peak regions for intestinal genes upregulated and downregulated by SHP binding are shown.

(B-D) Mice were re-fed for 2 h (B) or 4 h (C, D) after fasting overnight.

(B) CoIP of TFEB with SHP. Upper panel: Levels of TFEB detected by IB in SHP and IgG immunoprecipitated samples. Lower panel: levels of TFEB and SHP in input samples detected by IB. Similar results were obtained in a second experiment.

(C) Binding of the indicated factors at intestinal Ulk1 determined by ChIP (n=3 mice).

(D) Co-occupancy of TFEB and SHP at Ulk1 and Atgl determined by re-ChIP (n=3 mice).

(E) HT29 cells were transfected with siRNA for Shp and/or Tfeb and then, 24 h later, further transfected with indicated plasmids for 24 h, and the cells were treated with FGF19 (50 ng/ml) for 8 h. Luciferase activities normalized to β-galactosidase activity are plotted (n=3 culture plates).

(F, G) Protein levels detected by IB of ULK1 and ATGL in intestinal extracts from (F) C57BL/6 and SHP-KO mice or (G) FGF15-KO mice or littermates re-fed for 6 h after fasting overnight (n=3 mice).

(C-E) The mean and SD are plotted. Statistical significance was determined by (C, D) two-way ANOVA with the Tukey post-test or (E) one-way ANOVA with the Tukey post-test. P-values are indicated.
Feeding activates FGF15-SHP-TFEB-mediated lipophagy in the gut (2022)
Figure 7

Figure 7. TFEB is critical for FGF19-mediated lipophagic activation and decreased TG levels in HT29 cells.

(A) HT29 cells were transfected with GFP-LC3 and with Tfeb siRNA or Tfeb expression plasmids and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h as described in Materials and Methods. The control group (Veh) was treated with vehicle for FGF19 and transfected with control plasmid DNA and scrambled siRNA. The FGF19 groups were treated with control DNA or TFEB expression plasmid or with Tfeb siRNA or scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20 cells). Scale bar, 10 μm.

(B) HT29 cells were transfected with siRNA for Tfeb (siTFEB) or control scrambled siRNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle with or without 100 nM bafilomycin A1 (BAF) for 12 h. Levels of the indicated proteins determined by IB. LC3 II/I and p62/actin ratios are shown at the right (n=3-4 culture plates).

(C) HT29 cells were transfected with siRNA for TFEB (siTFEB) or control RNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle for 12 h. Protein levels detected by IB of ULK1 and ATGL (n=3 culture plates).

(D) HT29 cells were treated with 50 ng/ml FGF19 for 12 h. Levels of triglycerides (TG) in cell extracts (n=5 culture plates).

(E) HT29 cells were transfected with siRNA for Tfeb (siTFEB) or scrambled siRNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle for 12 h. ApoB48 and actin levels in cell extracts determined by IB (n=3 culture plates).

(A, B, D) The mean and SD are plotted. Statistical significance was determined by (A) one-way ANOVA with the Tukey post-test, (B) two-way ANOVA with the Tukey post-test, or (D) two-tailed Student's t-tests. P-values are indicated.
Feeding activates FGF15-SHP-TFEB-mediated lipophagy in the gut (2022)
Figure 8

Figure 8. Feeding-induced FGF15/19 signaling increases nuclear localization of both TFEB and SHP via PKC-mediated phosphorylation in mouse intestine and HT29 cells.

(A, B) C57BL/6 (A) and FGF15-KO mice or littermates (B) were re-fed for 6 h after fasting overnight. The cytosolic and nuclear fractions were isolated from intestine or liver, and the indicated proteins were detected by IB (n=2 mice). TBLN indicates tubulin.

(C-D) FGF15-KO mice or littermates were fasted overnight and re-fed for the indicated times. (C) The nuclear fraction was isolated and the nuclear levels of TFEB, SHP, and LAMIN were determined by IB.

(D) Levels of LC3, p62, and actin in cell extracts were determined by IB.

(E) HT29 cells were cultured overnight in serum free media and treated with vehicle or 50 ng/ml FGF19 for 30 min. The indicated proteins were detected by IB (n=3 culture plates).

(F) FGF15-KO mice or littermates were re-fed for 0, 2, or 3 h after fasting overnight. The indicated proteins in intestinal extracts were detected by IB (n=2 mice).

(G) HT29 cells were transfected with expression plasmids for flag-tagged TFEB or its mutants as indicated and 24 h later, cells were treated with FGF19 (50 ng/ml) for 12 h. ∆CT indicates the TFEB mutant with a C-terminal deletion of 15 amino acids. The indicated proteins were detected by IB (n=3 culture plates).

(H) Model of the opposite feeding regulation of autophagy in liver and intestine. Feeding activates intestinal lipophagy, paradoxically in the nutrient-rich fed conditions, limiting postprandial TG levels. Mechanistically, in mouse intestine and intestinal cells, postprandial FGF15/19 signaling increases nuclear translocation of both TFEB and SHP via PKCβ/ζ-mediated phosphorylation at S461/S462 in TFEB and PKCζ-mediated phosphorylation at T55 in SHP, leading to transcription activation of autophagy genes, Ulk1 and Atgl, in the late fed-state. In contrast, in the liver, in response to feeding-induced FGF15/19 signaling, TFEB is exported to the cytoplasm via mTOR/ERK-mediated phosphorylation at S211 and SHP is translocated into the nucleus via PKCζ-mediated T55 phosphorylation, leading to transcription repression of autophagy network genes, including Tfeb, Atg3, and Atg7.
Mechanosensor Piezo1 Mediates Bimodal Patterns of Intracellular Calcium and FAK Signaling (2022)
Figure 2

Figure 2. The Piezo1 dependent calcium influx upon LIS stimulation in HEK cells.

(A) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor before and after shockwave stimulation in HBSS Ca2+ medium with (yellow line, n = 6, N = 3) or without the Piezo1 expression (black line, n = 6, N = 3).

(B) The representative FRET/ECFP ratio images of calcium biosensor in Piezo1-expressed HEK cells before (top left) and after (top right) LIS stimulation. Phase image with laser initiating point (left) and fluorescent image indicating Piezo1 expression (right) were also shown on the bottom panels (Scale bar, 40 μm). Color scale bars in the figures are to show the FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively.

(C) The time course of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells before and after multiple shockwave stimulations in HBSS Ca2+ medium (n = 6, N = 3).

Data information: "n" means the total cell number. "N" means the number of individual experiments.
Mechanosensor Piezo1 Mediates Bimodal Patterns of Intracellular Calcium and FAK Signaling (2022)
Figure 3

Figure 3. The Piezo1-dependent FAK activation upon LIS stimulation in HEK cells. (A) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 overexpressed HEK cells before (left) and after (right) shockwave stimulation (Scale bar, 40 μm). Color scale bars in the figures are to show the ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively.

(B) The time courses of normalized ECFP/FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after shockwave stimulation in HBSS Ca2+ media with (green line, n = 16, N = 3) or without Piezo1 (black line, n = 10, N = 3).

(C) The percentage change of FRET ratio of the FAK FRET biosensor in HEK cells with or without Piezo1 after shockwave stimulation in HBSS Ca2+ medium. *P < 0.05 from student two-tailed t-test.

Data information: "n" means the total cell number. "N" means the number of individual experiments.
Mechanosensor Piezo1 Mediates Bimodal Patterns of Intracellular Calcium and FAK Signaling (2022)
Figure 4

Figure 4. The FAK and calcium activation upon Yoda1 stimulation in the Piezo1-expressing HEK cells.

(A) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively.

(B) The time courses of normalized FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after Yoda1 stimulation with (pink line, n = 10, N = 3) or without Piezo1 (black line, n = 11, N = 3). The grey line represents the DMSO control group (n = 8, N = 3).

(C) The representative FRET/ECFP ratio images of the calcium biosensor in the Piezo1-expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively.

(D) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells (purple line, n = 9, N = 3) before and after 25 μM Yoda1 stimulation in culture medium.

Data information: "n" means the total cell number. "N" means the number of individual experiments.
Mechanosensor Piezo1 Mediates Bimodal Patterns of Intracellular Calcium and FAK Signaling (2022)
Figure 7

Figure 7. SHP2 works as a molecular switch downstream of Piezo1 to regulate FAK. (A)&(B), The time courses (Mean ± SEM) (A) and representative images (Scale bar, 10 μm) of FRET ratio of SHP2 biosensor (Y542F) before and after 25 μM or 0.5 μM Yoda1 stimulation in cells with or without Piezo1 overexpression (red bar, n = 5, N=3; blue bar, n = 5, N=3; purple bar, n = 11, N=3; green bar, n = 5, N=3).

(C). The time courses of normalized FRET ratio (Mean ± SEM) of FAK FRET biosensor under different treatments. (From Top to the bottom, n=8, 10, 14 and 14 respectively; SHP 099, the cells were pretreated with 10 μM SHP099; SHP2 C495S, the cells overexpressing SHP2 protein with a non-functional catalytic domain).

(D). The percentage change of FRET ratio (Mean ± SEM) changes of FAK biosensor in the Piezo1-expressing cells in C at 5 minutes. (From left to right, n=8, 10, 14 and 14 respectively; one-way ANOVA, **P=0.006, ****P<0.0001, NS=not significant.)

(E). Proposed working model of FAK regulation by Piezo1 in which SHP2 works as a molecular switch.

Data information: "n" means the total cell number. "N" means the number of individual experiments.
Transfer of extracellular vesicle-microRNA controls germinal center reaction and antibody production (2020)
Figure 1

Figure 1. Mature IS formation between DICER-KO B-cells and OT-II T-cells promotes class switching, proliferation, and survival

DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA.

A. Representative confocal microscopy images of CMAC-stained conjugates formed between B lymphocytes (isolated from DICER-KO mouse splenocytes and pre-activated with a mixture of LPS plus IL-4) and CD4+ T cells (from OVA-specific OT-II transgenic mice); conjugates were formed either in the presence or in the absence of OVA. A representative experiment of at least 3 independent experiments is shown.

B-E. Quantification of 3 representative experiments as in (A), showing the percentage of B cells forming conjugates (B), the accumulation at the IS of the TCR (C) and actin (D) at the IS, and the distance from the CD4+ T cell MTOC to the contact site with the B cell (E). Conjugates were assessed using the Image J plug-in for IS analysis and MTOC-B-cell distance was measured with Imaris analysis software. Bar charts are representative of the mean of n ≥ 7 cells analyzed ± SEM from at least 3 independent experiments. Significance was assessed by unpaired Student t test; *P<0.05, **P<0.01, ***P<0.001.

F-J. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA, showing (F) IgG1 expression after 24h of co-culture, (G and H) B cell proliferation after 24h, 48h and 72h co-culture (Ki67 expression in live B cells and cell violet tracer dilution, respectively), (I) survival (staining with the live marker DAPI), and (J) apoptosis (cleaved Caspase-3 expression) 24h, 48h and 72h after IS formation.

Data information: Dot plots are representative of ≥ 4 independent experiments 48h after IS formation, and bar charts show mean values ± SEM of at least 3 independent experiments at the indicated time points. Significance was assessed by paired Student t test; *P<0.05, **P<0.01, ***P<0.001.