EMBO/soda-nel huggingface dataset:
Gastric stem cells promote inflammation and gland remodeling in response to H. pylori via Rsp3-Lgr4 axis (2022)
Figure 4

Figure 4 - Rspo promotes accumulation of nuclear p65.

A Immunofluorescence labeling for p65 and E-cadherin in the mouse antrum tissue of 2 weeks infected B6-Myh11CreErt2/Rspondin3fl/fl (Rspo3-/-) and control mice. Arrows in infected control mice indicate nuclear p65 that is not observed in Rspo3-/- mice. Scale bars: 50 µm.

B Quantification of p65+ area as percentage of total DAPI-stained cells of infected Rspo3-/- and control mice (n = 3 biological replicates per group, n = 20 glands per mouse).

C qPCR expression level of Cxcl1 and Cxcl2 in 2-week infected Rspo3-/- mice (n = 3) versus infected control mice (n = 4).

D qPCR expression level of Cxcl1 and Cxcl2 in 2-month infected Rspo3-/- mice (n = 5) versus infected control mice (n = 6).

E Myh11CreERT2/Rosa26Sor6(CAG-Rspo3) (Rspo3 KI) mice were infected for 6 weeks with H. pylori. Immunofluorescence labeling for p65, GSII and KI67 in uninfected and infected Rspo3 KI and control mice. Scale bars: 50 µm.

F Quantification of p65+ area as percentage of total DAPI-stained cells of uninfected and infected Rspo3 KI and control mice (n = 2-3 biological replicates per group, n = 20 glands per mouse).

G Quantification of GSII+ area of uninfected and infected Rspo3 KI and control mice (n = 2-3 biological replicates per group, n = 20 glands per mouse).

H Quantification of Ki67 as percentage of total DAPI-stained cells of uninfected and infected Rspo3 KI and control mice (n = 2-3 biological replicates per group, n = 20 glands per mouse).

I Immunofluorescence labeling for p65 acetyl K310 in infected Rspo3 KI and control mice. Insets show magnified images. Scale bars: 50 µm.

Data information: For B, C, D, F, G and H data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test for B, C and D, one-way ANOVA + Tukey´s multiple comparison test for F, G and H).
Gastric stem cells promote inflammation and gland remodeling in response to H. pylori via Rsp3-Lgr4 axis (2022)
Figure 5

Figure 5 - Lgr4 controls mucosal neutrophil infiltration.

A Immunofluorescence labelling for MPO in uninfected and 2-month infected Lgr4-/- and control mice (top panel) and 2-month infected Lgr5-/- and control mice (bottom panel). Asterisks indicate nonspecific staining. Scale bars: 50 µm.

B Quantification of MPO+ cells in uninfected and 2-month infected Lgr4-/- and control mice (n = 2 biological replicates per group, n = 5-10 FOVs per mouse) (left panel) and 2-month infected Lgr5-/- and control mice (n = 2 biological replicates per group, n = 6-8 FOVs per mouse) (right panel).

Data information: For B data are mean + SD. ** = p < 0.01; *** = p < 0.001; ns = not significant (one-way ANOVA + Tukey´s multiple comparison test).
Gastric stem cells promote inflammation and gland remodeling in response to H. pylori via Rsp3-Lgr4 axis (2022)
Figure 6

Figure 6 - Stem cells are the primary epithelial sensors of ADP heptose and promote pro-inflammatory signaling.

Antrum organoids from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose or 10 ng/ml TNF-α was added for 2 h to all organoid conditions prior to analysis.

A Bright-field images of organoids in respective conditions before treatment with ADP heptose or TNF-α. Scale bars: 200 µm.

B Immunofluorescence labeling for GSII and Muc5ac of organoids in respective conditions before treatment with ADP heptose or TNF-α. Scale bars: 50 µm.

C qPCR expression level of Lgr5 in untreated organoids in respective medium conditions (n = 4-5 biological replicates).

D qPCR expression level of Gif in untreated organoids in respective medium conditions (n = 4-5 biological replicates).

E qPCR expression level of Cxcl1, Cxcl2, Cxcl10, Ccl2 in organoids after treatment with ADP heptose compared to untreated controls within the respective medium conditions (n = 6 biological replicates per condition).

F qPCR expression level of Cxcl1, Cxcl2, Cxcl10, Ccl2 in organoids after treatment with TNF-α compared to untreated controls within the respective medium conditions (n = 2-3 biological replicates per condition).

Data information: For C, D, E and F data are mean + SD. * = p < 0.05; ** = p < 0.01; **** = p < 0.0001 (one-way ANOVA + Tukey´s multiple comparison test for C and D; t-test for E and F). Genes without asterisks are not significantly altered.
Gastric stem cells promote inflammation and gland remodeling in response to H. pylori via Rsp3-Lgr4 axis (2022)
Figure 7

Figure 7 - Activation of IKK-NF-κB pathway by ADP heptose requires Wnt and Rspo

Antrum organoids from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose was added for 2 h to all organoid conditions prior to analysis.

A Electrophoretic mobility shift assay (EMSA) and SDS-PAGE western blot from whole cell protein (WCL) lysates from gastric organoids untreated or treated with ADP heptose, and maintained either in FM in medium without Rspo, without Wnt and Rspo, or +BMP2. Representative images from n = 3 biological replicates.

B Quantification of EMSA based on n = 3 biological replicates using ImageJ with normalization to loading (ɑ-Tubulin).

C EMSA supershift from FM + ADP heptose organoids with the antibodies indicated. Blue arrow indicates NF-κB binding. Red arrow points to gel expected locations of supershifts from antibodies against p65, cRel, or RelB. Grey arrow points to supershift location on the gel from antibodies against p52 or p50. IgG lane serves as a negative control.

Data information: For B data are mean + SD. Significance is calculated by one-way ANOVA, F = 9.21.
Gastric stem cells promote inflammation and gland remodeling in response to H. pylori via Rsp3-Lgr4 axis (2022)
Figure 8

Figure 8 - NF-κB activity defines gland base compartment in the antrum.

A Bright-field images of antrum organoids from 129;129P2-ctnnb1tm(NFKBIAΔN)1Rsu (ΔN) mice and control mice, passage 0, day 6. Scale bars: 500 µm.

B, C qPCR expression levels of Lgr4, Lgr5, Gif, Pgc, Muc6 (B) and Cxcl1, Cxcl2 (C) of organoids (n = 3 biological replicates).

D Representative H&E staining and immunofluorescence labeling for GSII, Ki67 and MUC5AC in uninfected ΔN and control mice. Scale bars: 50 µm.

E Quantification of GSII and Ki67 as percentage of total DAPI-stained cells in ΔN and control mice (n = 5 biological replicates per group, n = 20 glands per mouse).

Data information: For B, C, and E data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test).

Expanded View Figure Legends:
FBXL4 deficiency increases mitochondrial removal by autophagy (2020)
Figure 1

Figure 1. Fbxl4 knockout mice that are viable at the postnatal stage display mild growth defects

A. Strategy to generate Fbxl4 knockout mice. Exon IV was targeted by loxP sites (orange arrows) and removed after Cre recombination. Black arrows indicate Frt sites.

B. Genotype distribution in litters from heterozygous matings (Fbxl4+/- x Fbxl4+/-) at embryonic stage 13.5 (E13.5) and of live animals at weaning. Data is presented as a percentage. Embryos n=79, weaned mice n=339. Dashed lines indicate the expected Mendelian ratios. Chi2 test vs expected Mendelian ratios; ***, p<0.001.

C. Representative images of wild-type (+/+), heterozygous knockout (+/-) and homozygous Fbxl4 knockout embryos (-/-) at E13.5.

D. Relative levels of mtDNA of Fbxl4 knockout (-/-) embryos and the corresponding wild type (+/+) embryos at E13.5. Data represent mean ± SEM, n=5. Student's t-test; *, p<0.05.

E. Representative images of male Fbxl4 knockout (-/-) and matched wild-type (+/+) animals at one year of age (left). A hunch-back phenotype was observed both in male and female knockout animals (right, indicated by arrow).

F-I. Whole body (F), heart (G), liver (H) and kidney (I) weights of 1-year old animals. Data represent mean ± SEM, n ≥ 5 animals. Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant (p ≥ 0.05).
FBXL4 deficiency increases mitochondrial removal by autophagy (2020)
Figure 2

Figure 2. Fbxl4 knockout mice have reduced amounts of mtDNA and mitochondrial proteins

A. Relative levels of mtDNA in tissues of 1-year-old Fbxl4 knockout mice determined by quantitative real-time PCR (normalized to controls). Data are presented as mean ± SEM, n=6 for controls and n=8 for knockout animals. Student's t-test; **, p<0.01; ***, p<0.001.

B. Transcript levels in indicated tissues of 1-year-old Fbxl4 knockout mice determined by quantitative real-time PCR. Relative mRNA levels in control animals were set as 1 (dashed line). Data are presented as mean ± SEM, n=6 for controls and n=8 for knockout animals. Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001.

C. Western blot analysis of protein steady-state levels in liver protein extracts from wild type (Fbxl4+/+) and Fbxl4 knock-out (Fbxl4-/-) animals.

D. Quantification of the western blot in (C). Signal normalized to the average of controls and presented as mean ± SEM, n=4 for both controls and knockout animals. Student's t-test; **, p<0.01; ***, p<0,001.

E. Quantitative TMT-based proteomic analysis of wild-type and Fbxl4 knock-out liver tissue protein extracts. Proteins from three control and three Fbxl4 knock-out liver protein extracts were TMT-labelled, quantified and analyzed separately as described in Materials and Methods; the data from control and knock-out animals were averaged after the analysis and presented as -log10 of the adjusted p-value vs log2-fold change (logFC). Mitochondrial proteins were selected according to mouse MitoCarta 2.0(Calvo et al, 2016) and highlighted in red; lysosomal proteins were selected according to hLGDB database(Brozzi et al, 2013) and highlighted in yellow.

F. Enrichment analysis of lysosomal and mitochondrial proteins according to the logFC median of each group of proteins. Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing the ±1.5x inter-quartile range. Proteins included in each group: total=1523, mitochondrial=324, lysosomal=50.
FBXL4 deficiency increases mitochondrial removal by autophagy (2020)
Figure 3

Figure 3. Mitochondrial alterations observed in FBXL4 patient-derived fibroblasts are consistent with the changes in knockout mice

A. Schematic representation of the human FBXL4 protein with amino acid positions of mutations found in patients (P1, P2 and P3). Relative positions of the F-box, leucine-rich repeat (LRR) and antagonist of mitotic exit network protein 1 (AMN1) domains are shown.

B. Relative mtDNA content in fibroblasts from patients and controls as determined by qRT-PCR using an ND6 probe and normalized to nuclear genomic DNA content using an 18S probe. Individual values are presented for each sample, as well as their mean ± SEM values.

C. Steady-state protein levels in three control and three FBXL4 patient-derived fibroblasts analyzed by western blotting.

D. Quantification of the western blot in (C). Signals from the control and FBXL4-mutated cells were considered as biological replicates, normalized to the average of controls and displayed as mean ± SEM (n=3 for each). Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001.

E. Quantification of nuclear and mitochondrial transcript levels in control and patient fibroblasts. The results from control and patient cells were averaged, normalized to controls and displayed as mean ± SEM (n=3 biological replicates for each condition).

F. Quantitative TMT-based proteomic analysis of control and patient fibroblasts. Proteins from three control and three patient fibroblast cell lines were TMT-labelled, quantified and analyzed separately as described in Materials and Methods; the data from control and patients were averaged after the analysis and presented as -log10 of the adjusted p-value vs log2-fold change (logFC). Mitochondrial proteins were selected according to human MitoCarta 2.0 (Calvo et al, 2016) and highlighted in blue; lysosomal proteins were selected according to hLGDB database (Brozzi et al, 2013) and highlighted in yellow.

G. Log2FC correlation analysis of Mouse Liver proteomic dataset and Fibroblast Proteomic dataset. Proteins with a log2FC > 0.5 are highlighted in black and lysosomal proteins are highlighted in yellow.
FBXL4 deficiency increases mitochondrial removal by autophagy (2020)
Figure 4

Figure 4. Lysosomal degradation of mitochondrial proteins is increased in FBXL4 knockout cells

A. Genomic DNA sequence from FBXL4 knockout (KO), and wild-type (WT) RKO cells was compared to the NCBI reference FBXL4 coding sequence (CDS) NM_001278716.2 and showed a frameshift-causing deletion in the knockout cells.

B. Steady-state protein levels in wild-type (WT) and FBXL4 knockout (KO) RKO cells.

C. (Upper panel) Pulse-chase labeling of mitochondrial translation products in wild type (WT) and FBXL4 knockout (KO) cells. The cells were incubated for 1 hour with a 35S methionine-cysteine labeling mix in the presence of anisomycin to inhibit cytosolic translation (pulse). After removal of the medium, the cells were incubated in complete growth medium without inhibitors and labeled amino acids for 16 or 24 hours (chase). A representative experiment of 4 biological replicates is shown. A Coomassie-stained part of the gel is shown as loading control. (Lower panel) Quantification of the MTCO1-ND4 signal presented as mean ± SEM; n=4 biological replicates; Student's t-test; *, p<0.05.

D. (Upper panel) Mitochondrial translation products were labeled in wild type (WT) and FBXL4 knock-out (KO) RKO cells for 60 minutes as in C, followed by 24 h chase in the presence of 5 μM epoxomicin (epoxo) or 20 mM ammonium chloride (NH4Cl). A representative experiment of n≥3 biological replicates is shown. A fragment of the Coomassie-stained gel is shown as loading control. (Lower panel) Quantification of the ND4-MTCO1 signal. n≥3 biological replicates, data are presented as mean ± SEM. Student's t-test; *, p<0.05.

E-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with NH4Cl (E), or epoxomicin (Epoxo, F).
Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion (2012)
f2

a, LUMIER binding assay: normalized ratio between luciferase activity bound to beads and present in lysates. Lysates of 293ET cells expressing NDP52, p62 or optineurin each fused to luciferase, and the indicated Flag-tagged galectins were incubated with anti-Flag beads. Flag-tagged proteins are further characterized in Supplementary Fig. 4a. b, Lysates of 293ET cells, expressing Flag-tagged proteins as indicated, were immunoprecipitated with anti-Flag beads. Lysates and immunoprecipitates (IP) were blotted for the presence of Flag-tagged proteins and endogenous NDP52. c, Confocal images of HeLa cells infected with S. Typhimurium for 1 h and stained with antisera against NDP52 and galectin 8. Arrowheads, bacteria shown in insets. d, Co-localization of NDP52 with galectin-positive bacteria in HeLa cells stably expressing YFP fused to the indicated galectins, infected with S. Typhimurium and stained with NDP52 antiserum 1 h after infection. Mean and s.d. of duplicate HeLa cultures, n > 100 bacteria per coverslip, representative of two independent experiments. Scale bar, 10 &mgr;m.
Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion (2012)
f3

a, Percentage of S. Typhimurium coated by the indicated galectins. HeLa cells stably expressing YFP-tagged galectins were treated with the indicated siRNAs. YFP-positive bacteria were counted by microscopy at 1 h p.i. siRNAs are further characterized in Supplementary Fig. 2. b, Analysis of HeLa cells treated with the indicated siRNAs and stained with NDP52 antiserum. NDP52-positive bacteria were counted by microscopy 1 h after infection with S. Typhimurium. siRNAs are further characterized in Supplementary Fig. 2. c, Percentage of bacteria coated by the indicated galectin 8 alleles. HeLa cells stably expressing the indicated galectin 8 alleles fused to YFP were infected with S. Typhimurium. YFP-positive S. Typhimurium were counted by microscopy at 75 min p.i. WT, wild type. d, Binding of galectin 8 to bacteria and HeLa cells. The indicated bacteria and HeLa cells were incubated with His-GST–ubiquitin (Ub), His-GST–galectin 8 or buffer as indicated, followed by murine anti-His antibody and PE-labelled anti-mouse serum. e, Percentage of S. Typhimurium coated by the indicated galectins. Wild-type CHO cells and mutant Lec3.2.8.1 cells stably expressing YFP-tagged galectins were infected with S. Typhimurium. YFP-positive bacteria were counted by microscopy at 1 h p.i. f, Confocal images of HeLa cells expressing the indicated YFP-tagged galectins. Cells were left untreated or were exposed to hypertonic conditions, with or without (w/o) PEG as indicated, followed by hypotonic shock. g, Fold replication of S. Typhimurium in HeLa cells expressing the indicated galectin 3 variants and transfected with the indicated siRNAs. At 2 h and 6 h after infection, cells were lysed and bacteria counted on the basis of their ability to form colonies on agar plates. Galectin 3 proteins are further characterized in Supplementary Fig. 4b. Mean and s.d. of duplicate coverslips (a–c, e) or triplicate HeLa cultures and duplicate colony counts (g). >100 bacteria counted per coverslip. Data are representative of at least two repeats. *P  0.05, Student’s t-test. Scale bar, 10 &mgr;m.
Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion (2012)
f4

a, Confocal micrograph of HeLa cells expressing GFP–LC3 and mCherry–galectin-8, stained for NDP52 1 h after infection with S. Typhimurium. The lower right panel contains a fluorescence line scan along the yellow line in the merge inset. Arrowheads, bacteria shown in insets. b, Percentage of GFP–LC3-positive S. Typhimurium at 1 h p.i. in HeLa cells treated with the indicated siRNAs. c, Percentage of bacteria positive for NDP52. HeLa cells expressing the indicated NDP52 variants fused to YFP were infected with S. Typhimurium. d, Percentage of bacteria positive for the indicated markers. HeLa cells, either wild type or expressing YFP–galectin-8 as indicated, were infected with S. Typhimurium. Ubiquitin was detected by antibody staining. e, HeLa cells treated with the indicated siRNAs were infected with S. Typhimurium and stained for NDP52 at the indicated time points. Fluorescent bacteria were counted by microscopy at the indicated time points. Graphs, representing at least two independent repeats, show mean and s.d. of duplicate coverslips for which >200 bacteria were counted. *P  0.05, Student’s t-test. Scale bar, 10 &mgr;m.
A dual role of human tRNA methyltransferase hTrmt13 in regulating translation and transcription (2021)
Figure 1

Figure 1. CCDC76 (hTrmt13) is a tRNA 2'-O-methyltransferase of position 4.

A. A representation of the primary structure of human CCDC76 (hTrmt13).

B. Schematic of the modified CLIP-seq approach employed to profile genome-wide hTrmt13-RNA interactions.

C. Autoradiograph of hTrmt13-RNA complexes in the MDA-MB-231 cells immunoprecipitated with an antibody against hTrmt13 (right lane) and IgG as a negative control (left lane). * RNA residing in the regions between marked lines was recovered for sequencing.

D. Pie chart showing distinct categories of RNA species bound to hTrmt13 identified by CLIP-seq.

E. Pie chart showing the different species tRNAs bound to hTrmt13 from CLIP-seq.

F. The capacity of HctRNAs to be methylated by hTrmt13. Error bars represent mean ± SD for at least three independent experiments.

G. A representative secondary structure of tRNA with Nm4 modification marked.

H. Mass chromatograms of the nucleosides, Um (Q1/Q3 = 259.1/113.1), Cm (Q1/Q3 = 258.1/112.1), and G (Q1/Q3 = 284.1/152.2) of HctRNAGly(GCC), HctRNAGly(CCC), HctRNAGly(UCC), and HctRNAPros isolated from WT and hTrmt13 KO cells. Um, 2′-O-methyluridine, Cm, 2′-O-methylcytidine. Q1/Q3: the mass of the precursor ion and the mass of the product ion. These endogenous tRNAs were purified separately from total cellular RNAs using sequence-specific probes.

I. Quantification of the Um/G and Cm/G ratios in endogenous tRNAs isolated from WT and hTrmt13 KO cells. Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards running in the same batch. Statistical analysis was performed using t-tests. Error bars represent mean ± SD for three independent experiments.
A dual role of human tRNA methyltransferase hTrmt13 in regulating translation and transcription (2021)
Figure 2

Figure 2. hTrmt13 impacts protein biosynthesis depending on its catalytic activity.

A. De novo protein synthesis measured by puromycin incorporation in shSCR, hTrmt13 knockdown cells (sh-1 and sh-2), and sh-1 with stable expression of wild type hTrmt13 (sh-1+hTrmt13) or enzymatically-inactive hTrmt13 (sh-1+E463A). Puromycin-labeled proteins were detected by immunoblotting. Representative images (left) and statistical analysis (right) are shown. Knockdown and overexpression efficiency of hTrmt13 in MDA-MB-231 verified by Western blots. See also Fig EV3D.

B. Northern blot analysis of substrate tRNAs of hTrmt13 from control and the hTrmt13 knockdown MDA-MB-231 cells. Statistical analysis is shown in Fig EV3F.

C. The in vivo aminoacylation level of hTrmt13 substrate tRNAs assayed by Northern blot. The samples were also deacylated (DA) by incubating with alkaline buffer (pH 9.0) to obtain fully uncharged tRNA control. Statistical analysis is shown in Fig EV3G.

D. RT-qPCR using a stem-loop primer to quantify the tRFs levels in shSCR, sh-1, and sh-2.

E. Quantification of the level of 5'-Gly-CCC in shSCR, sh-1, sh-2, sh-1+hTrmt13, and sh-1+E463A assayed by RT-qPCR.

F. Quantification of the Cm/G ratios of endogenous HctRNAGly(CCC) isolated from shSCR, sh-1, sh-2, sh-1+hTrmt13, and sh-1+E463A cells by UPLC-MS/MS analysis.

G. Quantification of de novo protein synthesis in cells treated with different concentration of tRFs as indicated in Fig EV3K.

H. Quantification of de novo protein synthesis in cells treated with different concentration of tRNAGly(CCC) as indicated in Fig EV3L.

Data information: In A, D-H: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments.