EMBO/sd-character-level-ner huggingface dataset:
Figure 1A Schematic diagram describing organoid culture derivation (AP, Anterior Prostate; DLP, Dorso-Lateral Prostate; VP, Ventral Prostate; SV, Seminal Vesicle; DHT, dihydrotestosterone). B Mouse prostate organoid forming efficiency. Efficiency at derivation (left; n = 6 biological replicates, data points are shown with crossing line representing mean value). Efficiency at passage 1-3 (right; n ≥ 3 biological replicates per organoid line/passage; data are presented as mean). C Representative longitudinal flow-cytometry analysis of dissociated organoid cells (Bas, Basal; LumP, Luminal Proximal; PrU, Periurethral; Lum Distal, Luminal Distal). D End-point RT-PCR analysis for selected marker genes (n = 3 biological replicates). E Bulk-RNAseq analysis (n=3 biological replicates; individual data points are shown with bar graphs representing mean value). F Representative Haematoxylin-Eosin (H&E) staining of mouse prostate tissue and organoid sections (scale bars = 50 μm). G Representative Immunofluorescence (IF) analysis for selected markers in mouse prostate tissue and organoid sections (scale bars = 50 μm). H IF staining for selected markers in mouse prostate tissue and organoid sections (scale bars = 50 μm).
Fig.Figure 9Engagement of the FcR by opsonized particles of multiple sizes and shapes (small or large beads, rod bacteria) commonly recruits the second messenger, NAADP, which activates TPC channels across the macrophage endo-lysosomal network. Local Ca2+ nanodomains around TPCs activate the Ca2+-dependent protein phosphatase, calcineurin (CaN), which dephosphorylates and thereby activates dynamin (Dyn-2). For clarity, only one dynamin-dependent event is shown (phagosome scission) but others are not excluded. TRPML1 Ca2+ nanodomains are only activated by large (6 µm) beads, a specialist secondary signal to additionally drive lysosomal exocytosis (via Synaptotagmin 7, Syn 7) and thereby provide supplementary lysosomal membrane (red-dashed lines) to the larger phagosome.
a) Representative confocal images of segment A3 muscle 6/7 synapses stained for both DVGLUT (green) and FasII (red), in late 2nd/early 3rd instar wild-type, Rae1EX28, Rae1EX28 presynaptic rescue (Rae1EX28; elav-Gal4/UAS–NTAP-Rae1) and Rae1EX28 wallenda suppression (Rae1EX28; wnd3) larvae.figf3(a) Representative confocal images of segment A3 muscle 6/7 synapses stained for both DVGLUT (green) and FasII (red), in late 2nd/early 3rd instar wild-type, Rae1EX28, Rae1EX28 presynaptic rescue (Rae1EX28; elav-Gal4/UAS-NTAP-Rae1) and Rae1EX28 wallenda suppression (Rae1EX28; wnd3) larvae. Scale bar represents 10 μm. (b,c) Quantification of bouton number (b) and size (c) in wild-type, Rae1EX28/Df, Rae1EX28, Rae1EX28 rescue, Rae1EX28; wnd3 suppression and hiwΔN larval NMJs (segment A3 muscle 6/7; n = 20, 21, 18, 20, 20 and 11, respectively, for bouton number; n = 271, 465, 548, 726, 486 and 826, respectively, for bouton size). There was no significant difference in either bouton number or bouton size between Rae1EX28 and Rae1EX28/Df (P > 0.1 for both comparisons). *P 0.001. Error bars denote s.e.m.
Endogenous PINK1 regulates Rab8A, Rab8B and Rab13 Ser111 phosphorylation.Figure 4PINK1 is essential for CCCP‐mediated Rab8A Ser111 phosphorylation. WT and PINK1 KO HeLa cells were transfected with either WT or Ser111Ala (S111A) mutant constructs of HA‐Rab8A, HA‐Rab8B or HA‐Rab13. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated. After transfection for at least 24 h, cells were treated with DMSO as a vehicle control or CCCP for 20 h. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with total PINK1 antibody to confirm PINK1 expression and with GAPDH as loading controls. Endogenous Rab8A Ser111 phosphorylation is PINK1 dependent. WT and PINK1 KO HeLa cells were treated with DMSO as a vehicle control or CCCP for 20 h. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated for at least 24 h before CCCP treatment. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A pre‐bound with protein A agarose followed by immunoblot with Rab8A phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot with anti‐total Rab8A antibody as loading controls.
a) Immunoprecipitation was performed with anti-PEX5 (left panel) and anti-PEX19 (right panel) in HEK293 cells and followed by immunoblotting for endogenous TSC1, TSC2, mTOR (negative control) and PMP70, and PEX1 (PEX19 cargo) or catalase (PEX5 cargo).figf6(a) Immunoprecipitation was performed with anti-PEX5 (left panel) and anti-PEX19 (right panel) in HEK293 cells and followed by immunoblotting for endogenous TSC1, TSC2, mTOR (negative control) and PMP70, and PEX1 (PEX19 cargo) or catalase (PEX5 cargo). (b) Schematic of the TSC2 C terminus (amino acids 1,517-1,807) showing the GAP- and PEX5-binding sequence (PxBS), naturally occurring mutations and the reintroduced SKL sequence. (c) Representative images of FAO cells transfected with DsRed-Del-ARL (deleted ARL sequence; red) and GFP-PTS1 (green; top panel). FAO cells were also transfected with DsRed-ARL (red) and stained for PMP70 or catalase (green) as indicated. Scale bars, 10 μm. (d) HEK293 cells expressing Flag-TSC2 wild type (WT) or Flag-TSC2 mutants (RQ, RW and RG) were co-immunoprecipitated using anti-Flag or anti-PEX5, and blotted for PEX5 and Flag-TSC2. (e) Representative experiment showing subcellular fractionation of HEK293 cells overexpressing Flag-TSC2 wild type and Flag-TSC2 mutants (RQ, RW and RG). Lysates from peroxisome (P) or membrane (M) fractions and whole-cell extracts (WCE) were immunoblotted with Flag or PMP70 antibodies. (f) Co-immunoprecipitation of HEK293 cells overexpressing Flag-TSC2 wild type, or Flag-TSC2 mutant (RQ) or Flag-TSC2 rescue mutant (RQ-9NT) using anti-Flag antibody or control IgG and immunoblotted with anti-Flag and anti-PEX5 antibodies. (g) Subcellular fractionation of TSC2−/− MEFs transfected with Flag-TSC2 wild type, or Flag-TSC2 mutants (RQ or RQ-9NT). β-integrin and catalase were used as subcellular markers for membrane and peroxisome fractions, respectively. WCE, whole-cell extract. The arrow indicates the position of overexpressed Flag-TSC2. (h) HEK293 cells co-transfected with Flag-TSC1 and Flag-TSC2 wild type (WT) or Flag-TSC2 G294E mutant (TSC2 mutant that cannot bind TSC1). Lysates were immunoprecipitated using anti-PEX19 and blotted for PEX19 and Flag. (i) Representative blots from subcellular fractionation of HEK293 cells overexpressing wild type (WT) or mutant (CaaX mutant) Flag-Rheb. LDH, lamin A/C and catalase were used as subcellular markers for the cytoplasmic (Cp), nuclear and peroxisome fractions, respectively. Uncropped images of western blots are shown in Supplementary Fig. S6.
Figure 1(A-B'') Nr2f1 (red) and Pax6 (green, retinal progenitors) immunofluorescences (IF) on sagittal sections of E10.5 and E12.5 mouse optic vesicles. Note the high-ventral to low-dorsal Nr2f1 gradient in the presumptive neural retina (pNR), and high Nr2f1 expression in both the presumptive dorsal and ventral optic stalk (pvOS and pdOS). Cr: crystal lens; Di, distal; Pr, proximal; presumptive retinal pigmented epithelium (pRPE). Scale bars: 50µm for mouse sections (C-D") IF on E13.5 mouse eye sagittal sections with Nr2f1 (red) and Sox2 (green, progenitors in C,C') or Brn3a (green, RGCs in D,D') showing Nr2f1 expression in NR progenitors and post-mitotic RGCs (insets in C'-D"). Scale bars: 50µm for mouse sections (E-F") Nr2f1 (red) and Brn3a (green) IF on E18.5 and P7 mouse eyes depicting Nr2f1 expression in virtually all RGCs in the ganglion cell layer (GCL). Scale bars: 50µm for mouse sections (G,G') Nr2f1 (red) and Sox2 (green) IF on P7 mouse retina illustrating low Nr2f1 expression in the inner nuclear layer (INL) and progenitors (NPs), and high levels in the GCL and ciliary marginal zone (CMZ). No expression in the outer nuclear layer (ONL). Scale bars: 50µm for mouse sections (H-K") IF on cross-sections of E13.5 optic stalks (OS), and E18.5 and P7 optic nerves (ONs) with Nr2f1 (red) and Glast (green, astrocytic progenitors in H-H''), NF1A (green, astrocytes in I-I'') or Sox10 (green, oligodendrocyte precursors in J-K'') showing Nr2f1 expression in both astrocytic and oligodendrocytic lineages. Arrowheads point to double labeled cells. Scale bars: 50µm for mouse sections (L-L") NR2F1 (red) and PAX6 (green; NR domain) IF on sagittal sections of human eye primordia at gestational week (GW) 11 illustrating high NR2F1 expression in both ON and NR cells. Scale bars: 100µm for human sections (M-N") NR2F1 (red) and S100β (green, astrocytes) IF on cross-sections of human GW11 and GW14 ONs. Higher magnifications (M'-N") show that most of S100β+ astrocytes co-express NR2F1. Scale bars: 100µm for human sections, (O-Q"') NR2F1 (red) and SOX2 (green in O-P') or BRN3a (green in Q-Q''') IF on sagittal sections of human GW14 eyes indicating high NR2F1 expression in virtually all NR progenitors (O''',P,P'), differentiating RGCs (Q-Q''') and in the majority of ON astrocytic progenitors (O'). Nuclei counterstaining (blue) was obtained with DAPI. Scale bars 100µm for human sections
PRSS35 is selectively upregulated in wound-induced tumours.Figure 3a. Log2 fold change of probe intensities of PRSS35 gene expression in different subsets of dermal fibroblasts observed in Affymetrix GeneChip® Mouse 430 2.0 array (n=3 per condition). b. Hexagonal triwise plot depicting the PRSS35 gene expression level. c. Relative mRNA levels of PRSS35 in lysates from normal skin (WT, n=4 mice), inflamed skin (InvEE, n=4 mice), wounds (d14pw, n=4 mice) and wound-induced papillomas (Pap, n=5 mice). (* p=0.0159; Mann-Whitney test). Data represent means of three technical replicates ± S.E.M. d. Wound healing dynamics of wild-type (WT, n=10) and PRSS35-/- (n=11) mice after full-thickness wounding with an 8 mm punch biopsy. Wound size expressed as percentage of initial wound size (ns; Wilcoxon matched-pairs signed rank test ). Data represent means ± S.E.M. e. Representative pictures of back skin of WT and PRSS35-/- mice at different days post-wounding (dpw). Scale bar: 1 cm. f. H&E-stained skin sections of WT and PRSS35-/- wounds at different days post-wounding. Scale bars: 100 µm.
Figure 3(A) XO8 GSCs and DFCs were differentially labeled with PKH67 (green) and PKH26 (red), respectively, and mixed before plated on laminine-coated plates. The cells were treated with 50 nM MG132 and subjected to fluorescent microscopy. Scale bar, 100 μM. (B) Quantitation of (A). Cell numbers from three independent experiments were normalized to those of control cells treated with DMSO and presented as means +S.D. (*p=0.0035, n=3). Student t-test (two-sided, one type) were used for statistical analysis. (C) The harvested cells from (A) were subjected to FACS analysis. (D) XO8 GSCs were treated with various PIs (salinosporamide A, epoxomicin, and PS341), followed by cell viability assays. Bars, means ±S.D. from three independent experiments. (E) XO6, XO8, and XO10 GSCs were treated with MG132 and subjected to cell viability assay. Bars, means ±S.D. from three independent experiments. (F) XO6, XO8, and XO10 GSCs were treated with MG132 and subjected to cell viability assay in serum-containing media. Bars, means ±S.D. from three independent experiments.
Figure 2A Impaired clearance of damaged lysosomes in cells expressing a p97 disease-mutant. Wild-type and p97R155H/+MEFs were LLOMe-treated for 1 h and released for indicated times. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD, Student's unpaired t-test). Scale bar, 25 µm. B Stable U2OS cell lines were doxycycline-induced to express myc-tagged p97 wild-type or disease-mutants as indicated. Induced parental cells served as control (U2OS -). Cells were treated with LLOMe for 1h and chased for indicated time before fixation. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments. Comparable expression levels were verified (see Figure EV2A). C Stable U2OS cell lines described in (B) were treated with LLOMe for 1 h and chased for indicated time. LC3 and GAPDH levels were analyzed by immunoblotting. Quantification is shown in Figure EV2B. D Accumulation of damaged lysosomes in affected p97 patient skeletal muscle. Patient biopsies immunostained for Gal3 and caveolin-3 (Cav3). Quantification from 4 random 10X fields for each biopsy and means for controls and patients, respectively. Patients carry p97 mutations R155H (#1-3) or R93C for (#4). Patients #2 and #3 are cousins. Scale bar, 100 µm.
Figure 1(A) HeLa cells were transfected with the indicated siRNAs for 48 h, and then were re-transfected with TFEB-EGFP. Twenty-four h after transfection, the cells were fixed. DAPI (blue) was used for nuclear staining. Cells were visualized using confocal microscopy. Scale bar, 5 µm. The quantification of TFEB localization in cytoplasm and nucleus is shown on the right side. The data from three independent experiments indicated the means ± S.E.M., **, p<0.01, one-way ANOVA.(B) Similar experiments as in (A) were performed in SH-SY5Y cells. Scale bar, 5 µm. The quantification data are shown on the right side. Data from three independent experiments represented as means ± S.E.M., **, p<0.01, one-way ANOVA. White arrowheads indicate nuclear localization of TFEB. (C) HEK 293 cells were similarly transfected as in (A). Lysates from the cells were separated into cytoplasmic and nuclear fractions and then subjected to immunoblot analysis using anti-GFP, GAPDH and Histone 2B antibodies. The relative densities are shown on the right side. The data from three independent experiments are presented as means ± S.E.M., **, p<0.01, one-way ANOVA.
Stat3 activation in an AD model is efficiently reduced using a conditional knockout strategy (A-B) To verify the specificity of Cx43-CreERT mice for astrocytes, mice were crossed to a tdTomato reporter line to induce Cre-dependent tdTomato expression after tamoxifen administration.Figure 1To verify the specificity of Cx43-CreERT mice for astrocytes, mice were crossed to a tdTomato reporter line to induce Cre-dependent tdTomato expression after tamoxifen administration. TdTomato fluorescence was enhanced with an antibody against red fluorescent protein, and astrocytes were stained with antibodies against GFAP or S100β (not shown). Merged images and quantification show efficient and widespread tdTomato expression specific for astrocytes (arrowheads) in 8-month and 11-month old animals. Expression was small in neurons (stained with NeuN, not shown) and negligible in NG2 cells and oligodendrocytes. Scale bars, 250 µm (upper panel) and 50 µm (lower panel). The majority of peri-plaque astrocytes in APP/PS1-Stat3WT were positive for activated/phosphorylated Stat3 (pStat3) in the cortex (Cx) and hippocampus (HC), whereas the fraction of pStat3-positive astrocytes was significantly lower in APP/PS1-Stat3KO mice in both regions (n = 8 mice (3 females and 5 males) per group; age, 8 months; Mann-Whitney test). The occurrence of pStat3 in WT-Stat3KO and WT-Stat3WT mice was negligible (n = 8 mice (4 females and 4 males) per group; age, 8 months; Mann-Whitney test). Examples of Stat3 activation in APP/PS1-Stat3KO and APP/PS1-Stat3WT. D, pStat3 (arrowheads) was abundantly present in reactive astrocytes (identified by GFAP) around plaques (identified by IC16). E, only few astrocytes were positive for pStat3 in APP/PS1-Stat3WT mice. Scale bars, 50 µm. in postmortem brain sections from AD patients, we detected nuclear pStat3 (arrows) in the majority of peri-plaque reactive astrocytes (identified by GFAP) in the cortex and hippocampus (plaques were stained with methoxy-XO4, arrowheads). Scale bar, 50 µm.
Figure 5IMR90 cells at day 25 post infection of lentiviral control or sgA#10 were synchronized by serum starvation and switched to 15% serum-containing medium and assayed by 53BP1/TelG immuno-FISH (A) at indicated time. Arrows denote telomeres that are positive for 53BP1 signals. Scale bar, 15 µm. (B) Percentage of cells containing ; 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test. C, D RPA/TRF2 co-immunofluorescence (C) revealed strongly increased telomeric replication stress in U87 cells at day 30 but not day 10 post lentiviral sgA#10 infection (D). Lentiviral control and sgA#10 infected U87 cells at indicated time post-infection were released following thymidine (Thy) synchronization and processed for RPA and TRF2 co-staining. Scale bar, 10 µm. (D) Percentages of cells with > 4 RPA/TRF2 co-localized foci are expressed as means ± s.d., N = 3, unpaired t test.
Figure 3Experimental scheme for hCDC14A-GFP expression combined with phospho-proteome (SILAC) analysis Molecular network of the hypo-phosphorylated proteins in ciliated hCDC14A-GFP RPE1 cells. Gene network was constructed in Cytoscape using the function prediction GeneMANIA app. GeneMANIA identifies the most related genes to a query gene set (hits) using a guilt-by-association approach. The plugin uses a large database of functional interaction networks from multiple organisms to construct a molecular network using the query genes (hits). Physical interactions are denoted by solid lines (edges), whereas the shared protein domain interactions are represented by broken lines. The extent of interactions (degree) is indicated by the Node size. The query proteins are marked with thick borders and the actin-related proteins are marked with Magenta colored nodes Gene Ontology (GO) analysis of the proteins that are at least two-fold hypo-phosphorylated in the heavy (hCDC14A overexpressed) samples. Values inside the parentheses indicate P values Substrate motif identified by analyzing the sequences of hypo-phosphorylated peptides in Perseus Molecular network of the neighbors of hCDC14A and hCDC14AC278S identified by proximity-dependent biotin identification (BioID) during ciliogenesis. Gene network was constructed in Cytoscape as described in B by considering the physical interactions only. Gene Ontology (GO) analysis of the proteins from which at least five peptides were two-fold enriched in the hCDC14A-BirA and hCDC14AC278S-BirA samples compared to BirA control
Figure 6Immortalized mouse fibroblasts were transduced with a lentiviral vector expressing different mDia2-targeting shRNAs (shmDia2 #1-3) or empty vector (sh-EV). The knock-down was confirmed by qRT-PCR (N=3), Western blot and immunofluorescence. Ki67 staining of mDia2 knock-down and control mouse fibroblasts. N=3. migration in scratch assays of mDia2 knock-down and control mouse fibroblasts. N=3. qRT-PCR for mDia1, mDia3, Fmn1 and Fmn2 and several CAF genes relative to Rps29 using RNA from control (sh-EV) and mDia2 knock-down (sh-mDia2) fibroblasts. N=3. Representative images of mDia2 knockdown and control mouse fibroblasts stained for p53 (left), and quantification of cells showing accumulation of p53 in the nucleus (right). N=3. Representative Western blots for p53, vinculin and histone H1 using nuclear and cytoplasmic fractions and total lysate of mDia2 knock-down (F) or activin A-treated fibroblasts (6h, 20 ng/ml) (G) and control mouse fibroblasts and quantification of the nuclear p53/histone H1 ratio. Vinculin and histone H1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. N=6. Representative Western blots for p53, mDia2 and GAPDH using total lysates of mDia2 knock-down and control mouse fibroblasts treated for 24 h with pifithrin-α (PFT-α) (15 μM). Representative p53 immunofluorescence images of mDia2 knock-down mouse fibroblasts treated with PFT-α or vehicle (DMSO) (top) and quantification of cells showing accumulation of p53 in the nucleus (bottom). N=3. qRT-PCR for mDia2, Postn, Mmp13, Fn1, Spp1 and Acta2 relative to Rps29 using RNA from mDia2 knock-down and control fibroblasts treated with PFT-α or vehicle. N=3. Colony size of SCC13 Act cells (clone 2) after co-culture with either immortalized mDia2 knockdown (sh-mDia2) or control (sh-EV) mouse fibroblasts. N=4. Representative pictures of 5-week-old tumors formed upon intradermal co-injection of SCC13 Act cells (clone 2) with sh-EV or sh-mDia2 knock-down immortalized mouse fibroblasts. Graph shows tumor volume at different timepoints of tumor development. N=3-5 tumors per group. Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 Act (clone 2) co-injected with sh-EV or sh-mDia2 fibroblasts, stained for E-cadherin (green) and periostin or p53 (red), counterstained with Hoechst (blue).
Transient inhibition of canonical Wnt/β‐catenin signaling activity decreases radio‐sensitivity of ISPCs in vivo A–G Two‐month‐old LGR5‐GFPki, mTerc+/+ mice were i.Figure 8A-G Two‐month‐old LGR5‐GFPki, mTerc+/+mice were i.v. injected with LRP6 neutralization antibody, and IgG serves as a negative control. Thirty‐six hours after injection, small intestinal tissue was collected for GFPimmunofluorescence microscopy staining. (A-F) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate cell positions in the crypts. Scale bar: 20 μm. (G) Quantification of GFP staining intensity of cells at indicated positions in basal crypts (n = 3 mice per group, fifty positively stained cells were measured per mouse). Note the GFP staining intensity was substantially reduced in anti‐LRP6 antibody‐injected group with intact maintenance of ISPCs at the crypt bottom as shown by DAPI staining. H-L Two‐month‐old mTerc+/+ mice were i.v. injected with anti‐LRP6 antibody 12 h before γ‐irradiation. Small intestinal tissue was collected at 24 h after IR (n = 3-4 mice per group). IgG serves as a negative control. (H-K) Representative pictures of PCNA staining of small intestine of non‐irradiated mice (H, I) or 6 Gy‐irradiated mice (J, K). Note the anti‐LRP6 antibody injection did not induce loss of CBC cells at position 1 and 2 in non‐irradiated mice. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (L) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts 24 h after IR with indicated doses or non‐irradiated mice. M-R Two‐month‐old LGR5‐GFPki, mTerc+/+ mice were i.v. injected with LRP6 neutralization antibody 12 h before 12 Gy γ‐irradiation. IgG serves as a negative control. Small intestinal tissue was collected at 24 h after IR (n = 4 mice per group). (M-P) Representative FACS plots of indicated treatments. (Q) Flow cytometry analysis of the number of LGR5+ ISPCs of irradiated mice compared to the number in non‐irradiated mice (NIR) set to 100%. (R) Quantification of the number of LGR5+, LGR5hi, and LGR5lo cells of mice with indicated treatment by flow cytometry. Note that the total LGR5+cell frequency was not significantly different between anti‐LRP6 antibody‐injected and control IgG‐injected mice, though the LGR5hi fraction was reduced in the LRP6 antibody‐injected group.