EMBO/soda-nel huggingface dataset:
SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland (2016)
Figure 2

Figure 2. Mammary ductal outgrowth during puberty is impaired in SHARPIN-deficient Sharpincpdm mice.

A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. A. Representative carmine-alum stained mammary gland whole mounts. Arrow indicates the inguinal lymph node. Scale bars represent 2 mm. B. Quantification of mammary ductal outgrowth area (n = 4 - 15 glands). C. Quantification of the number of ductal branch points per mammary gland (n = 7 - 9 glands, 7 weeks old mice). D. Cryosections from 7 weeks old wt or Sharpincpdm mouse mammary glands stained with HE (upper panel) or immunolabelled with the indicated antibodies. Scale bars represent 20 μm E. Representative carmine-alum stained images highlighting terminal end buds (TEBs) in 7 weeks old wt and Sharpincpdm mouse mammary glands and F. quantification of the number of TEBs per gland (n = 9 - 10 glands). Scale bar represents 500 μm. G. TEBs in paraffin sections from wt or Sharpincpdm mouse mammary glands stained with HE (upper panel) or immunolabelled with the indicated antibodies (lower panel). Scale bars represent 50 µm. Mean ± SEM.
SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland (2016)
Figure 3

Figure 3. Stromal SHARPIN regulates mammary gland ductal outgrowth during puberty.

A-C. Monitoring of mammary gland development following transplantation of wt or Sharpincpdm mouse mammary epithelium in the mammary fat pads (epithelium-free) of virgin wt mice. A. Representative carmine-alum stained images of mouse mammary glands generated from wt or Sharpincpdm epithelial tissue transplants. B. Quantification of transplant growth take-on-rate (n = 17). C. Quantification of fat pad filling rate of grown transplants (n = 7 - 10) in wt hosts. D. Monitoring of mammary gland differentiation during pregnancy following transplantation of wt or Sharpincpdm mouse mammary epithelium into wt hosts. Host animals were subsequently mated and mammary glands isolated at P15. Representative Carmine-alum stained mammary gland whole mounts generated from wt and cdpm mammary epithelial transplants (upper panel), and magnifications of the branched ductal epithelium (lower panel) are shown (n = 5 mice). Scale bars represent 1 mm. E. Representative images of carmine-alum stained mouse mammary gland whole mounts generated from 7 weeks old S100a4-Cre; Sharpinfl/fl conditional knockout mice and their littermate controls (S1004-Cre; Sharpin fl/+) F. Quantification of the normalized Sharpincpdm and S1004-Cre; Sharpinfl/fl mammary ductal outgrowth area relative to littermate control mice (wt and Sharpincpdm n = 10 glands; S100a4-Cre; Sharpin fl/+ and S100a4-Cre; Sharpinfl/fl n = 8 glands). G. Quantification of the number of TEBs per gland (n = 8 - 9 glands). Mean ± SEM.
SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland (2016)
Figure 4

Figure 4. SHARPIN deficiency causes aberrant collagen fibre organization and reduced tissue stiffness in the mouse mammary gland stroma.

A. Second harmonic generation (SHG) and unfiltered multiphoton (MP) imaging of collagen fibres surrounding TEBs (dashed line) in 6-7 weeks old wt and Sharpincpdm mouse mammary glands (n = 6 - 9 mice, 5 - 13 TEBs analysed per mouse; scale bars represent 100 µm). Magnified images of collagen fibres are also shown (scale bars represent 50 µm). B. Frequency of clear collagen bundles around TEBs per mouse (Mean ± SEM, n = 6 - 9 mice). C. AFM indentation analysis of stromal stiffness in 6-7 weeks old wt and Sharpincpdm mouse mammary gland tissue sections (n = 3 mice; 3 - 6 sample sections per mouse and 192 indentation curves per sample).
SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland (2016)
Figure 5

Figure 5. SHARPIN regulates collagen remodeling and traction force generation.

A. Ingenuity Pathway Analysis of differential gene expression (fold-change (FC) > 1.5, p < 0.05) in wt and Sharpincpdm MSFs (passage 0). The differentially expressed genes implicated in the most significantly altered canonical pathways (leukocyte adhesion and transmigration, and tissue fibrosis) are shown. Three independent cell isolation replicates were compared pairwise. FC colour scale represents a range from 0−4. B. Analysis of collagen degradation on cross-bow-shaped micropatterns coated with the DQ collagen substrate. Mean intensity projection images of the DQ collagen fluorescence (top panel; cell area is outlined by dashed line; original integer range 0 − 255) and single confocal images of the fibrinogen-Alexa647-marked micropatterns (bottom panel) are shown (n ≥ 53 from four independent experiments). Scale bars represent 10 μm. C-D. Collagen plug contraction assay using wt and Sharpincpdm MSFs or wt MSFs with SHARPIN siRNA silencing C. Representative light microscopy images of the collagen plugs three days post cell culture. D. Quantification of the average collagen plug area 1−3 days after seeding (n = 3-6). Scale bars represent 2 mm. E. SHARPIN protein level after silencing with negative control siRNA or SHARPIN siRNA in wt MSFs. Tubulin was used as loading control. F-I. Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen-coated matrices. F. Representative images of traction forces observed in wt and Sharpincpdm cells (colour scale units: Pa; cell boundaries are indicated by dashed lines). G. Quantification of strain energy per cell (n = 8 − 11 cells from two independent experiments). H. Representative images of the traction forces and mCherry fluorescence of mCherry-Ctrl transfected wt cells and mCherry-Ctrl or mCherry-SHARPIN transfected Sharpincpdm cells. Scale bars represent 20 µm. I. Quantification of strain energy per cell (wt mCherry control n = 26; Sharpincpdm mCherry-Ctrl n = 21; Sharpincpdm mCherry-SHARPIN n = 24 cells from five independent experiments). J-K. Focal adhesion dynamics. J. Visualization of GFP-Paxillin dynamics in a wt and a Sharpincpdm cell over time. Colour scale represents a range from early to late occurring focal adhesions. K. Quantification of focal adhesion average size, assembly rate and disassembly rate (wt n =17527, 5250, 5311 FAs; and Sharpincpdm n= 9052, 2790, 3264 FAs, respectively from two independent experiments). Mean ± SEM.
SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland (2016)
Figure 6

Figure 6. SHARPIN regulates the assembly of cell-derived ECM in vitro

A. Quantification of soluble collagen levels in wt and Sharpincpdm MSF cultures on days 2, 5 and 7 post serum removal (n = 6 from two independent experiments). B-C. Analysis of the collagen network in cell-derived matrices (CDMs) produced by wt and Sharpincpdm MSFs using COL1A1 immunolabelling. B. Maximum intensity projections of confocal images are shown. Scale bars represent 50 μm. C. Quantification of distinct collagen fibre bundles in wt and Sharpincpdm CDM samples (n = 5). Mean ± SEM.
R-loop formation during S phase is restricted by PrimPol-mediated repriming (2018)
Figure 1

Figure 1. Short (GAA) tracts cause BU-1 epigenetic instability in primpol cells.

A. Expression instability of the chicken BU-1 locus as a reporter for replication impediments formed by structure-forming DNA sequences. The leading strand of a replication fork entering the locus from the 3' end encounters a DNA sequence with structure-forming potential located 3.5 kb downstream of the transcription start site. In wild type cells this is a G4 motif, which is replaced by (GAA)n repeats in this study. Under conditions in which polymerase stalling is prolonged, e.g. loss of G4 processing enzymes or G4 stabilisation (Guilbaud et al., 2017, Sarkies et al., 2010, Schiavone et al., 2014), or repriming is defective (Schiavone et al., 2016), the persistence of a putative ssDNA gap leads to a zone of interrupted histone recycling and loss of parental histone modifications. If this loss of modifications involves a control region of the gene, e.g. the promoter, it can result in a change in expression.

B. Flow cytometry for Bu-1a expression in wild type cells with (GAA)n tracts of different length knocked into the BU-1A locus (in blue). DT40 cells are heterozygous and carry one BU-1A and one BU-1B allele. All experiments introducing repeats into BU-1A are carried out in cells in which the +3.5 G4 has been deleted from both A and B alleles, to avoid transvection between the alleles (Schiavone et al., 2014). Black outline: positive control (wild type cells); red outline: negative control (cells carrying a genetic disruption of BU-1).

C, D. Bu-1a fluctuation analysis of wildtype and primpol cells in which the endogenous +3.5 G4 has been deleted (ΔG4) or with (GAA)10 and (GAA)20 sequence orientated such that it is replicated as the leading (C) or lagging (D) strand template for a fork entering from the 3' end of the locus as shown in panel (A). At least two independent fluctuation analyses were performed. Circles represent the percentage of Bu-1a loss variants in at least 24 individual clones from these experiments, with mean ± SD reported. ****p < 0.0001, * p ≤ 0.05, ns = not significant; one-way ANOVA.
R-loop formation during S phase is restricted by PrimPol-mediated repriming (2018)
Figure 2

Figure 2. The repriming function of PrimPol is required to maintain expression stability of BU-1 harbouring a (GAA)10 repeat.

Human PrimPol, or mutants, tagged with YFP were expressed in primpol cells harbouring (GAA)10 sequence in the BU-1A locus. Bu-1a and YFP double positive cells were sorted and expanded for 2 weeks, and then analysed for Bu-1a expression variants. For each complementation, at least two independently derived clones were subjected to fluctuation analysis. As previously observed (Schiavone et al., 2016), expression of hPrimPol[AxA] and hPrimPol[ZfKO] is deleterious and unstable. Cells expressing these mutations and remaining YFP-positive at the end of the expansion period will have been through fewer divisions the other lines in this analysis. Pooled results from at least three independent fluctuation analyses are represented with mean ±SD indicated with red bar and whiskers. Statistical significance: **** p < 0.0001, *** p<0.001, * p ≤ 0.05, ns = not significant; Kruskal-Wallis test.
R-loop formation during S phase is restricted by PrimPol-mediated repriming (2018)
Figure 3

Figure 3. R-loops promote (GAA)10-dependent epigenetic instability of BU-1.

A. DRIP-qPCR analysis reveals accumulation of R-loops across the BU-1 locus in primpol cells. The DRIP signal was calculated as enrichment over RNase H-treated samples and was normalised to -0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired t-test was used to compare differences between matched amplicons in primpol BU-1A(GAA)10 and the other cell lines indicated. **** p ≤ 0.0001, ns = not significant.

B. DNA:RNA hybrids in primpol BU-1A(GAA)10:GgRNase H1 (see also Appendix Fig S5) and primpol BU-1A(GAA)10:hPrimPol. An unpaired t-test on three biological replicates was used to compare differences to primpol BU-1A(GAA)10 for each matched amplicon. The bar represent the mean and whiskers the SD. *** p ≤ 0.001, ** p ≤ 0.01, ns = not significant.

C. Overexpression of chicken RNase H1 prevents (GAA)10 induced BU-1A epigenetic instability in primpol cells. Fluctuation analysis was performed on three primpol BU-1A (GAA)10 clones. One-way ANOVA was used to calculate the significance of differences in BU-1 instability between primpol BU-1A ΔG4 and other cell lines. **** p ≤ 0.0001, ns = not significant.

D. Diagram of the RNase H1 hybrid binding domain (HBD) - mCherry fusion and flow cytometry expression profiles of the construct in four clones. Western blots of the same four clones are shown in Appendix Fig S6.

E. R-loop stabilisation induces epigenetic instability of BU-1. Bu-1a fluctuation analysis of wild type cells expressing HBD-mCherry. The scatter plots pool results from at least two different clones with matched HBD-expression. Mean ± SD reported. **** p ≤ 0.0001, *** p ≤ 0.001, ns = not significant; one-way ANOVA.
R-loop formation during S phase is restricted by PrimPol-mediated repriming (2018)
Figure 4

Figure 4. Loss of PrimPol leads to unscheduled S phase R-loop formation.

A . Expression of geminin-tagged chicken RNase H1-YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 (X-axis). RNase H1-YFP with or without the geminin degron protein is detected on the Y-axis. The RNase H1-YFP-geminin degron is degraded in G1. In contrast, RNase H1-YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replication.

B. Bu-1a fluctuation analysis of two independently derived primpol BU-1A(GAA)10: Gg RNase H1-YFP-geminin degron clones. Since the expression of the RNase H1-YFP-geminin degron construct is not stable (unlike the RNase H1-YFP construct without the degron), Bu-1a expression was assessed separately in the YFP +ve and YFP -ve cells within each clone. Statistical differences calculated the Kruskal-Wallis test. For all panels, at least 36 individual clones were analysed; mean ± SD reported. **** p ≤ 0.0001, *** p ≤ 0.001, ns = not significant.

C. DRIP-qPCR for R-loops around the engineered +3.5 (GAA)10 repeat in BU-1 in different phases of the cell cycle. The location of the qPCR amplicons is indicated in the map at the top of the panel. The BU-1 DRIP signal was normalised to -0.5 kb amplicon in G1-arrested cells (t = 0 h). See Fig EV4 for representative cell cycle synchronisation profiles. Black: wild type; red: primpol. Error bars = SD. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

D. Workflow for the S9.6-independent detection of newly synthesised R-loops. See Material and methods for details.

E. Validation of analysis of nascent DNA:RNA hybrid formation in BU-1 locus. Enrichment of 4SU labelled RNA moiety of DNA:RNA hybrids was calculated relative to input in three independent asynchronous wild type (black) or primpol (red) cells, with or without exogenous RNase H treatment. Error bars = SD. ** p ≤ 0.01, * p ≤ 0.05, ns = not significant; unpaired t-test.

F. Synchronisation and 4SU pulse labelling scheme to identify nascently formed DNA:RNA hybrids.

G. Newly synthesised R-loops in BU-1 during S phase in wild type (black) and primpol (red). Error bars represent 1SD of three biological repeats of the experiment. *** p ≤ 0.001, * p ≤ 0.05; unpaired t-test
R-loop formation during S phase is restricted by PrimPol-mediated repriming (2018)
Figure 5

Figure 5. PrimPol suppresses R-loop formation in association with DNA secondary structure-forming sequences across the DT40 genome.

A. Representative normalised DRIP-seq data in two genes COL22A1, spanning over 200 kb, and MYC. The locations of H-DNA and G4 motifs are shown below the gene map. Wild type in blue; primpol in red. The corresponding RNAse H-treated samples are dashed. See Methods for further details of graphic generation.

B. Metagene analysis of DRIP peak distribution in wild type and primpol DT40 cells compared with the distribution of the indicated features in the genome.

C. DRIP peak heights in wild type and primpol DT40 normalised to Drosophila S2 spike-in. n (wild type) = 41445; n (primpol) = 48648.

D. Correlation of normalised DRIP peak heights in the overlapping peaks between wild type and primpol. Blue line = 1:1 correlation; red line = linear regression through data.

E. Correlation between H-DNA forming sequences and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red).

F. Normalised DRIP peak heights in the genes identified as associating with H-DNA.

G. Correlation between G4 motifs ([G3-5N1-7]4) and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red).

H. Normalised DRIP peak heights in the genes identified as associating with G4 motifs ([G3-5N1-7]4).

Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).
R-loop formation during S phase is restricted by PrimPol-mediated repriming (2018)
Figure 6

Figure 6 PrimPol suppresses R-loop formation in association with DNA secondary structure-forming sequences in BOBSC iPS cells.

A. Representative normalised RNA DIP-seq data in the SKI locus. Wild type in blue; primpol in red. The locations of H-DNA and G4 motifs are shown below the gene map. The corresponding RNAse H-treated samples are dashed. Since so little material was recovered following RNase H treatment, all samples were pooled prior to library generation.

B. Metagene analysis of RNA-DIP peak distribution in wild type and primpol BOBSC cells compared with the distribution of the indicated features in the genome.

C. RNA DIP-seq peak heights in wild type and primpol BOBSC cells normalised to a DT40 spike-in. n (wild type) = 32740; n (primpol) = 33721.

D. Correlation of normalised RNA DIP-seq peak heights in the overlapping peaks between wild type and primpol. Blue line = 1:1 correlation; red line = linear regression through data.

E. Correlation between H-DNA forming sequences and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red).

F. Correlation between G4 motifs ([G3-5N1-7]4) and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red).

G. Normalised RNA DIP-seq peak heights in the genes identified as associating with H-DNA.

H. Normalised RNA DIP-seq peak heights in the genes identified as associating with G4 motifs ([G3-5N1-7]4).

Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).
Macrophagic AMPKa1 orchestrates regenerative inflammation induced by glucocorticoids (2022)
Figure 1

Figure 1. Glucocorticoid signaling through AMPK in macrophages

(A) Bone Marrow Derived Macrophages (BMDMs) were treated with either the AMPK activator 991, Dex or a combination for 1 h before analysis by western blot. (B, C) Phospho-AMPK (B) and phospho-ACC (C) were quantified. (D) WT and AMPKα1-/- BMDMs were treated with Dex for 1 h before analysis by western blot and GR phosphorylation at S211 was quantified. (E) WT and AMPKα1-/- BMDMs were treated with Dex for 24 h and analyzed by RNA-seq. Differentially expressed genes between any condition were clustered using c-means clustering. (F) WT and AMPKα1-/- BMDMs were treated with Dex for 24 h and anti-/pro-inflammatory markers assessed using immunofluorescence. (G,H) WT and AMPKα1-/- BMDMs were treated with Dex for 72 h, washed and conditioned medium was recovered after 24 h used to treat muscle stem cells. Proliferation (Ki67 immunostaining) (G) or differentiation (desmin immunostaining and counting the percentage of muscle cells with 2 nuclei or more) (H) were assessed in muscle stem cells. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 6 (B,C), 3 (D), 3-4 (F) of 4 biological replicates (G, H). * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using student's t-test (B, C) or ANOVA tests (D,G,H). Bar=25 µm (G,H).
Macrophagic AMPKa1 orchestrates regenerative inflammation induced by glucocorticoids (2022)
Figure 2

Figure 2. AMPKα1 in macrophages is required for glucocorticoid-dependent resolution of inflammation during cardiotoxin induced skeletal muscle damage.

(A-C) After cardiotoxin injection to damage the muscle, AMPKα1fl/fl (WT) and LysM-α1-/- mice were treated with a single dose of Dexamethasone (Dex) intra-peritoneal (i.p.) (0.1 mg/kg) at day 3 (D3) and Tibialis Anterior (TA) muscles were harvested at day 8 (D8) and 14 (D14) after injury. (A) Representative embryonic myosin heavy chain (EmbMHC) immunostaining on whole TA muscle section at D8 and D14 after injury. (B) Quantification of (A), results are expressed as percentage of positive myofibers expressing the EmbMHC on the whole muscle section. (C) Muscle mass at D14 after injury. (D-E) TA muscle from WT and AMPKα1-/- (D) or LysM-α1-/- (E) mice was damaged with cardiotoxin and Dex (or vehicle) was i.p. injected around 60 h after injury (~ 2.5 days) prior CD45pos cells were sorted with magnetic beads 15 h later (day 3). (D) CD45pos CD64pos Ly6Cpos, Ly6Cint and Ly6Cneg macrophage subsets were cultured with dead fluorescently labelled myoblasts for 6 h. Phagocytosis was evaluated by flow cytometry and expressed as the GeoMean fluorescence Intensity of the phagocyting cells. (E) The proportion of macrophages (CD64pos) expressing anti-inflammatory markers was assessed by flow cytometry. (F) Macrophage polarization was determined by immunofluorescence in vehicle, DAMPs and DAMPs+Dex treated WT and AMPKα1-/- BMDMs. Data information: results are displayed with box and whiskers in which the central band represents the median (B,C,D,E) or means ± SEM (F). N= 7-12 muscles (B, C), 5-6 biological replicates (D), 4-12 muscles (E) and 4-5 biological replicates (F). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 using ANOVA (B-F) tests. Bars = 500 µm (A), 25 µm (F).
Macrophagic AMPKa1 orchestrates regenerative inflammation induced by glucocorticoids (2022)
Figure 3

Figure 3. AMPKα1 in macrophages is required for glucorticoid dependent suppression of inflammation in endotoxin-induced lung injury.

AMPKα1fl/fl (WT) and LysM-α1-/- mice were treated with vehicle or with 10 mg/kg lipolysaccharide (LPS) or LPS + Dexamethasone (Dex, 1 mg/kg), before an intravenous injection of oleic acid was made to induce lung inflammation. (A) Hematoxylin-eosin staining of lung tissue after 24 h. (B) Broncho-alveolar lavage (BAL) cytokine content was measured by Luminex after 24 h. (C,D) After 24 h, mice were culled and cells infiltrating the lungs were analyzed. (C) The number of cells present in the BAL fluid was counted. (D) The number of pro-inflammatory macrophages (F4/80pos Ly6Cpos) was evaluated by flow cytometry as a percentage of CD45pos cells. (E) Survival curve of mice treated with vehicle or Dex. (F) Macrophage polarization was determined by immunofluorescence in vehicle, LPS and LPS+Dex treated WT and AMPKα1-/- BMDMs, data are expressed as percentage positive cells. Data information: Results are displayed with box and whiskers in which the central band represents the median (C,D) or means ± SEM (F). N= 5-10 (C), 5-7 (D), 3-7 (data below detection threshold were excluded from analysis) (B), 13-15 (E) or 3 (F) biological replicates. Statistical analysis by ANOVA (C,D,F) or Gehan-Breslow-Wilcoxon test (E). * p<0.05, ** p<0.01, *** p<0.001. Bar = 20 µm (A), 25 µm (F).