EMBO/soda-nel huggingface dataset:
miR-424/503 modulates Wnt/β-catenin signaling in the mammary epithelium by targeting LRP6 (2021)
Figure 1

Figure 1. Loss of miR-424/503 is associated with tumors with elevated Wnt/β-catenin activity. A) H&E and immunohistochemistry reveal that β-catenin accumulates in tumors emerging in one-year-old female miR-424(322)/503-/- mice. The panel is a representative image of n=5 independent tumors. The scale bar indicates 100 micrometers (µM).

B) Bar graphic showing that human breast cancers with deletion of the miR-424/503 locus have higher protein levels of β-catenin (RPPA). P-value is calculated based on one-sided Wilcoxon rank-sum test. In the box-plots in B and C the relative expression of β-catenin in the RPPA data from TCGA is shown. The yellow line indicates the median and the bars indicate the 75th percentile (Q3)+1.5 interquartile range (IQR).

C) The bar graph shows that human breast cancers with low expression levels of miR-424 and miR-503 have higher protein levels of β-catenin. P-value is calculated based on one-sided Wilcoxon rank-sum test.

D) GSEA indicating that tumors with deletion of the miR-424/503 locus present higher canonical Wnt-signaling activity. Independent analysis for TCGA and METABRIC data sets are shown. . P-values were obtained from the GSEA pipeline based on permutation tests.

E) The graphic shows the enrichment of miR-424/503 deletions in the BL2 and M subtypes of TNBC. The red line shows the overall fraction of miR-424/503 deletions in TCGA samples. The enrichment in subtypes is estimated by the binomial test. The asterisks indicate the columns that the p-values above are linked to.
miR-424/503 modulates Wnt/β-catenin signaling in the mammary epithelium by targeting LRP6 (2021)
Figure 2

Figure 2. The expression level of miR-424(322)/503 regulates Wnt/β-catenin activity.

A) The upper panel indicates the FACS strategy followed to purify the different mammary epithelial subpopulations found in the mouse mammary gland (basal, luminal HR-, luminal HR+, and MaSCs). The lower panel shows the normalized expression of miR-424(503) and miR-503 revealing that MaSCs express the highest levels. The error bars show the standard deviation. FACS was performed at two different stages of the ovarian cycle (estrus and diestrus). Carmine-red staining of the structure of the mammary tree at the time of purification is also provided. The scale bar indicates 5 millimeters (mm).

B) The bar graphs show the number of PROCR+ in 1-year-old female WT and miR-424(322)/503 KO mice during diestrus. The error bars show the standard deviation.

C) The bar graphs indicate the number of primary organoids formed when FACS sorted WT or miR-424(322)/503 mammary epithelial basal and luminal cells from female mice at diestrus (4-6 months old) were cultured as organoids. The error bars show the standard deviation.

D) The western blot shows the difference in nuclear accumulation of β-catenin when dox-induced (100ng/ml for 3 days) miR-cluster-Dox-MCF-10A cells (miR) and miR-control-Dox-MCF-10A cells (EV, empty vector) were exposed to of WNT3a ligand (100ng/ml for 6 hours). The numbers below the blots indicate the quantitation by densitometry of protein expression relative to EV without WNT3A. Histone H1 staining is shown to control for nuclear-cytosolic purification. The upregulation of miR-424 and miR-503 when dox was added to the media was quantified by QRT-PCR and is shown in the bar graph. The error bars show the standard deviation.

E) The panel shows the lack of induction of bona fide WNT targets in miR-over expressing cells from panel C compared with EV control. UT indicates samples untreated with WNT3.

Data information: For all panels, the results shown are the average of n≥3 or representative images of n≥3 independent biological experiments. The asterisks indicate a p-value <0.05 calculated by T-test and the bars show the standard deviation; n.s indicates no significant difference.
miR-424/503 modulates Wnt/β-catenin signaling in the mammary epithelium by targeting LRP6 (2021)
Figure 3

Figure 3. Mir-424/503 cluster targets the Wnt co-receptor LRP6.

A) The table shows putative miR-424(322)/503 targets selected from the TargetScan database and ranked based on their probability of conserved targeting. A schematic representation of the canonical Wnt pathway with arrows indicating the putative targets that were investigated is also shown.

B) The upper panel illustrates the location of the conserved and non-conserved (-nc) predicted microRNA binding sites in the 3'UTR of 3 selected target genes. The bar graph compares the normalized expression of a firefly luciferase reporter (luc) plasmid (EV) after transfection in 293T cells and the same luc reporter with a modified 3'UTR containing predicted microRNA binding sites. The values represent the ratio of luc expression when the reporter was co-transfected with synthetic miR-424 and miR-503 vs. a control non-targeting sequence. The bars with a diagonal line pattern indicate non-conserved sites. A known miR-424/503 binding site located in the 3'UTR of CDC25 is used as a positive control. The horizontal line indicates 25% of reduction in luminescence and it is included as a visual reference. The error bars show the standard deviation.

C) Luciferase assays showing that the effect of the miR-424 and miR-503 on luc expression is abrogated when the seed sequence in the microRNA binding sites was mutated. The study was performed in four sites statistically significant from panel B. The error bars show the standard deviation.

D) Western blot of LRP6 and TBLR1 in the miR-cluster-Dox-MCF-10A model upon upregulation of miR-cluster with 100 ng/ml of doxycycline for the specified number of days. The numbers below the blots indicate the quantitation by densitometry of protein expression relative to β-actin. The expression level of miR-424 and miR-503 is indicated in the bar graph below the blots. UN indicates samples untreated with doxycycline. The error bars show the standard deviation.

E) Western-blots of LRP6 and TBLR1 in parental MCF-10A cells after transfection with miRNA hairpin inhibitors against miR-424/503.

F) Western blot of LRP6 and TBLR1 expression in WT and miR-424/503-KO basal cell-derived organoids after 14 days in culture with and without Wnt3a (100ng/ml). The arrow shows the correct TBLR1 band.

G) IHC staining showing that LRP6 accumulates in the epithelium of female miR-424(322)/503-/- mice (1-year-old). The scale bar indicates 100 micrometers (µM).

Data information: For all panels, the results shown are the average of n≥3 or representative images of n≥3 independent biological experiments. The asterisks indicate a p-value <0.05 calculated by T-test and the bars show the standard deviation; n.s indicates no significant difference. The numbers under the blot indicate the quantification of the bands done by densitometry.
miR-424/503 modulates Wnt/β-catenin signaling in the mammary epithelium by targeting LRP6 (2021)
Figure 4

Figure 4. Regulation of LRP6 by miR-424/503 occurs through the Lrp6 mRNA 3' UTR.

A) Enrichment of the putative LRP6 binding sites bound to AGO2 in miR-cluster-Dox-MCF-10A cells after the miR-424/503 cluster is induced with 100ng/ml of dox for three days. A CDC25A-binding site is included as a positive control and a region of B2M and GAPDH are included as negative controls. The error bars show the standard deviation.

B) The WT-blot shows the expression of LRP6 upon upregulation of miR-424/503 in miR-cluster-Dox-MCF-10A cells (Dox+) when one or both of the conserved microRNA binding sites (#1 and #3) are blocked by target protection.

C) The WT-blot shows the expression of LRP6 and β-catenin in cells in miR-cluster-Dox-MCF-10A cells cultured in the presence of Dox and with and without WNT3a ligand (100ng/ml for 6 hours) when the microRNA binding sites #1 and #3 are blocked by target protection.

Data information: For all panels, the results shown are the average of n≥3 or representative images of n≥3 independent biological experiments. The asterisks indicate a p-value <0.05 calculated by T-test and the bars show the standard deviation; n.s indicates no significant difference. The numbers under the blot indicate the quantification of the bands done by densitometry.

Expanded View Figures
Miro proteins prime mitochondria for Parkin translocation and mitophagy (2018)
Figure 1

FIGURE 1. Miro1 and 2 are required for Parkin translocation to mitochondria and mitophagy

A. Representative pseudocolor images of PC6 cells transfected with EYFP-Parkin (green) and mitochondrially targeted CFP (red) with and without PINK1 and Miro1 shRNA and Miro2 shRNA. Zoomed images show translocation of EYFP-Parkin to mitochondria in PINK1-overexpressing PC6 cells.

B. Quantification of EYFP-Parkin translocation to mitochondria. The percentage of PC6 cells with EYFP-Parkin translocated to mitochondria in response to PINK1 overexpression is lower in the Miro shRNAs-expressing groups when compared with the scrambled shRNA-expressing group (scr shRNA). ****P < 0.0001, n = 6 dishes per group, 20 fields per dish, one-way ANOVA.

C. The kinetics of EYFP-Parkin translocation is different in PC6 cells transfected with scrambled or Miro shRNAs. Cells were plated in separate compartments of the same dish, enabling us to visualize Parkin translocation simultaneously over 3 h under similar conditions in response to antimycin with oligomycin treatment (A&O, both 10 µM). The spatial heterogeneity of EYFP-Parkin (coefficient of variation of the intensity of individual pixels) was estimated for individual cells for each time point (n = 39-47 cells; P < 0.0001 difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM).

D. Miro shRNAs mitigate the effect of A&O on EYFP-Parkin translocation. PC6 cells transfected with EYFP-Parkin with and without Miro shRNAs were treated with DMSO or A&O (both 10 µM) for 3h. The figure demonstrates that the percentage of cells with Parkin translocated to mitochondria was lower in the Miro shRNAs-expressing group when compared with the scrambled shRNA-expressing group. ****P < 0.0001, n = 4 dishes, 20 fields per dish, one-way ANOVA.

E. Miro shRNAs inhibit PINK1-induced mitophagy in PC6 cells. The figure shows the changes in the excitation spectra of mitochondrially targeted pH-sensitive Keima (ratio of 561 nm/458nm fluorescence intensities). ****P < 0.0001, n = 80 cells from 4 dishes, Kruskal-Wallis test.

F. Miro shRNAs inhibit basal mitophagy in primary cortical neurons expressing mitochondrially targeted Keima. Mitochondria in lysosomes (acidic pH) were counted manually, by a blinded observer (**P < 0.01, n = 9 dishes, 9 fields per dish, from 2 independent experiments, one-way ANOVA).

G. Miro shRNAs decrease the number of mitochondria co-localised with the autophagosome marker EGFP-LC3B in primary cortical neurons (**P < 0.01, n = 5 dishes, 9 fields per dish, one-way ANOVA).

Data information: Data are presented as means ± SEM.
Miro proteins prime mitochondria for Parkin translocation and mitophagy (2018)
Figure 3

FIGURE 3. Miro1-induced Parkin translocation to mitochondria does not require PINK1 and is independent from E3 ligase activity of Parkin

A. Miro1 induces EYFP-Parkin translocation in the presence of PINK1 shRNA. PC6 cells were transfected with EYFP-Parkin and Miro1 or treated for 3h with A&O (both 10 µM) in the presence of scrambled shRNA or PINK1 shRNA. Miro1 induced Parkin translocation also in the presence of PINK1 shRNA while A&O treatment did not. ****P < 0.0001 compared with the respective control group, n = 5-6 dishes, 20 fields per dish, one-way ANOVA.

B. Miro1 induces EYFP-Parkin translocation in PINK1 -/- MEFs. MEFs obtained from PINK1 +/+ and -/- mice were transfected with EYFP-Parkin and Miro1 or treated with A&O (both 10 µM for 3h). Miro1 induced Parkin translocation in both MEF lines but A&O only in PINK1 +/+ MEFs. ****P < 0.0001 compared with respective control group, n = 6 dishes, one-way ANOVA.

C. Mutated EYFP-Parkin is recruited to mitochondria in response to Miro1 overexpression. PC6 cells were transfected with wild type, T240R or C431N EYFP-Parkin and Miro1 or treated for 3 hours with A&O (both 10 µM). Miro1 induced translocation of all EYFP-Parkin mutants whereas A&O treatment was not able to induce equivalent translocation of Parkin mutants. **P < 0.01 and ****P < 0.0001 compared with respective control group, n = 3-6 dishes, 20 fields per dish, one-way ANOVA.

D. Parkin mutants T240R and C431N interact with Miro1 both in DMSO- or A&O-treated (both 15 µM for 3h) HEK cells. Note that the higher molecular weight bands of Miro1 were observed only in WT Parkin-expressing groups.

Data information: Data are presented as means ± SEM.
Miro proteins prime mitochondria for Parkin translocation and mitophagy (2018)
Figure 4

FIGURE 4. Prevention of Miro1 ubiquitination at K572 reveals two steps in Parkin recruitment to mitochondria

A. Miro1 K572R mutant interacts with HA-Parkin similarly to Miro1 WT. HEK cells overexpressing myc-Miro1 WT or K572R mutant were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. Myc-Miro1 was immunoprecipitated and probed for myc-Miro1 and HA-Parkin. Note the diminished degradation of Miro1 K572R in input and the additional band above Miro1 WT in the IP, which is missing in the case of the K572R mutant.

B. Miro1 K572R ubiquitination by Parkin is reduced. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1 K572R and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin-HA and myc-Miro1.

C. Representative images of PC6 cells overexpressing Miro1 K572R or Miro1 WT together with EYFP-Parkin. K572R mutant induces Parkin translocation similar to WT in basal conditions but not after treatment with A&O (both 7.5 µM) for 3 hours. Note the spatial heterogeneity values shown in the images.

D. Quantification of EYFP-Parkin signal heterogeneity in PC6 cells demonstrates that WT Miro1 overexpression amplifies Parkin translocation in response A&O (both 7.5 µM) treatment for 3 hours, but K572R mutation of Miro1 abolishes this effect. ***P < 0.001 and ****P < 0.0001, n = 152-172 cells from 3 dishes per group, Kruskal-Wallis test. Data are presented as means ± SEM.
Miro proteins prime mitochondria for Parkin translocation and mitophagy (2018)
Figure 5

FIGURE 5. Ca2+-insensitive Miro1 is less ubiquitinated in response to mitochondrial depolarisation

A. A Ca2+-insensitive mutant of Miro1 (Miro1-EF) interacts with Parkin. HEK cells overexpressing myc-Miro1 or myc-Miro1-EF and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. Myc-Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 (red) and Parkin (green). The high-contrast grayscale image from the green channel of this blot shows that endogenous Parkin co-immunoprecipitated with Miro1 in all conditions. The bar chart shows the quantification of co-immunoprecipitated Parkin EYFP from 4 different blots normalised to total EYFP-Parkin signal (*P < 0.05 and **P < 0.01, repeated measures ANOVA)

B. Miro1-EF is less susceptible to ubiquitination by Parkin. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1-EF and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin (red), Miro1 (green) and Parkin (green; representative images are from the same experimental material on separate SDS-PAGE-WB membranes). The high-contrast grayscale shows the green channel of this blot, and the bar chart shows quantification of co-immunoprecipitated Miro from 3 different blots (**P < 0.01 and ***P < 0.001, repeated measures ANOVA). Note that EYFP-Parkin co-immunoprecipitated with ubiquitin only after A&O treatment.

C. Mutation of Miro1 EF-hands diminishes Miro1 ubiquitination with S65-phosphorylated ubiquitin. HEK cells were transfected with EYFP-Parkin with either myc-Miro1-WT or myc-Miro1-EF with or without PINK1-V5. Cells were treated with either DMSO or A&O (both 10 µM) for 5 hrs with the exception of the PINK1-V5 group. Myc-Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 (red), S65-Phosphoubiquitin and PINK1-V5 (green; the weak bands marked with the asterisk are nonspecific).

Data information: Data are presented as means ± SEM.
Miro proteins prime mitochondria for Parkin translocation and mitophagy (2018)
Figure 6

FIGURE 6. Ca2+ insensitive Miro1 inhibits Parkin translocation to mitochondria

A. Kinetics of EYFP-Parkin translocation in response to A&O is different in control and Miro1-EF-expressing PC6 cells. The inset shows how different compartments of the same dish were transfected with EYFP-Parkin and control plasmid or Miro1-EF and followed over 3 hrs using a confocal microscope after addition of A&O (both 10 µM). The graph shows the average spatial heterogeneity of EYFP-Parkin fluorescence, reflecting the ratio of mitochondrial-to-cytosolic Parkin over time (n = 40-43 cells, difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM).

B, C The percentage of cells where EYFP-Parkin translocates in response to A&O (both 10 µM, B) or PINK1 overexpression (C) is decreased in Miro1-EF-expressing PC6 cells. **P < 0.01, ****P < 0.0001 compared with the indicated group (n = 3-6 dishes, 20 fields per dish, one way ANOVA for B and Welch's ANOVA for C).

D. Time course showing laser-induced EYFP-Parkin accumulation in the irradiated area of the neuronal soma of primary rat cortical neurons. Cells were transfected with CFP-mito and EYFP-Parkin and visualized using a confocal microscope. Mitochondria were irradiated in a 0.9 µm x 0.9 µm-frame using a 405-nm laser line (25 iterations using 100% laser power, pixel time 164 µs).

E, F Miro1-EF delays and reduces irradiation-induced EYFP-Parkin translocation. (E) The time course of translocation strength in control and Miro1-EF-expressing neurons. Difference between the curves P = 0.0016, two-way ANOVA. (F) Comparison at the 60-min time point (*P < 0.05, n = 6 dishes, at least 9 neurons per dish, t-test).

Data information: Data are presented as means ± SEM.
Miro proteins prime mitochondria for Parkin translocation and mitophagy (2018)
Figure 7

FIGURE 7. Glutamate-induced Ca2+ entry leads to Miro1-dependent Parkin translocation and mitophagy

A. EYFP-Parkin translocation requires Ca2+. PC6 cells plated in separated compartments of the same dish maintained during the experiment in Krebs containing 1 mM Ca2+ or in Ca2+ free Krebs containing 5 μM BAPTA-AM, were treated with A&O (both 10 μM). Images (left panels) demonstrate that EYFP-Parkin translocation was significantly weaker at zero Ca2+ at 195 min after A&O treatment (compared with 15 min; note the spatial heterogeneity values shown in photos). Spatial heterogeneity analysis (right panel) demonstrated that Parkin translocation was significantly decelerated in zero Ca2+ conditions (n = 25-27 cells; difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM).

B. EYFP-Parkin translocation accelerates in the presence of the L-type Ca2+ channel agonist Bay K 8644 (5 μM; n = 19-21 cells; difference between the curves p < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM).

C, D Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol, visualised by increased cytosolic GCaMP7b fluorescence. Glutamate-induced Ca2+ entry was not affected by Miro1 WT or Miro1 EF overexpression; n = 31-48 neurons per group; ns, Kruskal-Wallis test).

E, F Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) led to the appearance of a few Parkin-positive strong puncta co-localising with mitochondrial marker (white arrow), imaged 2.5 h after treatment (E). Further analysis demonstrated that the expression of Miro1-EF mutant decreased and Miro1 WT increased the number of neurons with EYFP-Parkin-positive mitochondria following glutamate treatment (F; n = 6-10; **P < 0.01 and ****P < 0.0001, one-way ANOVA).

G. Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) was associated with an increased number of mitochondrial co-localisation with the autophagosome marker EGFP-LC3B in neuronal soma at 5-6 h later, which was not observed in Miro1-EF-expressing neurons (n = 8 dishes; *P < 0.05, one-way ANOVA).

H. Glutamate treatment (30 μM glutamate and 1 μM glycine for 10 min) was also associated with a significant loss of mitochondrial density in axons at 6 h later, which was reversed by Miro1-EF but not by Miro1 WT (n = 60-90 axons from 6-9 dishes; **P < 0.01, ***P < 0.001 and ****P < 0.0001, Kruskal-Wallis test).

I. Glutamate treatment (100 μM glutamate and 10 μM glycine for 10 min) led to a decline, at 6-8 h, in cytosolic ATP measured using ATP/ADP sensor Perceval HR, which was reversed by Miro1-EF but not by Miro1 WT (n = 59-60 neurons from 6 dishes; **P < 0.01, Welch's ANOVA).

J. Glutamate (100 μM glutamate and 10 μM glycine for 10 min) induced excitotoxicity (24 h after treatment) that was partially reversed by Miro1-EF but not by Miro1 WT. Data is presented as a percentage of control neuron count (n = 10 dishes; ***P < 0.001, repeated measures ANOVA).

Data information: Data are presented as means ± SEM.
Bacterial protein domains with a novel Ig-like fold bind human CEACAM receptors (2020)
Figure 1

Figure 1: Human CEACAM1 binds to group B Streptococcus (GBS) via the surface-expressed β protein.

A, B) Binding of recombinant (r)CEACAM1 (CC1)-HIS (10 μg/mL) to (A) a panel of Gram-positive bacteria, namely Enterococcus faecalis, Enterococcus faecium, Group A Streptococcus (GAS), Group B Streptococcus (GBS), Group C Streptococcus (GCS), Group G Streptococcus (GGS), Streptococcus pneumoniae, Staphylococcus aureus, (B) an expanded collection of GBS isolates, with serotype and carriage of bac gene shown. rCC1 binding to live bacteria was quantified by a secondary anti-His-FITC monoclonal antibody (mAb). Mean and standard deviation (SD) values are reported for n = 3 independent experiments. In (B), GBS strains from left to right are A909, BS39, 515, BS22, H36B, BS29, 18RS21, BM110, COH1, SBL3066, NCTC10/84, SB35, SB10, SB20, BS26.

C) Correlation between rCC1-binding capacity and β protein expression in GBS strains, respectively. β protein expression was quantified using rabbit anti-β protein serum and a secondary PE-conjugated goat anti-rabbit-IgG. rCC1 binding to live bacteria was quantified by a secondary anti-His-FITC mAb. Mean binding values from n = 3 independent experiments.

D) Concentration-dependent binding of rCC1-HIS to wild type (WT), ∆bac and ∆bac + pLZ.bac complemented A909 strains. Mean and SD values are reported for n = 3 independent flow cytometry experiments.

E) Pull-down of human rCC1-HIS (30 μg/mL) by GBS strain A909 strain, detected using Western blot analysis.

F) Human rCC1-HIS (30 μg/mL) binding to GBS A909 and A909∆bac strains analyzed by pull-down experiments followed by Western blot analysis.

G) Western blot analysis of rCC1-HIS (30 μg/mL) binding to purified β protein, but not to control GBS surface proteins α and Rib. Panel on the left show staining of proteins after separation by SDS-PAGE.

H, I) Binding of purified β protein to dynabeads (DB) coated with rCC1 (DB.CC1) or control. Binding of β protein was detected using mouse anti-β serum or normal mouse serum (NMS), and PE-conjugated goat anti-mouse-IgG. (H) shows mean and SD values compiled from n = 3 independent replicates, and (I) shows representative flow cytometry plots using 10 μg/ml rCC1.

Data information: Fluorescence of bacteria (A, B, C and D) and DB (H and I) was measured by flow cytometry. Western Blot in (E,F and G) employed mouse anti-CC1 mAb (clone C5-1X/8) and HRP-conjugated goat anti-mouse-IgG pAb.