EMBO/soda-nel huggingface dataset:
Receptor interacting protein kinase 2–mediated mitophagy regulates inflammasome activation during virus infection (2013)
figf7

(a–c) Flow cytometry analysis of wild-type (WT) and Ripk2−/− BMDCs stained with the mitochondrial superoxide–specific stain MitoSOX during IAV or Listeria infection (a,b) and with the general mitochondrial stain MitoTracker Green during IAV infection (c). (d,e) Analysis of IL-18 production and immunoblotting to analyze caspase-1 (casp-1) p20 activation in IAV-infected BMDCs that had been pretreated with the ROS inhibitor NAC. (f) Immunoblot of casp-1 p20 in cell lysates of IAV-infected BMDCs pretreated with the mitochondrial superoxide inhibitor mitoTEMPO. (g) Confocal microscopy analysis of BMDCs stained for mitochondria (red) and LC3 (green) for colocalization as an indicator of mitophagy. Original magnification, ×400; inset (red boxed region) was digitally enlarged ×10. *P 0.01 (one-way ANOVA with Tukey's post-hoc test (d) and unpaired two-sided Student's t-test (g)). Data are representative of nine experiments (a), three experiments (b,d–g) or six experiments (c) (mean ± s.d.; n = 2–3 replicates per experiment).
Receptor interacting protein kinase 2–mediated mitophagy regulates inflammasome activation during virus infection (2013)
figf8

(a,b) IL-18 amounts (a), and caspase-1 (casp-1) p20 and LC3-II immunoblots (b) in PR8-infected wild-type (WT) BMDCs treated with the p38 and RIPK2 kinase inhibitor SB203580. (c) Immunoblots for phosphorylation of ULK1 at Ser555 in WT and Ripk2−/− BMDCs after PR8 infection. (d) Immunoblots for casp-1 p20 in untreated or NAC-treated WT and Ulk1−/− BMDCs after PR8 infection. (e) Flow cytometry analysis of MitoSOX staining in PR8-infected WT and Ulk1−/− BMDCs. (f) MitoTracker Green staining of PR8-infected WT and Ulk1−/− BMDCs. (g) Diagram of the NOD2-RIPK2–dependent mechanism for mitophagy and subsequent NLRP3 inflammasome regulation. *P 0.05 (unpaired two-sided Student's t-test). Data are representative of three experiments (a–c) or two experiments (d–f) (mean ± s.d.; n = 2–3 replicates per experiment).
Reprogramming-derived Gene Cocktail Increases Cardiomyocyte Proliferation for Heart Regeneration (2016)
Figure 1

Figure 1. Early reprogramming process of CMs and NCs.

A. Flow cytometry of cells isolated from heart tissue when scaled against TMRM staining and also FSC and SSC parameters of flow cytometry.

B. Cardiac troponin I (cTnI) and α-Sarcomeric actinin (α-SA) staining of the TMRM++ population (CMs) and Vimentin staining of the TMRM+ population (NCs). The scale bar represents 100 µm.

C. Schematic showing early reprogramming processes of CMs and NCs isolated from triple transgenic mice.

D. EGFP expression in iPSC-like colonies derived from CMs or NCs isolated from triple transgenic mice.

E. EGFP expression combined with immunofluorescence staining of SSEA-1 and DAPI of iPSC-like colonies derived from CMs isolated from triple transgenic mice.

F. Morphological changes of isolated CMs and NCs at different time points before AP positive colony formation at day 6. The scale bar represents 100 µm.
Reprogramming-derived Gene Cocktail Increases Cardiomyocyte Proliferation for Heart Regeneration (2016)
Figure 3

Figure 3. Gene screening for CM proliferation by mimicking early reprogramming.

A. RNA expression ratio (D2-to-D0) of candidates selected from day 2 microarray data.

B. Timeline followed for proliferation examination of neonatal CMs in vitro.

C. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with control (Ctrl), or combinations of a FoxM1 up-regulation adenovirus (F), Id1 up-regulation adenovirus (I), Hmgb2 up-regulation adenovirus (H) or a small molecule Jnk3 inhibitor (Ji).

D. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with Ctrl or combination of FI with Jnk3-shRNAs in place of the small molecule (Ji). The arrow head indicates Ki67+/α-MHC+ proliferated CMs.

E. Morphology and quantification of FIJs-treated CMs undergoing cytokinesis by H3P and Aurora kinase B (AURKB) staining.

F. Timeline followed for adult reprogrammable mice administered the control or doxycycline in vivo.

G. RNA expression ratio of Oct4, FoxM1, Id1, or Jnk3 in doxycycline-injected versus control adult CMs isolated from reprogrammable mice.

H. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from control or doxycycline injected mice.

The scale bar represents 50 µm. Data are represented as mean ± SEM. Sample size is indicated in the bar chart. Statistical analysis – unpaired T-test. *, P < 0.05, **, P < 0.01, and ***, P < 0.001.
Reprogramming-derived Gene Cocktail Increases Cardiomyocyte Proliferation for Heart Regeneration (2016)
Figure 4

Figure 4. The defined gene cocktail induces CM proliferation in neonatal and adult mice.

A. Timeline followed for neonatal P1 mice administered the Ctrl or FIJs.

B. The size of hearts isolated from Ctrl or FIJs-treated mice; heart/body weight ratio of Ctrl or FIJs-treated mice.

C. H3P, cTnI, and DAPI staining and quantification of heart tissues in Ctrl or FIJs-treated mice. The arrowhead indicates H3P+/cTnI+ proliferated CMs and the scale bar represents 50 µm.

D. Timeline followed for adult mice administered the adeno-Ctrl or adeno-FIJs.

E. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from adeno-Ctrl or adeno-FIJs injected mice.

F. Quantification of BrdU+/EGFP+ population % in isolated CMs from adeno-Ctrl or adeno-FIJs injected M/Z mice by flow cytometry.

The arrowhead indicates H3P+/cTnI+ proliferated CM and the scale bar represents 50 µm. Data are represented as mean ± SEM. Sample size is indicated in the bar chart. Statistical analysis – unpaired T-test. *, P < 0.05, ***, P < 0.001, and n.s., no significance.
Reprogramming-derived Gene Cocktail Increases Cardiomyocyte Proliferation for Heart Regeneration (2016)
Figure 5

Figure 5. Enhancement of CM proliferation by delivery of a gene cocktail after MI.

A. Timeline followed to analyze proliferation of CMs and heart improvement in mice subjected to MI followed by administration of FIJs treatment.

B. RNA expressional changes of FoxM1, Id1, and Jnk3 after adeno-Ctrl or adeno-FIJs treatment after MI.

C. Immunofluorescence staining of BrdU or H3P and cTnI or EGFP and quantification in the heart tissues after MI. The arrow head indicates cTnI+/BrdU+, cTnI+/H3P+ or EGFP+/BrdU+, EGFP+/H3P+proliferated CMs. The scale bar represents 50 µm.

D. Ejection fraction and fraction shortening percentage in Ctrl or FIJs-treated mice 21 days post-MI.

E. Catheterization in Ctrl or FIJs-treated mice 21 days post-MI under baseline or inferior vena cava occlusion condition.

F. Trichrome staining and quantification showing percentage of fibrotic tissue (blue) in Ctrl or FIJs-treated mice 21 days after MI.

Data are represented as mean ± SEM. Sample size is indicated in the bar chart. Statistical analysis – unpaired T-test. *, P < 0.05 and n.s., no significance.
Reprogramming-derived Gene Cocktail Increases Cardiomyocyte Proliferation for Heart Regeneration (2016)
Figure 6

Figure 6. Cell cycle effect of each gene or combination of the cocktail on CMs.

A. RNA expression of mitosis checkpoint genes in CMs with FoxM1 or Id1 overexpression, Jnk3-shRNA, or combined treatment.

B. Transcriptional expression of cyclin-dependent kinases in FoxM1 or Id1 up-regulated, Jnk3 down-regulated, or combined treated CMs.

C. Real-time RT-PCR analysis of CDK inhibitor expression in CMs with FoxM1 or Id1 overexpression, Jnk3-shRNA or combined treatment.

D. Schematic diagram demonstrating the proposed mechanism for FIJs-driven CM proliferation.

Data are represented as mean ± SEM. n=3. Statistical analysis – unpaired T-test. *, P < 0.05, **, P < 0.01 and ***, P < 0.001.
Monocytopenia, morphological anomalies and hyperinflammation characterise severe COVID-19 in type 2 diabetes (2020)
Figure 1

Figure 1. Type-2 diabetes is associated with decreased monocyte frequency and phenotypic alterations in COVID-19 patients.

A Flow cytometric quantification of lymphocytes, monocytes, B-cells, natural killer (NK) cells, dendritic cells (DCs) and granulocytes in peripheral venous blood samples from non-diabetic (ND) and type-2 diabetic (T2D) patients with COVID-19.

B Quantification of monocyte subpopulation phenotypes as classical (CD14Hi CD16-), intermediate (CD14Hi CD16+) or non-classical (CD14Lo CD16+).

C Principle component analysis of lymphocyte and monocyte population frequencies in ND and T2D COVID-19 patients and in T2D patients without COVID-19.

Data information: Data are presented as mean +/- SEM. Differences between groups were evaluated with unpaired t-test. See also Appendix Figure S1 and Appendix Tables S1-3. Sample size and exact p-value in Appendix Table S5.
Monocytopenia, morphological anomalies and hyperinflammation characterise severe COVID-19 in type 2 diabetes (2020)
Figure 2

Figure 2. Morphologically altered monocytes in type-2 diabetic COVID-19 patients are associated with an aberrant inflammatory response and increased disease severity.

A Quantification of CD14 expression and size (FSC) in monocyte populations from non diabetic (ND) and type-2 diabetic (T2D) COVID-19 patients.

B Frequency of conventional (FSCLo) and large (FSCHi) monocytes in ND and T2D COVID-19 patients.

C Expression of CD16, CD14 and HLA-DR in FSCLo and FSCHi monocytes from COVID-19 patients.

D Proportions of classical, intermediate and non-classical monocytes within FSCLo and FSCHi monocytes from COVID-19 patients.

E Correlative analyses of IL8 expression in peripheral blood mononuclear cells (PBMCs) to monocyte FSC.

F IL8, IL6 and CCL2 mRNA expression in PBMCs from ND and T2D COVID-19 patients.

G Monocyte size quantified in ND and T2D COVID-19 patients treated in general wards (Gen) or in intensive care unit (ICU).

Data information: Data are presented as mean +/- SEM. Differences between groups were evaluated with unpaired t-test (except for c. and d. where paired by patient). Analyses of variance (ANOVA) or covariance (ANCOVA) were used for multiple group comparisons. For correlatiive analysis Spearman's test was carried out calculating a 2-tailed p-value. See also Appendix Figure S2. Sample size and exact p-value in Appendix Table S5.
Monocytopenia, morphological anomalies and hyperinflammation characterise severe COVID-19 in type 2 diabetes (2020)
Figure 3

Figure 3. IRF5 expression is associated with monocyte activation and morphological adaptation in COVID-19 patients

A Quantification of IRF5 and IFNB1 mRNA expression in peripheral blood mononuclear cells of non-diabetic (ND) and type 2 diabetic (T2D) COVID-19 patients (A).

B Histograms of IRF5 expression on different populations analysed by flow cytometry on venous blood samples.

C IRF5 median fluorescence intensity (MFI) in monocytes of ND and T2D patients with COVID-19.

D IRF5 expression overlaid onto monocyte phenotypic gating.

E IRF5 expression (MFI) in monocyte subtypes from ND and T2D COVID-19 patients.

F HLA-DR and IRF5 expression in monocytes from COVID-19 patients.

G IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted to intensive care unit (ICU) or treated exclusively in general wards (Gen) .

H IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted ICU or treated exclusively in Gen.

Data information: Data are presented as mean +/- SEM. Differences between groups were evaluated with unpaired t-test. Analyses of variance (ANOVA) was used for multiple group comparisons. For correlative analysis Spearman's test was carried out calculating a 2-tailed p-value. See also Appendix Figure S3. Sample size and exact p-value in Appendix Table S5.
Modeling by disruption and a selected-for partner for the nude locus (2020)
Figure 1

Figure 1. FOXN1 disrupts eye and bristle development in flies.

(A, B) Flies form well-organized compound eyes when they carry GMR-GAL4 without a target gene (A) but develop rough eyes when GMR-GAL4 drives expression of UAS-Foxn1 (B). Panel B shows the typical severity of the rough-eye phenotype.

(C, D) Bristles are pigmented and properly shaped when flies carry hs-GAL4 without a target gene (C) but are often white, bent, and shortened when hs-GAL4 drives expression of UAS-Foxn1 (D). Flies in panels C and D were raised at 25°C. Arrows denote examples of white bristles.

Data information: Representative phenotypes are shown in all panels. Scale bars, 100 μm.
Modeling by disruption and a selected-for partner for the nude locus (2020)
Figure 3

Figure 3. The Lilli homolog AFF4 is positioned to interact with FOXN1 in the skin and thymus.

(A) A schematic diagram indicates sequence features of Lilli and the AFF proteins (Nilson et al, 1997; Wittwer et al., 2001). A correspondence is seen between the C-terminal half of Lilli and the C-terminal two-thirds of the AFFs (the Lilli-like segment). Scale bar indicates length in amino acids (a.a.). pSer, poly-serine domain; NLS, nuclear localization sequence; CHD, C-terminal homology domain; POU, POU transactivation domain; AT, AT-hook DNA-binding motif; NHD, N-terminal homology domain; ALF, AF4/LAF4/FMR2 homology domain.

(B-E) AFF4 (green) and FOXN1 (red) are shown by immunofluorescence in the following wild-type tissue samples: (B) murine hair follicles, P9; (C) developing murine epidermis, E16.5; (D) human epidermis, adult; (E) murine thymus, P0, outer region. DNA is stained by Hoechst dye 33258 (blue). Co-localization of AFF4 and FOXN1 generates yellow color, visible in cell nuclei of all samples. In panel C, the fluorescent signal in the periderm (P) is nonspecific, as it is present when primary antibodies are omitted from the staining. Inner regions of the murine thymus (not shown) yielded the same extensive overlap of FOXN1 and AFF4 staining as that shown in panel E. Arrowheads mark the dermal/epidermal border (C, D). CH, cortex of the hair; CT, cortex of the thymus. Scale bars, 20 µm.
Modeling by disruption and a selected-for partner for the nude locus (2020)
Figure 4

Figure 4. AFF4 and FOXN1 play similar roles in the skin.

(A) A schematic diagram shows the design of the Aff4flox allele and the strategy for knocking out AFF4. The deletion of exons 10 and 11 ablates AFF4's entire Lilli-like segment.

(B-N) Representative skin phenotypes of wild-type (WT), Foxn1nu/nu (Nude), or KRT14-cre; Aff4flox/flox (Aff4 cko) mice are shown macroscopically (B-G) or histologically after hematoxylin and eosin staining (H-N). The phenotypes compared are as follows: (B-D) hair coats, 4-month-old adults; (E-G) vibrissae, P7; (H) hair follicles with growing hairs, juveniles; (I-K) regressing hair follicles, juveniles; (L-N) epidermis, P9. In panel H, micrographs from left to right are WT, Nude, and Aff4 cko. In panels I-K, HF marks examples of hair follicles. In panels L-M, arrowheads indicate the dermal/epidermal border; arrows mark examples of bent hair shafts. Scale bars: H, L-N, 20 µm; I-K, 40 µm.
Modeling by disruption and a selected-for partner for the nude locus (2020)
Figure 5

Figure 5. AFF4 and FOXN1 play cooperative roles in the thymus.

(A-C) Thymi from E18.5 embryos are shown: (A) intact with genotypes indicated, or (B, C) after sectioning, immunofluorescent staining for DLL4 (red), and counterstaining of DNA (blue). Sectioned samples are wild-type (B) or Aff4ko/ko; Foxn1+/- (C). Scale bars: A, 1 mm; B-C, 50 µm.
Modeling by disruption and a selected-for partner for the nude locus (2020)
Figure 6

Figure 6. AFF4 and FOXN1 have similar effects on gene expression.

(A) Venn diagrams summarize global changes in gene expression caused by the inactivation of Foxn1 or Aff4 and detected by RNA-seq in murine skin at P6. The upregulated or downregulated gene populations of each mutant - and the extents to which they overlap - are indicated.

(B) Gradient bars (green) signify the ranking of downregulated genes by fold-change, with genes ordered from most downregulated (gene #1) to least downregulated. Fold-change was determined by RNA-seq. Separate rankings were performed for each mutant (Foxn1 null or Aff4 cko). Pie charts indicate whether genes of a given rank in one mutant were downregulated in the other mutant. Percentages indicate the overlap in the mutants' downregulated gene populations.

(C-E) KRT86 (red) is shown by immunofluorescence in skin sections from wild-type (WT), Foxn1 null, or Aff4 cko mice at P9. DNA is stained blue. HS marks examples of hair shafts. Scale bar, 40 µm.
Modeling by disruption and a selected-for partner for the nude locus (2020)
Figure 7

Figure 7. AFF4 and FOXN1 cooperatively activate transcription.

(A) Murine Krt86 is shown schematically with exons as rectangles and the transcription start site marked by an arrow. Sites assayed in the pre-mRNA or ChIP studies of panels B-G are indicated.

(B-G) Graphs present pre-mRNA (B, C) or ChIP (D-G) assays of Krt86 using skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P6 (B, C) or P7 (D-G). ChIP assays were performed with antibodies to FOXN1 or AFF4 as indicated. All values are derived from 3 independent experiments and presented as mean ± S.D. Pre-mRNA measurements were analyzed statistically using two-way ANOVA with post-hoc Sidak's multiple comparison test at a 95% confidence interval. ChIP assays were analyzed statistically using one-way ANOVA with post-hoc Tukey's multiple comparison test at a 95% confidence interval. ****, p ≤ 0.0001.
Modeling by disruption and a selected-for partner for the nude locus (2020)
Figure 8

Figure 8. AFF4 and FOXN1 cooperatively promote pre-initiation complex formation.

(A) Murine Krt86 is shown schematically as in Fig 7A. Sites assayed in ChIPs of panels B-G are indicated.

(B-G) Graphs present ChIP assays of Krt86 using antibodies to total (B, C), P-Ser5 (D, E), or P-Ser2 (F, G) Pol II and skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P7. All values are derived from at least 3 independent experiments and presented as mean ± S.D. Assay results were analyzed statistically using one-way ANOVA with post-hoc Tukey's multiple comparison test at a 95% confidence interval. ****, p ≤ 0.0001.

Expanded View Figure Legends
Autophagy regulates neuronal excitability by controlling cAMP/Protein Kinase A signaling at the synapse (2022)
Figure 1

Figure 1 | ATG5 deficiency in excitatory and inhibitory neurons results in differential neuronal excitability.

(A, B) Immunoblot analysis of ATG5-ATG12 protein levels in cortical brain lysates of 13-week-old Atg5flox:CamKIIα-Cre WT/KO mice (WT set to 100%, KO: 28.13±8.18%; p=0.0005, nWT=3, nKO=3, one-tailed unpaired t-test).

(C,D) Immunoblot analysis of ATG5-ATG12 protein level in striatal brain lysates of 13-week-old Atg5flox:Slc32a1-Cre WT/KO mice (WT set to 100%, KO: 21.71±0.41%; p<0.0001, nWT=3, nKO=3, one-tailed unpaired t-test).

(E, F) Protein levels of p62 are significantly increased in cortical lysates of Atg5flox:CamKIIα-Cre KO mice compared to the WT set to 100% (KO: 4855± 587.4%; p<0.0001, n=4, one-tailed unpaired t-test).

(G, H) Protein levels of p62 are significantly increased in striatal lysates of Atg5flox:Slc32a1-Cre KO mice compared to the WT set to 100% (KO: 2956± 839.3%; p=0.0072, n=4, one-tailed unpaired t-test).

(I, J) Startle response analysis of Atg5flox:CamKIIα-Cre WT/KO ((I), WT: 0.88±0.125, KO: 1.56±0.176; p= 0.0204, nWT=8, nKO= 9) and Atg5flox:Slc32a1-Cre WT/KO mice ((J) WT: 1.11±0.111, KO: 2.00±0.000; p<0.0001, nWT=9, nKO=11, two-tailed Mann-Whitney test).

(K, L) Behavioral seizure analysis in Atg5flox:CamKIIα-Cre KO and heterozygous (HET) mice (K), as well as in Atg5flox:Slc32a1-Cre KO/HET mice (L). Mice were observed twice a week during 4 months after birth (the number animals and the age of seizure onset are indicated in the graph). No seizures were detected in WT mice.

(M, N) Examples of electrocorticogram (ECoG) traces (top) and corresponding wavelet spectrograms (bottom, warm colors indicate high power) from Atg5flox:CamKIIα-Cre KO mice during a spontaneous generalized tonic-clonic seizure (M), or during a focal seizure associated with repetitive grooming and head-nodding (N). Representative example from n=4 recordings for each genotype. All data represent mean ± SEM.

Data information: *P < 0.05; **P < 0.01; ***P < 0.001. All data represent mean ± SEM. All n represent biological replicates.
Autophagy regulates neuronal excitability by controlling cAMP/Protein Kinase A signaling at the synapse (2022)
Figure 3

Figure 3 | PKA R1α/β subunits of the PKA complex are enriched at the synaptic compartments of autophagy-deficient neurons.

AMIRA-based 3D-visualization of PKA R1α distribution in Atg5flox:CamKIIα-Cre WT/KO cell soma and neuropil in the cortex. Scale bar 20 µm. Drawn contours indicate somata of WT or KO neurons.

(B-D) Immunohistochemical analysis and surface rendering of Amira 3D reconstructions of PKA R1α and bassoon colocalization in Atg5flox:CamKIIα-Cre WT (B, C) and KO (D) cortical neuropil. In 3D analysis, the PKA R1α neuropil staining is represented by 3D reconstruction of its surface in red and PKA R1α /bassoon contacts are color coded, with the cold to warm colors spreading from 0- to 250-nm distance between the surface of either bassoon-positive synapses and/or PKA R1α-positive neuropil (see color-coded horizontal bar for the distance definition). Scale bar 5 µm in dual-channel, 1 µm in 3D-reconstruction zoom.

(E) Mander's overlap coefficient of PKA R1α and bassoon colocalization in Atg5flox:CamKIIα-Cre WT/KO cortex (WT: 0.32±0.023, KO: 0.43±0.018; p=0.0002, two-tailed unpaired t-test). nWT= 33; nKO= 39 from n=3 mice.

(F) Mander's overlap coefficient of PKA R1β and bassoon colocalization in Atg5flox:CamKIIα-Cre WT/KO cortex (WT: 0.20±0.014, KO: 0.34±0.021; p<0.0001, two-tailed unpaired t-test). nWT= 27; nKO= 27 from n=3 mice (see also Appendix Figure S5A).

(G) Immunohistochemistry for PKA R1α and postsynaptic density marker PSD95 on horizontal brains sections from Atg5flox:CamKIIα-Cre WT/KO mice. White rectangular boxes indicate areas magnified below with arrows indicating PKA R1α and PSD95 colocalization. Scale bar: 20µm, 5µm (insert).

(H) Mander's overlap coefficient of PKA R1α and PSD95 colocalization in Atg5flox:CamKIIα-Cre WT/KO hippocampus (WT: 0.29±0.009, KO: 0.40±0.012; p<0.0001, two-tailed unpaired t-test). nWT= 31; nKO= 38 from n=3 mice.

(I) Electron microscopy-based analysis of immunogold-labeled PKA R1α/β on Tokuyasu cryosections of the CA1 neuropil area of the hippocampus of Atg5flox:CamKIIα-Cre KO mice. No immunogold labelling was detected in samples where the PKA R1α/β antibody were omitted (negative control). Scale bar: upper row 50 nm, 500 nm lower picture. (J) PKA R1α/β is enriched at mitochondria (WT: 1.10± 0.266, KO: 2.73± 0.515; p=0.0082, two-tailed unpaired t-test) and synapses (WT: 0.52± 0.131, KO: 1.14± 0.221; p=0.0234, two-tailed unpaired t-test) of KO mice. Values display the mean number of gold particles counted per structure in nWT=21 and n KO=22 pictures per genotype.

Data information: *P < 0.05; **P < 0.01; ***P < 0.001. All data represent mean ± SEM. All n represent biological replicates.