EMBO/soda-nel huggingface dataset:
Macrophage mitochondrial bioenergetics and tissue invasion are promoted by an Atossa-Porthos axis in Drosophila (2022)
Figure 1

Figure 1. CG9005 acts in macrophages to spur pioneer cell infiltration into the germband tissue.

A-C Confocal images of Stage 12 embryos from control, P{GT1}CG9005BG02278 P-element mutant (CG9005PBG), and CG9005PBG with CG9005 expression restored in macrophages. Macrophage: red. Phalloidin to visualize embryo: green. Germband edge: dotted white line.

D Quantification of macrophages that have penetrated the germband from genotypes in (A-C) and from CG9005PBG over two deficiencies (Df) that remove the gene. n=35, 56, 25, 9, 18 embryos respectively, p<0.0001 for control vs CG9005PBG, Df1, or Df2; p=0.98 for control vs mac>CG9005 rescue, p=0.91, 0.90 for CG9005PBG vs Df1 or Df2.

E Macrophage-specific knockdown of CG9005 by UAS-RNAi lines. n=22, 20, 21, 23, 35, 28 embryos. p<0.0001 for all comparisons.

F Stills from two-photon movies of control and CG9005PBG mutant embryos showing macrophages (nuclei, red) migrating starting at Stage 10 from the head towards the germband and invading the germband tissue. Elapsed time indicated in minutes. Germband edge (white dotted line) detected by yolk autofluorescence. For quantification of migration parameters in movies see (G-L).

G-H Macrophage migration speed (G) in the head or (H) between the yolk sac and the germband edge. For (G): control n=8 movies, CG9005PBG mutant n=3; control n=360 tracks, mutant n=450, p=0.65. For (H): control n=7 movies, mutant n=3; control n=46 tracks, mutant n=19, p=0.62

I The time required for the first macrophage nucleus to enter into the extended germband. Control n=7 movies, mutant n=5. Time to entry: control=23 min, CG9005PBG=38 min, p<0.0001.

J-L The migration speed of the (J) 1st, (K) 2nd, or (L) 3rd-5th macrophages along the first 25-30µm into the germband between the mesoderm and ectoderm. In schematics, analyzed macrophages--light blue, other macrophages--red, ectoderm--green, mesoderm--purple, and yolk--beige. For (J): control n=6 movies, mutant n=5, p=0.012. For (K): control n=5 movies, mutant n=5, p=0.03. For (L): control n=5 movies, mutant n=4, p=0.17.

Data information: Scale bars: 50µm (A-C), 30µm (F). Throughout paper mac> indicates GAL4 driven expression of a UAS construct specifically in macrophages by srpHemo-GAL4. N for movies represents imaging from different embryos. Throughout this work, embryos with stomadeal invagination and germband retraction away from the anterior of less than 29% were defined as Stage 10, 29%-31% Stage 11, and 35%-40% Stage 12. (D) One-way ANOVA with Tukey. (E and G-L) Unpaired t-tests. Graphs show mean±SEM; ns=p>0.05, *p<0.05, ****p<0.0001.

See Source Data 1 and 2 for Figure 1.
Macrophage mitochondrial bioenergetics and tissue invasion are promoted by an Atossa-Porthos axis in Drosophila (2022)
Figure 2

Figure 2. CG9005/Atossa requires conserved domains linked to transcriptional activation to enhance tissue invasion, a function maintained by its mammalian orthologs.

A Deduced protein structure of Drosophila CG9005/Atossa (Atos) and its murine orthologs, mFAM214A-B, highlighting conserved domains. FAM214A-B are 44-45% identical to Atos.

B Macrophages (red) near the germband in Stage 11/12 embryos. Atos tagged at C terminus with HA (HA-antibody, green) and expressed under direct control of macrophage specific promoter. Nucleus stained by DAPI (blue).

C-D Confocal images or (D) quantification of the macrophages in germband in Stage 12 embryos from the control, atosPBG, and atosPBG expressing Atos itself or variants lacking particular domains. Transgene expression directly from macrophage specific promoter (mac-). For control (n=32 embryos) vs atosPBG mutant (n=56) p<0.0001; vs rescue with mac-atos (n=18) p>0.99; vs rescue with mac-atosDUF- (n=17) p=0.0003; vs rescue with mac-atosChrSeg-(n=21) p=0.0003; vs rescue with mac-atosDUF-/ChrSeg-(n=19) p=0,00014; vs rescue with mac-atosTAD1-/ TAD2- (n=25) p=0.0009, atosPBG mutant vs. rescue with mac-atos p=0.0031.

E Confocal images of atosPBG rescued by expressing Atossa's murine orthologs, mFAM214A or B (mFAMA-B) in macrophages,

F Quantification of macrophages in the germband in Stage 12 embryos from the control, atosPBG, and atosPBG embryos expressing mFAM214A or B specifically in macrophages (mac-). For control (n=24 embryos) vs. atosPBG mutant (n=56) p<0.0001; vs mac-atos rescue (n=18) p=0.7; vs mac-mFAMA rescue (n=22) p=0.6; vs mac-mFAMB rescue (n=25) p=0.086. For atosPBG mutant vs mac-atos rescue p=0.0006; vs mac-mFAMA rescue p=0.0002; vs mac-mFAMB rescue p=0.0043.

Data information: Germband edge: dotted white line. Mac- indicates direct expression from the srpHemo promoter. (C, E) macrophage nuclei (red), actin by Phalloidin staining (green). (D,F) One-way ANOVA with Tukey. Mean±SEM, ns=p>0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars: 5µm (B), 50µm (C, E). See Source Data 1 and 2 for Figure 2.
Macrophage mitochondrial bioenergetics and tissue invasion are promoted by an Atossa-Porthos axis in Drosophila (2022)
Figure 3

Figure 3. Atos leads to higher mRNA levels of a Ddx protein and metabolic enzymes required for germband invasion.

A A selection of genes down-regulated in atosPBG mutant macrophages compared to the control, chosen for their >5 fold change in expression as well as an identified biological function.

B-D Confocal images of early Stage 12 embryos from the control and lines expressing RNAis against (B) porthos, (C) GR/HPR or (D) LKR/SDH, specifically in macrophages (red). Germband edge: dotted white line.

E-G Quantification of the number of germband macrophages in embryos from control and upon RNAi knockdown of the genes in B-D. In (E) control (n=36 embryos) vs. porthos RNAi (n=28) p<0.0001. In (F) сontrol 1 (n=18 embryos) vs. GR/HPR RNAi 1 (n=18) and control 2 (n=21) vs. GR/HPR RNAi 2 (n=24), both p<0.0001; control 3 (n=15 embryos) vs. GR/HPR RNAi 3 (n=23) p=0.08. In (G) сontrol 1 (n=18 embryos) vs. LKR/SDH RNAi 1 (n=17) and control 2 (n=21) vs. LKR/SDH RNAi 2 (n=23), both p<0.0001.

H Schematic: Glyoxlate reductase/Hydroxypyruvate reductase (GR/HPR) catalyzes the reduction of glyoxylate into glycolate and converts hydroxypyruvate into D-glycerate by oxidation of the cofactor NAD(P)H.

I Schematic: Lysine α-Ketoglutarate Reductase/Saccharopine Dehydrogenase (LKR/SDH) catalyzes the first two steps of the lysine catabolism pathway, resulting in the production of glutamate and acetyl-CoA, a TCA substrate, through several downstream enzymatic reactions.

Data information: Glu: Glutamate, α-KG: α-Ketoglutarate, AASA: α-Aminoadipate δ-semialdehyde. Mean±SEM and ns=p>0.05, ****p<0.0001. One-way ANOVA with Tukey (E-G). Scale bar: 50µm (B-D). See Source Data 1 for Figure 3.
Macrophage mitochondrial bioenergetics and tissue invasion are promoted by an Atossa-Porthos axis in Drosophila (2022)
Figure 4

Figure 4. The nucleolar Ddx protein, Pths, acts as a key downstream target of Atos to promote pioneer macrophage germband invasion.

A Macrophages (cytoplasm, red) near the germband in Stage 11/12 embryos show partial colocalization of the HA-antibody labeling Porthos (green) with the nucleus (DAPI, blue).

B Stills starting at Stage 11 from two-photon movies of control embryos and those expressing porthos (pths)-RNAi in macrophages (nuclei, red), migrating from the head mesoderm towards and into the germband at the indicated time points. White dotted line: germband edge.

C-H Quantification of macrophage migration parameters from movies as in (B). Macrophage migration speed (C) in the head or (D) between the yolk sac and the germband edge. (C) control n=4 movies, pths RNAi n=6; control n=507 tracks, pths RNAi n=859; p=0.56. (D) control n=5 movies, pths RNAi n=5; control n=40 tracks, pths RNAi n=51; p=0.45. (E) The time required for the first macrophage nucleus to enter into the extended germband: control=23 min, CG9005PBG=38 min, p<0.0001. The migration speed of the (F) 1st, (G) 2nd, or (H) 3rd-5th macrophages along the first 25-30µm into the germband between the mesoderm and ectoderm: control=21.5 min, n=6, pths RNAi=36.2 min, n=4, p<0.0001.

I Confocal images of early Stage 12 embryos from control, atosPBG, and atosPBG expressing atos::FLAG::HA (mac>atos) or pths::FLAG::HA (mac>pths) in macrophages (red). Embryo detected by phalloidin staining (green). White dotted line: germband edge..

J Quantification of macrophages in the germband of an atosPBG mutant rescued by expressing pths::FLAG::HA in macrophages. For control (n=15 embryos) vs atosPBG mutant (n=22) and mac>atos rescue (n=27) both p<0.0001, vs mac>pths rescue (n=30) p=0.0007.

Data information: Movies in each analysis set are from independent embryos. Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Unpaired t-test (C-H); one-way ANOVA with Tukey (J). Scale bars: 50µm (A,I), 30µm (B). See Source Data 1 for Figure 4.
Macrophage mitochondrial bioenergetics and tissue invasion are promoted by an Atossa-Porthos axis in Drosophila (2022)
Figure 6

Figure 6. Pths increases the translational efficiency of nuclear-encoded mitochondrial proteins.

A-B Pths increases the translation of RNAs involved in (A) mitochondrial function and (B) metabolic pathways, along with transcription, translation, signal transduction, immune responses as well as redox processes as shown in Appendix Fig S1. See also Dataset EV1.

C-F RT-qPCR analysis of mRNA from ribosomal protein fractions for control (black) and pths KD (gray) transcripts from S2R+ cells. RNA was isolated individually from fractions and pooled into five categories: RNP, 40S/60S, monosome, low polysome (di- and trisome), and high polysome (remaining fractions). The data were plotted relative to the amount present in the monosome fraction for each transcript. The mRNA levels of subunits of mitochondrial complexes (C) I, (D) III and (E) V were reduced in low and high polysome fractions in pths KD cells compared to the control cells, while (F) mRNA levels of GAPDH in the same fractions, used as an internal control, were not changed (n=3 independent biological replicates for control and pths KD). For (C,E-F) low polysome fraction for control vs. pths KD: (C) p=0.057, (D) p=0.0039, (E) p=0.09, (F) p=0.5. For (C,E-F) high polysome fraction for control vs. pths KD: (C) p=0.039, (D) p=0.046, (E) p=0.041, (F) p=0.66.

G-I Western blots and their quantifications of protein extracts from control and pths KD S2R+ probed with (G) MT-ND1 (for mitochondrial complex I) n=4, p<0.0001), (H) ATPsynt-β (for mitochondrial complex V) n=3 p=0.034 and (I) tubulin β antibodies p=0.56) (n=3). Profilin serves as loading control. N=biological replicates.

Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. Unpaired t-tests (C-F, G-I). See Source Data1 and 2 for Figure 6, Appendix Fig S1.
Macrophage mitochondrial bioenergetics and tissue invasion are promoted by an Atossa-Porthos axis in Drosophila (2022)
Figure 7

Figure 7. Porthos boosts mitochondrial respiration, which is required in macrophages to power their germband tissue invasion.

A Schematic of mitochondrial energetic profiling with a Seahorse efflux assay.

B The Oxygen Consumption Rate (OCR, pmols O2/min) assessed as a representative parameter of OxPhos in control and pths-KD S2R+ cells by a Seahorse efflux assay. n≥3 independent biological experiments each with n>6 technical replicate.

C Distinct respiration parameters calculated from relative OCR values obtained in experiments shown in B. p<0.0001 for all comparisons.

D-F Confocal images (D) and quantification of macrophages (E) in the germband or (F) on the yolk next to the germband from control embryos and those expressing a dominant negative c-ring of ATP synthase (CV-DN) in macrophages. (D-H) from Stage 12 embryos and germband edge shown with dotted white line. For (E) control (n=24 embryos) vs CV-DN (n=20) p=0.0032. For (F) control (n=21 embryos) vs CV-DN (n=17) p=0.003.

G-H (G) Confocal images and (H) quantification of germband macrophages in control embryos and those expressing different RNAis against mitochondrial OxPhos Complex III (or an RNAi against Complex V. For (H) control (n=34 embryos) vs Complex III RNAi 1 (n=20) p=0.0001; vs. Complex III RNAi 2 (n=18) p=0.027; vs. Complex III RNAi 3 (n=16) p<0.0001; vs. Complex V RNAi (n=14) p<0.0001.

I Single plane confocal microscope image during germband entry from control or atosPBG embryos, or those expressing pths-RNAi or CV-DN in macrophages. Antibodies against S293-phosphorylated inactivated Pyruvate Dehydrogenase (pPDH, green) or total PDH (magenta) in macrophages (red). Higher pPDH levels are usually found when ATP/ADP levels are high and input into the TCA cycle is being downregulated (Patel et al., 2014).

J Quantification of normalized pPDH/PDH levels calculated from fluorescence intensities in macrophages from the genotypes in (I) during initial germband invasion. The pPDH/PDH ratio is significantly reduced, arguing that decreased function of CV, Atos or Pths in macrophages results in lower cellular ATP/ADP ratios compared to the control. N=3 independent experiments. Ctrl 1 (n=10 embryos) vs CV-DN (n=9) and vs. atos mutant (n=13) both p=0.0002; Ctrl 2 (n=7 embryos) vs pths RNAi (n=8) p=0.0001.*** shown above columns in J are for comparison to relevant control.

Data information: D-I show Stage 12 embryos. G-H Complex III RNAis target Cyt-c1, UQCR-cp1-2, for Complex V, ATP synthase F1F0. Scale bars: 50µm (D,G) 10µm (I). Mean±SEM, **p<0.01, ***p<0.001, ****p<0.0001. Unpaired t-tests (B,E,J), two-way ANOVA with Sidak's test (C), and one-way ANOVA with Tukey's (H). See Source Data 1 for Figure 7.
Macrophage mitochondrial bioenergetics and tissue invasion are promoted by an Atossa-Porthos axis in Drosophila (2022)
Figure 9

Figure 9. Mitochondrial metabolism is enhanced by Atos

A Schematic depicting ATP-generating pathways in eukaryotic cells: glycolysis, the Pentose Phosphate Pathway (PPP), fatty acid oxidation (FAO), the TCA cycle, and the mitochondrial respiratory electron transport chain (ETC). Blue stars mark pths targets. Green indicates individual metabolites with statistically significant upregulation in atosPBG compared to the control.

B-G Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. (B) Normalized ATP/ADP ratio values are decreased in atosPBG compared to control embryos (p=0.028). (C-D,F-G) Heatmap of non-targeted metabolites in atosPBG compared to wild-type embryos shown with average log2fold change (FC) in the non-targeted LC-MS/MS analysis reveals (C) an increase in atosPBG in substrates of the dGR/HPR enzyme, including 4-hydroxy α-ketoglutarate and hydroxyproline (HLP) and a smaller decrease in its products, glycolate and glycerate, (D) a significant increase in some dipeptides including those containing hydroxyproline. (E) Quantification shows an increase in atosPBG in the pyruvate/glucose ratio (p=0.035), but none for the lactate/glucose ratio (p=0.65). (F) We observe increases in atosPBG in intermediates of mitochondrial fatty acid β-oxidation (FAO), including different carnitine-conjugated lipids, and (F-G) a significant increase in ketone body substituents compared to the control.

Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. Values in heat maps are obtained from untargeted metabolomic analysis, unpaired t-test (B-G). See Source Data 1 for Figure 9 and Dataset EV1 for metabolomics source data.
Dynamic chromatin association of IκBα is regulated by acetylation and cleavage of histone H4 (2021)
Figure 1

Figure 1. Specific interaction of nucleosome particles with phosphorylated IκBα.

(A) Electrophoretic analysis (under non-denaturing conditions) of the association between the indicated IκBα species association and reconstituted nucleosome core particles (NCP).

(B) Electrophoretic analysis in agarose gels of in vitro generated p-IκBα and reconstituted nucleosome in absence or presence of anti-IκBα antibodies.
Dynamic chromatin association of IκBα is regulated by acetylation and cleavage of histone H4 (2021)
Figure 2

Figure 2. Preferential association of IκBα with N-terminal acetylated histone H4.

(A) PD experiment with biotinylated peptides of H4 (1-23aa) and total lysates of HCT-116 cells.

(B) Quantification of the IκBα 60 kDa band (upper panels) and the 37 kDa band (lower panels) is shown as average and s.d. of three independent experiments.

(C) PD experiments with GST-SUMO IκBα fusion protein and serial dilutions of histone-enriched HEK-293T lysates. Quantification of three independent replicates is shown.

(D) Venn diagrams representing the overlap between IκBα and acetylated H4 peaks obtained in ChIP-seq experiments from HCT-116 cells.

(E) Peak distribution of acetylated H4, H4K12ac and total histone H4 obtained in IκBα target genes and non-IκBα targets relative to the transcription start site (TSS).

Data information: WB in A and C are representative of three biological replicates performed. In B, p values were derived from unpaired two-tailed t-test of triplicates, ***p-value<0.001, **p-value<0.01, * p-value<0.05, n.s. no significant.
Dynamic chromatin association of IκBα is regulated by acetylation and cleavage of histone H4 (2021)
Figure 3

Figure 3. Stem cell compartments contain acetylated H4 species that are lost following histone cleavage.

(A) Immunofluorescence (IF) analysis with the indicated antibodies in sections from murine small intestine of 2-month-old WT mice.

(B) Double IF analysis with the indicated antibodies in sections from murine small intestine of 2-month-old Lgr5-GFP transgenic mice.

(C) Western Blot analysis of chromatin extracts from isolated intestinal villus and crypt cells.

(D) Western Blot analysis of chromatin extracts from isolated intestinal villus and crypt cells in several 2-month-old WT mice (each # represents a mouse).

(E) Western Blot analysis of soluble (Sol) and chromatin (Chr) extracts from isolated intestinal villus and crypt cells in 2-month-old mCherry-H4 transgenic mice.

(F) Western Blot analysis of chromatin fraction from mCherry-H4 transfected HCT-116 cells.

(G) Pulldown assay using GST-SUMO IκBα protein and chromatin lysates from 2-month-old WT mice villi.

Data information: Scale bars in A and B, 25μm. Red arrowheads in C-G are showing the location of truncated histone H4.
Dynamic chromatin association of IκBα is regulated by acetylation and cleavage of histone H4 (2021)
Figure 4

Figure 4. Cleavage of the N-terminal tail of H4 is mediated by chymotrypsin and trypsin activity present in the intestinal villus.

(A) Western Blot analysis of the crypt-derived chromatin fractions incubated with soluble lysates from villi or crypts for the indicated times.

(B) Table showing the proteases differentially expressed in the villus compartment as determined by RNA-seq analysis of intestinal crypt (purified EphB2 high cells) and villus (EphB2 negative/low) cells. Predicted cleavage sites for each protease in the histone H4 sequence was determined using the PeptideCutter-ExPASy and PROSPER software.

(C) Western blot analysis of mCherry-H4 with the indicated mutations transfected in HCT-116 cells. Quantification of relative amounts of truncated H4 relative to the WT.

(D, E) Western Blot analysis of mCherry-H4 transfected in HCT-116 cells treated for 16 hours with TLCK (D) or AdaAhx₃L₃VS (E) at the indicated concentrations. (F) Western Blot analysis of a cleavage experiment incubating modified H4 peptides (P1-P9) with soluble lysates from villi or crypts in the presence (+) or absence (-) of the commercial protease inhibitors cocktail. Lanes indicated as P correspond to the control peptide without lysate incubation. Notice the electrophoretic shift of peptides incubated with villus lysates (compared with control peptides) suggestive of post-translational modifications or binding to proteins absent from crypt lysates.

(G) Quantification of the relative cleavage of specific H4 peptides incubated with villus-derived lysates from 3 independent experiments performed.

Data information: Red arrowheads in A, C-F indicate truncated histone H4.
Dynamic chromatin association of IκBα is regulated by acetylation and cleavage of histone H4 (2021)
Figure 5

Figure 5. Functional impact of IκBα chromatin binding dynamics in cell differentiation.

Western blot analysis of soluble and chromatin extracts from HT29 cells at different differentiation stages (days post-confluence).

(B, C) qPCR (B) and WB analysis (C) of the indicated genes or proteins in parental or IκBα KO HT29 cells obtained at pre-confluence or 7 days post-confluence. Bars represent mean values ± standard deviation of 3 technical replicates from 3 biological replicates performed.

(D, E) qPCR (D) and WB analysis (E) of MUC5AC in HT29 cells treated with the indicated protease inhibitors. Bars represent mean values ± standard deviation from 3 biological replicates performed

(F, G) qPCR (F) and WB analysis (G) of MUC5AC in HT29 cells transduced with sh-RNAs against trypsin (T1, T2) or chymotrypsin (C1, C2). Bars represent mean values ± standard deviation of 3 technical replicates from 3 biological replicates performed

(H) Western blot analysis of cells transfected with mCherry-H4 and the indicated sh-RNAs.

(I) Model for gene regulation by chromatin-associated IκBα. In brief, dynamic dissociation of IκBα from the chromatin at specific genetic loci promotes transcription of genes involved in stem cell maturation or differentiation (upper panel). Absence of dynamic binding (middle panel) or cells IκBα deficient (lower panels) fail to activate IκBα-dependent transcription thus imposing a differentiation/maturation blockage.

Data information: Images in A-F are representative of three independent replicates performed. p values were derived from unpaired two-tailed t-test, ***p-value<0.001, **p-value<0.01, * p-value<0.05, n.s. no significant.

EXPANDED VIEW FIGURE LEGENDS
Developmental and Functional Heterogeneity of White Adipocytes within a Single Fat Depot (2018)
Figure 1

Figure 1. Depot-specific Immortomouse Cell Lines Retain the Molecular Signatures of the Depot of Origin

A) Expression level of Shox2, Tbx15, HoxC8, and HoxC5, mRNA was compared using quantitative real-time PCR (qPCR) of RNA isolated from different fat depots. Two subcutaneous depots (inguinal and scapular white), three intra-abdominal fat depots (perigonadal, perirenal, and mesenteric), and interscapular brown adipose tissue of 8 week-old male C57BL/6 mice. Data are shown as mean ± SEM of six samples.

B) Expression level of Shox2, Tbx15, HoxC8, and HoxC5, mRNA was compared using qPCR of RNA isolated from cultured stromovascular cells derived from adipose tissue of the Immortomouse™. The depot-specific cell lines were made from inguinal, scapular white, perigonadal, perirenal, mesenteric, and interscapular brown adipose tissue of 8 week-old male mice. Data are shown as mean ± SEM of cell lines from three individual mice.

C) Bright-field image of depot-derived cell lines after in vitro adipogenic differentiation and Oil Red O staining (Lee et al., 2017a). Original photographs were taken at 10x magnification (left panel). Scale bar = 100 μm.

D) qPCR analysis for adiponectin (Adipoq), peroxisome proliferator-activated receptor gamma (Pparγ2), and fatty acid synthase (FAS) in RNA isolated from depot-derived cell lines after in vitro differentiation. Data are shown as mean ± SEM of 3 cell lines/group.

Data information: Bars indicates significant differences in expression (p < 0.05; two‐way ANOVA).