EMBO/SourceData huggingface dataset:
Figure 2(D) Expression of phenotypic markers used for UMAP projection.(E) Supervised gating strategy for moDC and moMac at each time point (gated on live CD11b+ CD115+ cells).(F) Supervised gating strategy to identify monocyte-derived cells.
Figure 3(E) Transcription factor activity was calculated with Dorothea and Viper. Differentially activated regulons between clusters are shown.
Figure 4(A) Monocytes were cultured with M-CSF, IL-4 and TNFα for 5 days. Protein quantification by Western Blot. Actin was used as loading control. Representative results are shown (n=6 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor.Monocytes were cultured with M-CSF for 5 days. IRF1 expression was silenced using a lentivirus containing shRNA. (B) Protein quantification by Western Blot after 5 days. Actin was used as loading control. Representative results are shown (n=8 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor.Monocytes were cultured with RPMI medium (R10), or combinations of M-CSF, IL-4 and TNFα in the presence or absence of B18R (type I interferon inhibitor). (G) ISG expression after 9 hours was analyzed by RTqPCR. Each symbol represents an individual donor (n=5 biological replicates).Monocytes were cultured with combinations of M-CSF, IL-4 and TNFα in the presence or absence of B18R (type I interferon inhibitor). (H) Monocyte differentiation after 5 days. One representative donor is shown (n=5 biological replicates). Proportion of mo-Mac and mo-DC after 5 days. Each symbol represents an individual donor (n=5 biological replicates). Paired t-test.
Figure 5(B) ZNF366 expression across clusters.Monocytes were cultured with M-CSF, IL-4 and TNFα for 5 days. (C) Protein quantification by Western Blot. Actin was used as loading control. Representative results are shown (n=6 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor. Paired one-way Anova.Monocytes were cultured with M-CSF, IL-4 and TNFα for 5 days. ZNF366 expression was silenced using a lentivirus containing shRNA. (D) Protein quantification by Western Blot after 5 days. Actin was used as loading control. Representative results are shown (n=4 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor. Paired one-way Anova.Monocytes were cultured with M-CSF, IL-4 and TNFα for 5 days. (G) Protein quantification by Western Blot. Actin was used as loading control. Representative results are shown (n=6 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor. Paired one-way Anova.MAFF expression was silenced using a lentivirus containing shRNA. (H) Protein quantification by Western Blot after 5 days. Actin was used as loading control. Representative results are shown (n=4 biological replicates). Quantification was performed by densitometry. Each symbol represents an individual donor. Paired one-way Anova.MAFF expression was silenced using a lentivirus containing shRNA. (I) Mo-mac and mo-DC differentiation after 5 days was assessed by flow cytometry. One representative donor is shown (n=7 biological replicates). DN = double negative. Proportion of mo-DC, mo-Mac and DN cells after 5 days. Median is shown (n=7 biological replicates). Paired one-way Anova.
Figure 1(A, B) Fluorescence confocal microscopy images show TMRE staining and GFP::MIRO-1 in WT and fzo-1 mutants animals. Dashed lines outline regions where fluorescence intensity was quantified.(C) Immunoblot shows protein levels of MIRO-1 in WT and fzo-1 mutant animals, with tubulin and COX-4 serving as loading controls.(D) Fluorescence confocal images show GFP::MIRO-1 and mito::mKate2 in WT and fzo-1 mutants. (E) Quantification of MIRO-1::GFP levels in WT and fzo-1 mutants. n≥33, biological replicates.
Figure 2(A, B) Fluorescence confocal microscopy images show TMRE staining and GFP::MIRO-1 in WT animals with and without needle wounding. Dashed lines outline regions where fluorescence intensity was quantified.(C) Representative confocal images showg GFP::MIRO-1 and mito::mKate2 before and 2 hours after wounding.(D) Immunoblot shows protein levels of MIRO-1 before and 2 hours after wounding, with tubulin and COX-4 serving as loading controls.
Figure 3(A) Representative confocal images show the TMRE and mito::GFP signals in WT, miro-1(tm1966), miro-1(ΔEF) (EF-hand domain mutation), and miro-1(OE, overexpression) animals. (B) Quantitation of TMRE and mito::GFP signal (normalized ratio of TMRE fluorescence to mito::GFP fluorescence). n > 200 mitochondria.(D) ATP content of crude isolated mitochondria from WT and miro-1(tm1966) mutants as measured with a commercial luminescence kit. n = 3, biological replicates.
Figure 4(D) Western blot analysis of the interaction between MIRO-1 and VDAC-1 by coimmunoprecipitating with GFP nanobody-conjugated beads in GFP::MIRO-1 animals and detected by GFP and VDAC-1 antibodies.(E) Western blot analysis of the interaction between MIRO-1 and VDAC-1 by coimmunoprecipitating with VDAC-1 antibody-conjugated beads in GFP::MIRO-1 animals and detected by GFP and VDAC-1 antibodies.(F and G) Immunoblot showing the immunoprecipitation of purified GST::MIRO-1 and VDAC-1::His with GST-beads and His-beads, respectively.(H) BN-PAGE and immunoblot analysis of the total worm lysate of GFP::MIRO-1 (KI) by GFP and VDAC-1 antibodies. Green shading highlights the complex that contained MIRO-1 and VDAC-1 proteins.
Figure 5(A) Schematic of the vdac-1 transgene by CRISPR-Cas9 method. The images below show the young adult stage worms of WT and vdac-1(zju247) mutants. (B) Representative confocal images showing TMRE staining and mito::GFP in L4440, vdac-1 RNAi, vdac-1(zju247), and vdac-1 OE animals.(E) ATP content in crude isolated mitochondria of WT animals and vdac-1(zju247) mutants. Quantification is conducted with a commercial luminescence kit. n = 3, biological replicates.
Figure 6(B) Immunoprecipitation of GFP::MIRO-1 and GFP::MIRO-1(E473G) with GFP nanobody-conjugated beads.(F) Immunoprecipitation of GFP::MIRO-1 in fzo-1 mutant with GFP nanobody-conjugated beads. The interaction between GFP::MIRO-1 and VDAC-1 is increased in fzo-1 mutants.(G) BN-PAGE and immunoblot analysis of VDAC-1 oligomerization crosslinked with BMH in WT and miro-1(tm1966) mutants using VDAC-1 antibody.
Figure 1(B-C) Representative images (B) and quantification (C) of Oil Red O staining of hypoxia- and normoxia-cultured cells, 9 days after induction of adipogenesis. n=5 biologically independent replicates.(D-E) Representative images (D) and quantification (E) of Alizarin Red S staining of hypoxic, normoxic and reversed hypoxic (R_2% O2) cells, 12 days after induction of osteogenesis. Cells were exposed to 21% O2 for 7 days and then moved back to 2% O2, where osteogenesis was induced after 4 days. n=3 for hypoxic cells and n=5 biologically independent experiments for normoxic and reversed-hypoxic cells and merged results are shown in (E).
Figure 3(C-D) Representative images (C) and quantification (D) of Mean Fluorescence Intensity (MFI) after immunostaining against H3ac and H3 of hypoxic and normoxic cells. Mean Fluorescence Intensity (MFI) of histone H3 was used as internal control, for normalization. Nuclei were stained with DAPI. Quantification of H3ac/H3 MFI from n=67 hypoxic and n=56 normoxic individual cells from a representative experiment out of 3 biologically independent experiments is shown in D. Distribution of data points in (D) is shown as a violin plot, where the mean is indicated by a solid line and the quartiles are indicated with dashed lines.(J) Representative immunoblots for ACS and ACLY in hypoxia- and normoxia-cultured cells. α-tubulin was used as loading control.
Figure 4(A-B) Representative images after immunostaining of hypoxia- and normoxia-cultured cells against acetyl-Lysine and TOMM20 (A), and assessment of the acetyl-Lysine signal localization, after manual assignment into three categories: exclusively nuclear, exclusively mitochondrial and nuclear/mitochondrial (B). Nuclei were stained with DAPI. n=3 biologically independent experiments. Results are shown as mean ± S.E.M and statistical significance was determined using two-sided unpaired t-test. In magnified insets intensity of the acetyl-Lysine signal was adjusted similarly to all samples, for visualization purposes.
Figure 5(B-C) Representative images (B) and quantification (C) of Nile Red staining of hypoxic control and BTA-treated cells and of normoxic control and acetate-treated cells. Treatments were done for three days with 1mM BTA or 5mM acetate.Representative images after immunostaining of hypoxic control and BTA-treated cells and normoxic control and acetate-treated cells against acetyl-Lysine and TOMM20 (D), assessment of the acetyl-Lysine signal localization, as described above (E) Nuclei were stained with DAPI. In magnified insets intensity of the acetyl-Lysine signal was adjusted similarly to all samples, for visualization purposes.Representative images after immunostaining of hypoxic control and BTA-treated cells quantification of nuclear acetyl-lysine MFI (F). Nuclei were stained with DAPI. Quantification of nuclear acetyl-Lysine MFI from n=67 hypoxic, n=71 hypoxic_BTA, n=44 normoxic and n=63 normoxic_acetate individual cells from a representative experiment out of 3 biologically independent experiments is shown in (F). In magnified insets intensity of the acetyl-Lysine signal was adjusted similarly to all samples, for visualization purposes.(G-H) Representative images and quantification of Alizarin Red S staining of control hypoxic and normoxic cells and of normoxic acetate-treated cells, 12 days after induction of osteogenesis. Merged results of n=5 biologically independent experiments for hypoxic and normoxic cells and n=4 biologically independent experiments for acetate-treated normoxic cells are shown in (H).
Figure 1Montage of Atoh7+ progenitor division generating an RGC (magenta dot) and a PRpr (cyan dot). Dashed line indicates the apical side, arrowhead points to RGC axon. atoh7:GFP-CAAX (Atoh7, grey). Scale bar 10 µm.Montage of Atoh7+ progenitor division generating an HC (orange dot) and a PRpr (cyan dot). Dashed line indicates the apical side. atoh7:GFP-CAAX (Atoh7, grey). Scale bar 10 µm. Montage of Atoh7+ progenitor division generating an AC (yellow dot) and a PRpr (cyan dot). Dashed line indicates the apical side, arrowhead points to basal dendrites. atoh7:GFP-CAAX (Atoh7, grey). Scale bar 10 µm.Event plot of all divisions analysed in (H).
Figure 2Two examples of retinas at 48 hpf in (left) control and (right) Prdm1a morphant (MO). Atoh7+ cells (magenta), inhibitory neurons (yellow) and photoreceptors (cyan) are labelled. Scale bar 50 µm. A') Close up of Crx (cyan) signal (upper panel) and DAPI (grey, lower panel) from Fig A, for controls (left) and Prdm1a morphant (right). Scale bar 20 µm. Staining for the photoreceptor cell marker zpr-1 at 72 hpf in (left) control and (right) Prdm1a knockdown. Atoh7+ cells (magenta), inhibitory neurons (yellow), photoreceptors (cyan) and zpr-1 (grey). Scale bar 50 µm. Arrowheads indicate zpr-1 staining. B') Close up of Atoh7 (magenta) and Crx (cyan) signal (upper panel), together with Zpr-1 (grey, lower panel) from Fig B, for controls (left) and Prdm1a morphant (right). Scale bar 20 µm. Measurements of retinal diameter in control (black dots) and Prdm1a knockdown (empty dots) at 24, 36, 48 and 72 hpf. N and p values are found in Table 2.**** for p < 0.0001, Two-way Anova with Bonferroni correction. Mean and single values are indicated.Layer thickness analysis in control and Prdm1a knockdown embryos at 72 hpf. N = 4 embryos (control), 6 embryos (Prdm1a morphant). Total thickness comparison: p = 0.0055; GCL comparison, p < 0.0001; INL comparison, ns; ONL comparison, p = 0.0369. Two-way Anova with Bonferroni correction. Mean and SD are indicated, as well as single values.
Figure 3Retina at 72 hpf in (top left) control, (top right) Atoh7 knockdown, (bottom left) Ptf1a knockdown and (bottom right) Atoh7 and Ptf1a knockdown. Atoh7+ cells (magenta), inhibitory neurons (yellow) and photoreceptors (cyan). Scale bar 50 µm, 20 µm in close-up panels.Layer thickness analysis in control and morphant embryos measured at 72 hpf. N = 9 embryos (control), 7 embryos (Atoh7 morphant), 7 embryos (Ptf1a morphant), 7 embryos (Atoh7+Ptf1a morphant). p values for thickness measurements are found in Table 3. Mixed effects analysis with Bonferroni correction. Mean and SD are indicated, as well as single values.Number of PH3+ cells per retina in control and morphant conditions at 28, 32 and 36 hpf. N = 4 to 10 embryos per condition, mixed effects analysis with Dunnet's correction. All comparisons are statistically non-significant. Mean and SD are indicated, as well as single values.Montage of neurogenic progenitor division upon Atoh7 and Ptf1a knockdown, generating two PRpr (cyan dots). Dashed line indicates the apical side. atoh7:GFP-CAAX (Atoh7, grey). Scale bar 10 µm. Montage of neurogenic progenitor division upon Atoh7 and Ptf1a knockdown, generating a BC (blue dot) and a PRpr (cyan dot). Dashed line labels the apical side, yellow arrow points at BC apical process, white arrows point at BC basal process. atoh7:GFP-CAAX (Atoh7, grey). Scale bar 10 µm.
Figure 4Distribution of each Atoh7+ division in time (Event plots) from 28 hpf in Atoh7 morphants, Ptf1a morphants and double Atoh7/Ptf1a morphants. Coloured violin plots indicate different fates, grey violin plots show all neurogenic divisions analysed. Time in hpf.
Figure 6A. Western blot analysis of phosphorylation levels of tau and expression levels of indicated proteins in the cortical and hippocampal regions of the brain in APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). The membranes analysed in (A) are identical to those in Fig 5F.B. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for phosphorylated tau expression levels. Anti-tau (phospho Ser199) antibody (red colour) was applied for staining tau phosphorylation on Serine-199 and Hoechst 33342 (blue colour) for nuclear counterstaining in the brain sections. Scale bars = 200 μM.C. Western blot analyses of phosphorylated RIPK3 expressions in primary cultured astrocytes treated with Nec-1 (lower). The membranes analysed in (C) are identical to those in Fig 1G and 5F.C. Western blot analyses of phosphorylated RIPK3 expressions in cortex and hippocampus of APP/PS1mice injected with Nec-1 (upper). The membranes analysed in (C) are identical to those in Fig 1G and 5F.D, E. ThT assays for the inhibition of tau aggregation (D) and for disaggregation of tau (E). Fluorescence intensity was normalized to tauaggregates (100%, day 5).F. Surface plasmon resonance sensorgrams of Nec-1 targeting tau (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.
Figure 1(A) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 11-15 pigmented or grey HFs from 4 different donors. (B) Representative images of p-S6 immunofluorescence. Only anagen VI HFs were investigated and analyzed immediately after surgery. Analyses were performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.(C) Quantitative analysis of TSC2 protein levels. N = 11-14 pigmented or grey HFs from 4 different donors. (D) Representative images of TSC2 immunofluorescence. Only anagen VI HFs were investigated and analyzed immediately after surgery. Analyses were performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.
Figure 3Quantitative analysis of TSC2 mRNA (A) N = 8-10 anagen VI HFs from 4 different donors treated with siTSC2 or non-targeting oligos (NTO) for 6 days. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001.Quantitative analysis of TSC2 protein expression (B). N = 8-10 anagen VI HFs from 4 different donors treated with siTSC2 or non-targeting oligos (NTO) for 6 days. (C) Representative images of TSC2 immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Samples from each donor are represented by a different color. Nuclei stained with DAPI.(D) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 2-3 anagen VI HFs from 1 donor treated with siTSC2 or non-targeting oligos for 6 days. (E) Representative images of p-S6 immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Samples from each donor are represented by a different color. Nuclei stained with DAPI.(F) Quantitative histomorphometry of melanin production. N = 9-10 anagen VI HFs from 4 different donors treated with siTSC2 or non-targeting oligos for 6 days. (G) Representative bright field microscopic images of Masson-Fontana histochemistry. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Samples from each donor are represented by a different color.(H) Quantitative analysis of tyrosinase activity. N = 8-9 anagen VI HFs from 4 different donors treated with siTSC2 or non-targeting oligos for 6 days. (I) Representative images of tyrosinase activity immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Samples from each donor are represented by a different color. Nuclei stained with DAPI.(J) Quantitative analysis of α-MSH expression. N = 8-9 anagen VI HFs from 4 different donors treated with siTSC2 or non-targeting oligos for 6 days. (K) Representative images of α-MSH immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Samples from each donor are represented by a different color. Nuclei stained with DAPI.
Figure 4(A) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 9-10 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (B) Representative fluorescence images of p-S6 immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.(C) Hair cycle staging was performed using Ki-67 and Masson-Fontana histochemistry. Mean ± SEM; N = 16-17 grey HFs per groups from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (D) Representative fluorescence images of Ki-67 and bright field microscopic images of Masson-Fontana. for C-D where all HFs were analyzed) were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.(E) Quantitative histomorphometry of melanin production by Masson-Fontana histochemistry. N = 7-9 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (F) Representative pictures of Masson-Fontana histochemistry. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mann-Whitney U-test(G) Quantitative analysis of tyrosinase activity. N = 7 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (H) Representative images of tyrosinase activity immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mann-Whitney U-test Nuclei stained with DAPI.(I) Quantitative analysis of α-MSH expression in defined reference area within the bulb. N = 5-7 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (J) Representative images of α-MSH immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mann-Whitney U-test Nuclei stained with DAPI.
Figure 1(B) GALpr-Dfm1-BirA-Flag and GALpr-BirA-Flag levels were measured by western blotting with α -FLAG at 0 (uninduced) vs. 5 hours post-galactose induction (3 biological replicates; n=3).(C) Dfm1-BirA is still functional and able to degrade Hmg2-GFP. dfm1Δ+Hmg2-GFP strains containing DFM1-BIRA, empty vector, or BIRA only addbacks were grown to log phase and degradation was measured by CHX. After CHX addition, cells were lysed at the indicated times and analyzed by SDS-PAGE and immunoblotted for Hmg2-GFP with α-GFP.(D) Yeast strains expressing Dfm1-Bira and BirA only negative control were incubated with different amounts of biotin: 0, 0.1, and 1 mM. dfm1Δ+Hmg2-GFP. Microsomes were isolated from each strain and subjected to streptavidin pulldown (3 biological replicates; n=3). Flow-through and pull-down fractions were detected by western blotting for Dfm1-BirA with α-Flag and Cdc48 with α-Cdc48 antibodies.
Figure 2(A) Dfm1-GFP binding to Lcb2-RFP and Orm2-RFP were analyzed by co-IP. As negative control, cells not expressing Dfm1-GFP were used (3 biological replicates; n=3).
Figure 3(A) Indicated strains were spotted 5-fold dilutions on synthetic complete (SC) plates , and plates were incubated at room temperature, 30oC, and 37oC (3 biological replicates, 2 technical replicates; n=5). WT, dfm1∆, tsc3∆, and dfm1∆tsc3∆ were compared for growth in the dilution assay. Arrowhead indicates growth phenotype of tsc3∆ cells; open circle indicates growth phenotype of dfm1∆tsc3∆ cells. (B) dfm1∆tsc3∆ confers resistance to myrocin. WT, dfm1∆, tsc3∆, and dfm1∆tsc3∆ strains were grown to log-phase in YPD medium, and 5-fold serial dilutions of cultures were spotted on (SC) plates containing either drug vehicle alone, 1 µM of myriocin and 10 μM of PHS (3 biological replicates, 2 technical replicates; n=5). Arrowhead indicates growth phenotype of tsc3∆ cells; open circle indicates growth phenotype of dfm1∆tsc3∆ cells.(D) Indicated strains were spotted 5-fold dilutions on SC plates in 3 biological replicates and 2 technical replicates (n=5), and plates were incubated at room temperature, 30oC, and 37oC (n=3). Upper panel: WT, orm1∆, tsc3∆, and orm1∆tsc3∆ were compared for growth by dilution assay. Middle panel: WT, orm2∆, tsc3∆, and orm2∆tsc3∆ were compared for growth by dilution assay. Bottom panel: WT, orm1∆, orm2∆, tsc3∆, and orm1∆orm2∆tsc3∆, were compared for growth by dilution assay. Upper panel: arrowhead indicates growth phenotype of tsc3∆ cells; open circle indicates growth phenotype of orm1∆tsc3∆ cells. Lower panel: arrowhead indicates growth phenotype of orm2∆ cells; open circle indicates growth phenotype of orm2∆tsc3∆ cells.