EMBO/soda-nel huggingface dataset:
Interferon-mediated repression of miR-324-5p potentiates necroptosis to facilitate antiviral defense (2022)
Figure 6

Fig 6 IFN-mediated repression of miR-324-5p enhances necroptosis

A U937 cells were transfected with NC, siMLKL-3'UTR, or miR-324-5p. After 48 h, cells were exposed to 100 ng/ml IFN-γ plus 20 μM z-VAD for 24 h. Identical concentrations were used in later experiments unless otherwise stated. Cell viability was determined by measuring ATP levels.

B qPCR analysis for the expression of MLKL (left) and miR-324-5p (middle) in PBMC-derived macrophages that were treated with PBS or IFN-γ for 24 h. The corresponding western blotting analysis of MLKL and β-actin (right).

C, D PBMC-derived macrophages (C) and HT-29 cells (D) were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were exposed to S+Z or IFN-γ+S+Z for 24 h. Cell viability was determined by measuring ATP levels (left). The corresponding western blotting analysis of MLKL and β-actin (right).

E qPCR analysis for the expression of miR-324-5p in miR-324-5p knockout HT-29 cells (miR-324-5p-/- cells) . Altered miR-324-5p DNA sequences were shown in miR-324-5p-/- clone 19# and 36#.

F MiR-324-5p+/+ HT-29, miR-324-5p-/- -19# , and miR-324-5p-/- -36# cells were treated with T+S+Z for the indicated times. Cell viability was determined by measuring ATP levels.

G, H MiR-324-5p+/+ HT-29, and miR-324-5p-/- HT-29 cells were treated with IFN-β (G) or IFN-γ (H) plus 20 μM z-VAD for 24 h. Cell viability was determined by measuring ATP levels.

Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.
Interferon-mediated repression of miR-324-5p potentiates necroptosis to facilitate antiviral defense (2022)
Figure 7

Fig 7 MiR-324-5p inhibition sensitizes IAV-infected cells to necroptosis and reduces viral replication

A qPCR analysis for the expression of IFN-β (left) and IFN-γ (right) in PBMC-derived macrophages that were infected with IAV (H1N1 strain PR8) at an multiplicity of infection (MOI) of 0.2 for 24 h.

B qPCR analysis for the expression of MLKL (left) and miR-324-5p (right) in PBMC-derived macrophages that were treated with the 300nM ruxolitinib for 2 h prior to the infection with IAV (MOI=0.2) for 24 h.

C PBMC-derived macrophages were transfected with NC, siMLKL, or miR-324-5p. After 48h, cells were treated with S+Z in the presence or absence of IAV (MOI=0.2) for 24 h. Cell viability was determined by measuring ATP levels.

D qPCR analysis for the expression of M gene of IAV in PBMC-derived macrophages that were transfected with NC, siMLKL or miR-324-5p for 48h, followed by the infection with IAV (MOI=0.2) for 24 h.

E HT-29 cells were infected with IAV (MOI=0.2) for 24 h. qPCR analysis for the expression of IFN-β.

F HT-29 cells were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were treated with DMSO, S+Z in the presence or absence of IAV (MOI=0.2) for an additional 24 h. Cell survival was determined by measuring ATP levels.

G MiR-324-5p+/+ and miR-324-5p-/- HT-29 cells were infected with IAV (MOI=0.2) for 24 h. Cell viability was determined by measuring ATP levels.

H MiR-324-5p+/+ and miR-324-5p-/- HT-29 cells were infected with IAV (MOI=0.2) with or without z-VAD for 48 h (n = 4, biological replicates). IAV CCID50 analysis for the virus titer in the cell culture supernatant.

Data information: In (A-G), data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.
ATP citrate lyase controls hematopoietic stem cell fate and supports bone marrow regeneration (2022)
Figure 1

Figure 1. The HSC potential is changed during BM regeneration after the administration of 5-FU

(A) Kinetics of cell numbers in Lineage-EPCR+CD150+CD48- (L-ESLAM) HSCs, Lineage-EPCR+CD150+CD48+ (CD150+CD48+) progenitor cells, and Lineage-EPCR+CD150-CD48+ (CD150-CD48+) progenitor cells after the administration of 5-FU (250 mg/kg, i.v.). The small graph is an enlargement of 0 to 8 days after the administration of 5-FU. (* or **) in the small graph represents the statistical analysis of HSC between 3 and 4 or 7 days, respectively. Data are shown as means ± S.D. (n ≥4, two or more independent experiments, *p<0.01, **p<0.05 by the t-test).

(B) The enrichment plots of genes related to cell cycle in the comparison of the steady state with the early (upper) or late phase (lower). NES and q values represent normalized enrichment scores and FDR, respectively.

(C) The frequency of EdC+ cells within L-ESLAM HSCs 24h after the administration of EdC (150 mg/kg, i.p.). Data are shown as means ± S.D. (n ≥4, two independent experiments, *p<0.01 by the t-test).

(D) A principal component analysis (PCA) using the transcriptome data of L-ESLAM HSCs derived from untreated (steady state) or 5-FU-teated mice at 6 (early phase) or 10 days (late phase).

(E) Comparison of gene expression patterns in HSCs between the early (6 days) and late phases (10 days) on GSEA. The graph shows normalized enrichment scores (NES). Bold and italic mean gene sets show (p<0.05, FDR< 0.05) or (p<0.05), respectively.

(F) The frequencies of phenotypic HSCs (Orange gate) and CD48+ progenitor cells (blue gate) after a 4-day culture of HSCs derived from 5-FU-treated mice (at 6 to 9 days). Data are shown as means ± S.D. (n=4, two independent experiments, *p<0.01 by the t-test).
ATP citrate lyase controls hematopoietic stem cell fate and supports bone marrow regeneration (2022)
Figure 4

Figure 4. HSCs showed enhanced mitochondria-Acly activity leading to histone acetylation in the early phase after the administration of 5-FU.

(A) Global H3K27ac in HSCs during BM regeneration after the administration of 5-FU. Data are shown as means ± S.D. (n=4, two independent experiments, *p<0.01, **p<0.05 by the t-test).

(B) The relationship between the TCA (tricarboxylic acid cycle) cycle within mitochondrial metabolism, Acly, and histone acetylation.

(C-E) mRNA (C), protein (D), and phosphorylation levels of Ser455 (E) in Acly in HSCs after the administration of 5-FU. Data are shown as means ± S.D. (n≥4, two independent experiments, *p<0.01, N.S.; not significant by the t-test).

(F) The potential for glucose uptake by HSCs obtained from untreated or 5-FU-treated mice was estimated using 2-NBDG. Data are shown as means ± S.D. (n ≥3, two independent experiments, *p<0.01, N.S., not significant by the t-test).

(G-I) The levels of intracellular Glu (G), citrate (H) and total acetylated lysine (I) in HSCs obtained from untreated or 5-FU-treated mice. Data are shown as means ± S.D. (n ≥3, two independent experiments, *p<0.01, **p<0.05, N.S., not significant by the t-test).

(J) ΔΨm in L-ESLAM HSCs after the administration of 5-FU. Histograms represent the fluorescence intensity of JC-1 Red, which is polymer-derived fluorescence generated in response to ΔΨm. The graph depicts normalized ΔΨm by the ratio of the fluorescence intensity of JC-1 Red to JC-1 Green, which indicates monomer-derived fluorescence. Data are shown as means ± S.D. (n ≥4, more than two independent experiments, *p<0.01, **p<0.05 by the t-test).

(K) The enrichment of the gene set "HALLMARK_OXIDATIVE_PHOSPHORYLATION" within differentially expressed genes of the steady state vs the early phase. NES and q values represent normalized enrichment scores and FDR, respectively.
ATP citrate lyase controls hematopoietic stem cell fate and supports bone marrow regeneration (2022)
Figure 5

Figure 5. Acly-mediated histone acetylation was essential for HSC expansion in the early phase after the administration of 5-FU.

(A) Schema of experimental designs and the targets of inhibitors. Serial treatments with CTPI-2 (50 mg/kg/shot/day, i.p.), BMS303141 (100 mg/kg/shot/day, p.o.), A-485 (20 mg/kg/shot/day, i.p.), or TV-3664 (10 mg/kg/shot/day, p.o.) were performed 3, 4, and 5 days after the administration of 5-FU.

(B-C) The effects of CTPI-2 (B) or BMS303141 (BMS) (C) on H3K27ac in HSCs in the early phase after the administration of 5-FU. Data are shown as means ± S.D. (n ≥3, two independent experiments, *p<0.01, **p<0.05 by the t-test).

(D-E) The effects of CTPI-2 (D), BMS303141 (BMS) (E), A-485 (D), and TV-3664 (E) on HSC expansion in the early phase after the administration of 5-FU. Numbers in the dot plots represent the frequencies of the indicated fractions within total BM cells. Data are shown as means ± S.D. (n≥4, two independent experiments, *p<0.01, **p<0.05 by the t-test).

(F and G) Average plot (top) and heatmap (bottom) of H3K27ac reads in the cis-regulatory regions of genes belonging to the gene set "HALLMARK_MITOTIC_SPINDLE" (F) or "HALLMARK_G2M_CHECKPOINT" (G) in HSCs in the steady state or early phase.

(H) The enrichment of the gene set "GOBP_FATTY_ACID_BIOSYNTHETIC_PROCESS" within differentially expressed genes of the steady state vs the early phase.

(I) Average plot (top) and heatmap (bottom) of H3K27ac reads in the cis-regulatory regions of genes having the GO term "fatty acid biosynthetic process" in HSCs in the steady state or early phase.
ATP citrate lyase controls hematopoietic stem cell fate and supports bone marrow regeneration (2022)
Figure 7

Figure 7. CD48+ progenitor cells in the late phase show increased H3K27ac levels within accessibility-increased regions in HSCs from the early to late phase.

(A-D) Global H3K27ac (A), Acly levels (B), acetylated lysine (C), and ΔΨm (D) in CD48- HSCs and CD48+ progenitor cells (HPCs)in the late phase after the administration of 5-FU. Data are shown as means ± S.D. (n=4, two independent experiments, *p<0.01 by the t-test).

(E) The enrichment of the gene set "HALLMARK_OXIDATIVE_PHOSPHORYLATION" within differentially expressed genes between CD48- HSCs and CD48+ progenitor cells. NES and q values represent normalized enrichment scores and FDR, respectively.

(F) Average plot (top) and heatmap (bottom) of H3K27ac reads in CD48- HSCs and CD48+ HPCs in the late phase within cis-regulatory regions related to genes with lower (left) or higher (right) expression levels in CD48+ HPCs than in CD48- HSCs.

(G) ATAC peaks of HSCs in the early or late phase, and H3K27ac peaks in CD48- HSCs or CD48+ HPCs in the late phase within the promoter and enhancer regions in the CD48 locus.

(H) CD48 mRNA levels in CD48- HSCs and CD48+ HSCs in the late phase after the administration of 5-FU. Data are shown as means ± S.D. (n=4, two independent experiments, *p<0.01 by the t-test).

(I) Average plot (top) and heatmap (bottom) of H3K27ac reads in CD48- HSCs and CD48+ HPCs in the late phase within accessibility-decreased (left: Green dot in Fig 3E), -unchanged (center: Blue dot in Fig 3E), or -increased cis-regulatory regions (right: brown dot in Fig 3E) in HSCs in the late phase.

(J) Within accessibility-increased cis-regulatory regions in the late phase (brown dot in Fig 3E), 192 regions showed more than 2-fold increases in H3K27ac levels in CD48+ progenitor cells than in maintained HSCs (Venn diagrams). The GSEA plot represents the enrichment of the gene set related to these H3K27ac-increased regions within differentially expressed genes between CD48- HSCs and CD48+ progenitor cells. NES and q values represent normalized enrichment scores and FDR, respectively.
ATP citrate lyase controls hematopoietic stem cell fate and supports bone marrow regeneration (2022)
Figure 8

Figure 8. The mitochondria-Acly activity regulates the production of CD48+ progenitor cells during BM regeneration after the administration of 5-FU.

(A and B) ΔΨm (A) and Acly levels (B) in HSCs derived from 5-FU treated mice in the late phase before and after a 1-day culture. Data are shown as means ± S.D. (n≥3, two independent experiments, *p<0.01 by the t-test).

(C) The frequencies of the CD150+CD48- and CD48+ fractions within the lineage-EPCR+ fraction after a 4-day culture of HSCs in the late phase with or without BMS303141 (BMS; 100 μM), SB204990 (SB; 100 μM), an alternative Acly inhibitor, or CTPI-2 (50 μM). Data are shown as means ± S.D. (n=4, two independent experiments, *p<0.01, by the t-test).

(D and E) Total cell number (D) or CD48+ cell number (E) after the 4-day culture of HSCs in the late phase with or without BMS303141 (BMS), SB204990 (SB), or CTPI-2. Numbers within the graph represent the inhibitory effects of each treatment (fold decrease by an inhibitor treatment). Data are shown as means ± S.D. (n=4, two independent experiments, *p<0.01, by the t-test).

(F) Global H3K27ac in HSCs in the late phase before or after a 1-day culture with or without BMS303141 (BMS), SB204990 (SB), or CTPI-2. Data are shown as means ± S.D. (n≥4, two independent experiments, *p<0.01, by the t-test).

(G) The frequencies of CD150+CD48- and CD48+ fractions within lineage-EPCR+ after HSCs in the late phase were cultured for 4 days with or without A-485 (1 μM), an inhibitor of histone acetyltransferase. Data are shown as means ± S.D. (n=4, two independent experiments, *p<0.01 by the t-test).

(H and I) Total cell number (H) or CD48+ cell number (I) after a 4-day culture of HSCs in the late phase with or without A-485. Numbers within the graph represent the inhibitory effects of each treatment (fold decrease by an inhibitor treatment). Data are shown as means ± S.D. (n=4, two independent experiments, *p<0.01, by the t-test).

(J) The frequencies of CD150+CD48- and CD150+CD48+ fractions within lineage-EPCR+ after a serial CTPI-2 treatment (50 mg/kg/shot/day) following the administration of 5-FU in mice transplanted with 15000 HSCs without irradiation. Data are shown as means ± S.D. (n=4, two independent experiments, **p<0.05 by the t-test).

SUPPLEMENTAL FIGURE LEGENDS
PSMC3 variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress (2020)
Figure 1

Figure 1. Family pedigree and cDNA analysis

(A) Family pedigree. Variant segregation analysis of PSMC3. Electropherogram of a part of intron 10 of PSMC3 encompassing the identified variation (c.[1127+337A>G];[1127+337A>G], p.[(Ser376Arg15*)];[(Ser376Arg15*)]) in the affected individuals, their unaffected parents and siblings. The variation was found at the homozygous state in the affected individuals (II.2, II.4, II.7), at the heterozygous state in the parents (I.1, I.2, I.3, I.4, I.5, I.6) and was either at the heterozygous state (II.5) or absent in the unaffected siblings (II.1, II.3, II.6).

(B) Face (up) and profile (down) photographs for patients II.4 (a: 8 yo, b: 16 yo), II.2 (c: 6 yo, d: 14 yo) and II.7 (e: 1 yo, f: 7 yo) over time. One can observe prominent supraciliay arches, synophris, sunken cheeks, short philtrum and retrusion in the malar region.

(C) Sub cutaneous calcifications found only on knees (g: 9 yo and h: 16 yo) on elbows (i: 9 yo) of patient II.4. White hair were present only on the legs of the 3 patients as illustrated for patient II.4 (j: 16 yo).

(D) Temporal bone CT-scan from patient II.7 (left column) and a control (right column) showing malformation of the semicircular canal. The left ear is shown on the upper panels while de right ear on the lower panels.

(E) Amplification of the cDNA fragment between exons 9-10 and 11 of PSMC3 showing the abnormally spliced RNA fragment. One band at 180bp representing the normal allele is seen for the control and two bands for the individual II.4 (pathologic allele at 300bp).

(F) Schematic representation for the incorporation of the 114bp intronic sequence resulting from the c.1127+337A>G deep intronic variation on the mRNA. Sanger sequencing of the fragment between exons 9-10 and 11 of the PSMC3 cDNA obtained from patient II.4 fibroblasts' RNA, showing the insertion of the 114bp cryptic exon. As a comparison, the schematic representation and sequence from a control individual are shown below.
PSMC3 variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress (2020)
Figure 2

Figure 2. Effect of the deep intronic PSMC3 variant on the proteasome function

(A) Anti-ubiquitin western blot in control and patient fibroblasts and total amount of proteins loaded (stainfree) showing increased ubiquitination in patient cells (lane 4).

(B) Histogram showing the quantification of ubiquitin with western blot assays. The data shown correspond to the sum of all bands detected by the anti-ubiquitin antibody expressed as a percentage of the amount of ubiquitin in "Control 1" cells. Bars show mean of ten independent experiments +/- SD (n=10, t-test *: p<0.01, **: p<0.05).

(C) Mass spectrometry results from the co immunoprecipitation with PSMC3 are displayed as the normalized total number of spectra count of each protein computed as the mean from 3 controls (x axis) vs the mean of patient II.4 triplicate. Proteasome subcomplexes are colored according to the displayed legend and standard ratio lines are drawn.
PSMC3 variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress (2020)
Figure 3

Figure 3. Fibroblasts derived from patient carrying the c.1127+337A>G homozygous PSMC3 variation exhibit an increased amount of both proteasome complexes and ubiquitin-protein conjugates.

(A) Whole-cell lysates from control and patient (case index, CI) fibroblasts were assessed by 3-12% native-PAGE gradient gels with proteasome bands (30S, 26S and 20S complexes) visualized by their ability to cleave the Suc-LLVY-AMC fluorogenic peptide.

(B) Ten micrograms of control and patient cell lysates were tested for their chymotrypsin-like activity by incubating them with 0.1 mM of the Suc-LLVY-AMC substrate at 37°C over a 180-h period of time in quadruplicates on a 96-well plate. Indicated on the y-axis are the raw fluorescence values measured by a microplate reader and reflecting the AMC cleavage from the peptide. Bars show the mean of 4 independent experiments +/- SD.

(C) Proteasome complexes from control and patient fibroblasts separated by native-PAGE were subjected to western-blotting using antibodies specific for α6, Rpt5 (PSMC3) and PA28-α, as indicated.

(D) Proteins extracted from control and CI PSMC3 were separated by 10 or 12.5% SDS-PAGE prior to western-blotting using primary antibodies directed against ubiquitin and several proteasome subunits and/or components including α6, β1, β; β5, β5i, Rpt2 (PSMC1), Rpt5 (PSMC3), Rpt3 (PSMC4), Rpt4 (PSMC6) and PA28-α, as indicated. For the PSMC3 staining, two exposure times are shown. Arrow indicates an additional PSMC3 species corresponding to the expected size of the truncated PSMC3 variant. Equal protein loading between samples was ensured by probing the membrane with an anti-α-Tubulin antibody.
PSMC3 variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress (2020)
Figure 4

Figure 4. Patient fibroblasts carrying the c.1127+337A>G homozygous PSMC3 variation exhibit an exhausted TCF11/Nrf1 processing pathway which prevents them to upregulate proteasome subunits in response to proteotoxic stress.

Control and patient (index case, IC PSMC3) fibroblasts were exposed to a 16-h treatment with 30 nM of the proteasome inhibitor carfilzomib or left untreated (as a negative control). Following treatment, cells were collected and subjected to RIPA-mediated protein extraction prior to SDS-PAGE and subsequent western-blotting using antibodies specific for ubiquitin, TCF11/Nrf1, Rpt1 (PSMC2), Rpt3 (PSMC4), Rpt5 (PSMC3), Rpt6 (PSMC5), β1, β2, β5, β5i, β1i, PA28-α and α-Tubulin (loading control) as indicated. For the TCF11/Nrf1 staining, two exposure times are shown.
PSMC3 variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress (2020)
Figure 5

Figure 5. Overexpression of wild-type PSMC3 in patient fibroblast restores intracellular protein homeostasis and prevents the loss of proteasome subunit expression in response to proteasome inhibition.

(A) Control and/or patient (index case, IC PSMC3) fibroblasts were subjected to a 24-h transfection with pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3 prior to RIPA-mediated protein extraction and subsequent western-blotting using antibodies specific for ubiquitin, PSMC3 (i.e. Rpt5) and α-Tubulin (loading control).

(B) Densitometry analysis showing the relative ubiquitin contents detected by western blotting in patient fibroblasts exposed to either pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3, as indicated. The y-axis represents the percent changes in densitometry measurements (of pixel intensities using Image J) which are set as 100 % for cells transfected with the pcDNA3.1/empty vector (mock) at 24 post-transfection (n=4, *: p<0.05, t-test). Bars show the mean +/- SEM.

(C) Patient (index case, IC PSMC3) fibroblasts transfected with either pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3 were exposed to a 30-nM treatment of carfilzomib or left untreated (as a negative control). After 16 hours, cells were collected and subjected to RIPA-mediated protein extraction prior to SDS-PAGE and subsequent western-blotting using antibodies specific for ubiquitin, TCF11/Nrf1, Rpt1 (PSMC2), Rpt3 (PSMC4), Rpt5 (PSMC3), Rpt6 (PSMC5), β1, β2, β5, β5i, PA28-α and α-Tubulin (loading control), as indicated. For the TCF11/Nrf1 staining, two exposure times are shown.
PSMC3 variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress (2020)
Figure 6

Figure 6. psmc3 morphants and F0 mosaic zebrafish exhibit cataract and show abnormalities during the semicircular canal development in the ear.

(A) Scheme of a zebrafish eye.

(B-B'). Cataract detection revealed abnormal lens reflection in psmc3 morpholino (MO)-mediated knockdown but not in controls (uninj, ctrl-mo). Similarly, abnormal lens reflection was also observed in embryos injected with sgRNA + Cas9 but not in sgRNA injected embryos without Cas9 (sgRNA2). Co-injection of wt psmc3 mRNA with either psmc3-mo or sgRNA2 + Cas9 reduced the number of embryos presenting abnormal lens reflection. Scale bar = 50 µm. (B') Quantification of embryos with abnormal lens reflection.

(C) Representative image of a zebrafish ear at 4 dpf. kc= kinocilia.

(D-D') bright field images of inner ear development (lateral position). (D) Epithelial projections were fused and formed canal pillars in 4-day-old uninjected and control injected fish (ctrl-mo, sgRNA2) but not in morphants (mo) and crispants (sgRNA2+Cas9). Co-injection of wt psmc3 mRNA with psmc3-mo or sgRNA + Cas9 reduced the number of embryos presenting abnormal ear phenotype. Black asterisks indicate fused pillars. Red arrowheads mark unfused projections. Scale bar = 100 µm. (D') Quantification of embryos with abnormal projection outgrowth.

(E-E') An anti-acetylated tubulin antibody (green) staining revealed an abnormal amount of kinocilia in psmc3 crispants (sgRNA2+Cas9) compared to uninjected and control injected embryos (sgRNA2). Nuclei are stained in blue with DAPI. Representative images show kinocilia of the lateral cristae. Scale bar = 20 µm. (E') Quantification of embryos with an abnormal amount of kinocilia.