EMBO/soda-nel huggingface dataset:
YY1 regulates skeletal muscle regeneration through controlling metabolic reprogramming of stem cells (2018)
Figure 6

Figure. 6. YY1 loss leads to a defect in glycolytic switch during SC activation via Hif1α destabilization. (A) An equal number of FACS-isolated SCs from C57 mice were cultured for 24 hrs and treated with 0, 1, 10, or 20mM 2-DG (glycolytic inhibitor) or NaN3 (respiration inhibitor) for 3 hrs before measurement of ATP production. The relative ATP levels normalized to the 0 mM values are plotted. N=3 mice. (B) SCs from C57 mice were treated with 10mM 2-DG or 10mM NaN3 for 36 hrs before measurement of proliferation rate by MTS assay (n=3 mice, each). (C) SCs from C57 mice were treated with 10mM 2-DG for 24 hrs and subject to EdU labeling for 6 hrs. The percentage of Pax7+/EdU+ cells over the total number of Pax7+ cells was quantified (n=3 mice, each). (D) Ctrl and YY1iKO ASCs were stained with 60 ug/mL 2-NBDG for 45 mins and the fluorescence intensity (MFI) of 2-NBDG was measured by flow cytometry (n=3 mice, each). (E-G) An equal number of Ctrl and YY1iKO cells were cultured for 36h; basal extracellular acidification rate (ECAR) level (E), Lactate concentration (F) and ATP production (G) were measured and normalized with cell numbers. N=3 mice. (H) Genomic snapshots depict RNAseq profiles of the selected glycolytic genes (Hk2, Glut1, Ldha, Pdk1, Eno1) down-regulated in YY1iKO vs Ctrl ASCs. (I) Expression of the selected glycolytic genes was quantified by RT-qPCR. Hsp90ab1 was used as the normalization control. (J) Re-expression of YY1 by transfecting YY1iKO ASCs with a YY1 WT expressing plasmid led to up-regulation of glycolytic genes including Glut1, Pfkl, Gapdh, Eno1, Pgam1, Pkm. Hsp90ab1 was used as the normalization control. (K) FACS-isolated SCs from Pax7-nGFP mice were transfected with Hif1α or control siRNAs and EdU labeled for 5 hrs. The percentage of MyoD+EdU+ cells over the total number of MyoD+ cells was quantified. Scale bar = 100 µm. N=3 mice. (L) The expression of glycolytic genes and cell cycle related genes was detected by RT-qPCR in the above transfected cells. β-actin was used as the normalization control. (M) Hif1α protein level was detected by Western blotting in YY1iKO vs Ctrl ASCs. β-actin was used as the loading control. (N) Left: Image of flow cytometric analysis of Hif1α protein or IgG control in YY1iKO vs Ctrl ASCs. Right: Relative mean fluorescence intensity (MFI) of Hif1α protein is shown. N=3 mice. (O) YY1iKO ASCs were transfected with empty YY1 WT viruses; the relative expression level of Hif1a protein was detected by the above described flow cytometric analysis N=3 mice. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.
YY1 regulates skeletal muscle regeneration through controlling metabolic reprogramming of stem cells (2018)
Figure 7

Figure. 7. YY1 stabilizes Hif1α protein through direct interaction. (A-B) Ctrl or YY1iKO ASCs were treated with CHX (Cycloheximide) for 90 mins and Hif1α protein was measured by flow cytometry. The numbers indicate the degradation rate after CHX treatment. N=3 mice. (C) The relative degradation rate was calculated by the ratio of degradation rate in YY1iKO (11.14% ± 1.71%) vs Ctrl (5.08% ± 0.796%). N=3 mice. (D) Lysates from C2C12 myoblasts (GM) were subjected to co-immunoprecipitation (Co-IP) assays with anti-YY1 or anti-IgG antibodies and blotted with anti-Hif1α or anti-YY1 antibodies. (E) The above lysates were also IPed with anti-Hif1α or anti-IgG antibodies and blotted with anti-YY1 or anti-Hif1α antibodies. (F) ASCs from YY1iKO mice were infected with pRlenti-Hif1α (P402A/P564A) (Hif1α-DM) viruses to overexpress a non-degradable Hif1α protein or GFP-expressing viruses. ASCs from Ctrl mice were also infected with the GFP viruses. 36 hrs after infection, the expression of target glycolytic genes was detected by RT-qPCR. Hsp90ab1 was used as the normalization control. (G) SCs were isolated from Ctrl or YY1iKO mice and infected with the above viruses. EdU labeling was then performed for 5.5 hrs. The percentage of MyoD+EdU+ cells over the total number of MyoD+ cells was quantified. Scale bar = 100 µm. N=3 mice. (H-I) Heat maps indicating gene expression levels (Log2[FPKM]) of pentose cycle enzymes (H) and amino acid transporter (I) in YY1iKO vs Ctrl. (J) YY1(WT) or YY1(S365D) mutant was expressed in YY1iKO ASCs and EdU labeled for 6 hrs. (K) Similarly, YY1 (WT) or YY1 (1-394) mutant was expressed and EdU labeled for 6hrs. A GFP expressing plasmid was used the negative control. The percentage of Pax7+/EdU+ cells over the total number of Pax7+ cells was quantified. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.
DJ-1 depletion prevents immunoaging in T-cell compartments (2021)
Figure 1

Figure 1. DJ-1 deficiency diminishes immunoaging in CD8 T cells of an index patient.

a-c, Expression of CD45RO and CCR7 (a), CD45RO and CD27 (b), CD27 and CD28 (c) on peripheral blood CD8 T cells of three participants [P1 (heterozygous mutation), P2 (homozygous mutation) and P3 (heterozygous) for the c.192G>C mutation in the DJ-1 gene].

d-g, Expression of CD57 (d), PD-1 (e), EOMES (f) and T-bet (g) on peripheral blood CD8 T cells of three participants. The enlarged number in the corresponding gate represents the corresponding percentage out of the parent population.

h-k, mRNA expression of senescence related genes (h), exhaustion related genes (i), KIR or KLR genes (j) and cyclin-dependent kinase inhibitor genes (k) in sorted CD8 T cells of the peripheral blood from three participants.

l-m, Comparison of the lower bound of TCR-beta repertoire (l) and richness (m) of naïve CD8 T cells among three participants.

n, The sample clonality index of TCR repertoire of naïve CD8 T cells among three participants.

o, Serology detection of CMV infection for three participants. The threshold of 11 was decided according to the manufacture instruction. The data in panel o represents as mean of technical replicates ± standard deviation (s.d.).

Data information: The legends P1_Naive CD8, P2_Naive CD8 and P3_Naive CD8 refer to the panels (h-n).
DJ-1 depletion prevents immunoaging in T-cell compartments (2021)
Figure 2

Figure 2. Dj-1 depletion reduces signs of immunoaging in murine CD8 T cells.

a, Representative flow-cytometry plots of CD44 and CD62L expression on total CD8 T cells of 45-wk-old Dj-1 KO and age- and sex-matched WT mice.

b-c, Percentages of CD44low CD62Lhigh (Tn) (b) and CD44high CD62Llow (Tem) (c) cells among total CD8 T cells of spleen and pLNs from young and 45-wk-old Dj-1 KO and WT littermates (young KO, n=5; young WT, n=5; 45-wk-old KO, n=8; 45-wk-old WT, n=6; for 45-wk-old mice, data pooled from 2 independent experiments).

d, Representative histogram overlay of PD-1 expression among total CD8 T cells in spleen of 45-wk-old mice (left panel) and percentages of PD-1+ cells among total CD8 T cells (right panel).

e, Percentages of Ki-67+ cells among total CD8 T cells.

f, Percentage of Ki-67+ cells among splenic CD8 Tn, Tem and Tcm in 45-wk-old Dj-1 KO and age- and sex-matched WT mice.

g, Representative flow-cytometry plots of Ki-67 and PD-1 among splenic CD8 Tem of 45-wk-old mice.

h, IFN-γ production in CD8 T cells of spleen and pLNs after in vitro stimulation using 50 ng/ml of PMA and 750 ng/ml of ionomycin for 5 h.

i, Represenative flow-cytometry plots of CD44 and CD49d among blood CD8 T cells of 60-wk-old mice.

j, Percentages of Tn (top), Tvm (middle), Tmem (bottom) among 60-wk-old or young mice (young KO, n=4; young WT, n=3; 60-wk-old KO, n=4; 60-wk-old WT, n=5).

k, Represenative histogram of CD31 among blood CD8 T cells of 60-wk-old mice.

l, CD31 MFI of total CD8 T cells (young KO, n=4; young WT, n=3; 60-wk-old KO, n=4; 60-wk-old WT, n=5).

Data information: SP and LN represents spleen and lymph nodes, respectively. Results represent at least four (b-h) or two (i-l) independent experiments. Data are mean of biological replicates ± s.d. Each dot/symbol represents the measurement from one mouse. The P-values are determined by a two-tailed non-paired Student's t-test. n.s. or unlabeled, not significant, *P<=0.05, **P<=0.01 and ***P<=0.001.
DJ-1 depletion prevents immunoaging in T-cell compartments (2021)
Figure 3

Figure 3. Dj-1 depletion inhibits the expression of key immunoaging markers KLRG1 and PD-1 in a hematopoietic- and CD8-Tn-intrinsic manner.

a, Schematic of the experimental setup of BM transplantation. A total of 10E6 of bone marrow cells from 45-wk-old Dj-1 KO mice (CD45.2+) and WT mice (CD45.1+) (1:1 mix) were transferred into lethally-irradiated young Dj-1 KO or WT recipients (CD45.2+) by i.v. injection. Mice stably engrafted with donor cells were sacrificed for flow cytometry (FCM) analysis later.

b, Percentages of KLRG1+ CD8 T cells derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO (n=4) or WT recipients (n=5).

c, Percentages of KLRG1+ among CD8 Tem derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO or WT recipients.

d, Percentages of PD-1+ population among CD8 T cells derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO (n=4) or WT recipients (n=5).

e, Percentages of CD8+ CD44highCD62Llow (Tem) cells derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO or WT recipients.

f, Ratios between CD8 Tn and Tem cells developed from two types of BM cells within young Dj-1 KO or WT recipients.

g, Percentages of CD8+ CD44lowCD62Lhigh (Tn) cells derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO or WT recipients.

h, Schematic of the experimental setup of CD8 Tn adoptive transfer. 2.5E5 naïve CD8 T cells isolated from young or 55-wk-old Dj-1 KO and WT littermates were injected into Rag-1 deficient mice by i.v. injection. 6 weeks later, mice were sacrificed for FCM (flow cytometry) analysis.

i, Representative flow-cytometry plots of KLRG1+ population among total CD8 T following adoptive transfer of CD8 Tn from 55-wk-old mice.

j, Percentage of KLRG1+ population among total CD8 T cells (55-wk-old KO, n=4; 55-wk-old WT, n=5; young KO, n=4; young WT, n=4).

k, Percentage of KLRG1+ population among CD8 Tem cells.

l, Percentage of KLRG1+PD-1+ population among total CD8 T cells (55-wk-old KO, n=4; 55-wk-old WT, n=5; young KO, n=4; young WT, n=4).

m, Percentage of KLRG1+PD-1+ population among CD8 Tem cells.

Data information: Results from BM transfer and adoptive transfer of CD8 Tn represent two independent experiments. Data are mean ± s.d. The P-values are determined by a two-tailed paired (b-g) or non-paired (j-m) Student's t-test. n.s. or unlabeled, not significant, *P<=0.05, **P<=0.01 and ***P<=0.001.
DJ-1 depletion prevents immunoaging in T-cell compartments (2021)
Figure 4

Figure 4. Dj-1 depletion compromises TCR sensitivity of CD8 Tn in young mice but enhances TCR sensitivity of CD8 Tn in 45-wk-old mice in a dose-dependent manner.

a, Representative flow cytometry data of CD69 and Celltrace violet (CTV) staining on gated living CD8 T cells. The purified CD8 Tn were isolated from young mice and stimulated by different doses of anti-CD3 ab for 72 hours. Enlarged number representing the percentage of the corresponding gate.

b, the percentages of proliferating cells among living CD8 singlets (CD8 Tn were from young mice).

c, the percentages of CD69+ cells among living CD8 T singlets (CD8 Tn were from young mice).

d, Representative flow cytometry data of CD69 and CTV staining on gated living CD8 T cells. The purified CD8 Tn were isolated from 45-wk-old mice and stimulated by different doses of anti-CD3 ab for 72 hours. Enlarged number representing the percentage of the corresponding gate.

e, the percentages of proliferating cells among living CD8 singlets (CD8 Tn were from 45-wk-old mice).

f, the percentages of CD69+ cells among living CD8 T singlets (CD8 Tn were from 45-wk-old mice).

Data information: Results represent at least two independent experiments (a-f). Data are mean± s.d. All the CD8 Tn cells were first pooled from 3-4 mice of the same group before exposing to different doses of anti-CD3 ab. The error bar (s.d.) here essentially refers to technical replicates. The P-values are determined by a two-tailed non-paired (b-c, e-f) Student's t-test. Unlabeled, not significant, *P<=0.05, **P<=0.01 and ***P<=0.001.
DJ-1 depletion prevents immunoaging in T-cell compartments (2021)
Figure 5

Figure 5. Dj-1 depletion impairs OXPHOS essentially in resting CD8 Tn.

a-b, Comparison of splenic CD8 Tn mitochondrial mass [Mitotracker (MT) green MF, a] and mitochondrial potential (MT Deep Red, b) of young and 45-wk-old Dj-1 KO and WT mice.

c-d, Comparison of CD8 Tem mitochondrial mass (c) and mitochondrial potential (d) of young and 45-wk-old Dj-1 KO and WT mice.

e-h, Oxygen consumption rate (OCR) of unstimulated (e) and stimulated CD8 Tn (f) by 2ug/ml of anti-CD3 and 1ug/ml of anti-CD28 for 24 hours. The CD8 Tn were isolated from 45-wk-old mice. OCR of unstimulated (g) and stimulated CD8 Tn (h) by anti-CD3 and -CD28 for 24 hours. The CD8 Tn were isolated from young mice. Left of each OCR measurement panel, representation plots following Mito stress test. Right of each panel, quantitation of basal and maximum respiration under Mito stress test conditions The dashed line indicates the time when the corresponding modulator Oligomycin (Oligo), Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) or Rotenone and Antimycin (AA/Rot) was added.

i-k, Extracellular acidification rate (ECAR) of unstimulated and stimulated CD8 Tn by anti-CD3 and -CD28 antibodies for 24 hours (i). The CD8 Tn were isolated from 45-wk-old mice. ECAR of unstimulated (j) and stimulated (k) CD8 Tn by anti-CD3 and -CD28 for 24 hours. The CD8 Tn were isolated from young mice. Left of each ECAR measurement panel, representation plots following Glycolysis stress test. Right of each panel, quantitation of glycolysis and glycolytic capacity under Glycolysis stress test conditions. The dashed line indicates the time when the corresponding modulator glucose, Oligomycin (Oligo) or 2-deoxy-glucose (2-DG) was added.

Data information: Of note, except for the results within the same subfigure (e.g., j), the values are not necessarily fully comparable due to the measurements at different days and not-fully-identical cell numbers between unstimulated and stimulated conditions from young and 45-wk-old mice. Results represent two independent experiments. Data are mean ± s.d. All the CD8 Tn cells were first pooled from 4-5 mice of the same group before being stimulated or not. The error bar (s.d.) here essentially refers to technical replicates. The P-values are determined by a two-tailed Student's t-test. ns or unlabeled, not significant, *P<=0.05, **P<=0.01 and ***P<=0.001.

Expanded View Figure legends:
HP1 maintains protein stability of H3K9 methyltransferases and demethylases (2022)
Figure 1

Figure 1. HP1 interaction-defective Suv39h1 and Setdb1 proteins are degraded via the proteasome pathway.

A) Schematic illustration of Suv39h1 protein. ΔN-Suv39h1 lacks the 41 N-terminal amino acids but contains a chromodomain.

B) Co-immunoprecipitation analysis to elucidate the interaction between Suv39h1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Suv39h1 or 3xFlag-ΔN-Suv39h1 were subjected to immunoprecipitation with the anti-Flag tag antibody followed by immunoblotting with the indicated antibodies. IgG represents the heavy or light chain oFigG.

C) Protein decay analysis of HP1 interaction-defective Suv39h1 mutant. mESC lines stably expressing 3xFlag-Suv39h1 or 3xFlag-ΔN-Suv39h1 were treated with CHX and MG132 for the indicated times. p53 was used as the positive control for the protein decay analysis. Stacked bar plots show the summarized protein amounts of Suv39h1 (bottom). Data represented as mean ± SD (n = 4, biological replicates). **p < 0.01 (Tukey's honestly significant difference test).

D) Schematic illustration of Setdb1 protein with putative HP1-binding motifs. Setdb1-5A and Setdb1-3A carry substitutions of amino acids from 344PMVLL348 to AMALA and from 617PLLVPL622 to ALLAPA, respectively.

E, F) Co-immunoprecipitation analysis to elucidate the interaction between Setdb1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Setdb1 or 3xFlag-Setdb1 mutants were subjected to immunoprecipitation with the anti-Flag tag antibody (E) or the anti-HP1β antibody (F). The immunocomplex was analyzed by immunoblotting with the indicated antibodies.

G) Protein decay analysis of HP1 interaction-defective Setdb1 mutant. mESC lines stably expressing 3xFlag-Setdb1 or 3xFlag-Setdb1-3A were treated with CHX and MG132 for the indicated times. Stacked bar plots show the summarized protein amounts of Setdb1 (right). Data represented as mean ± SD (n = 4, biological replicates). **p < 0.01 (Tukey's honestly significant difference test).
HP1 maintains protein stability of H3K9 methyltransferases and demethylases (2022)
Figure 2

Figure 2. HP1 regulates protein stability of Suv39h1/2, Setdb1, and G9a/GLP complex.

A) Strategies to establish HP1 conditional triple-KO mESC lines named HP1-cTKO. After the disruption of HP1β/γ by CRISPR/Cas, an expression construct of loxP-flanked Myc-hHP1β was introduced into an mESC line, TT2. Next, we disrupted the HP1α allele by CRISPR/Cas and subsequently introduced an expression construct for mER and Cre fusion protein (MerCreMer). When HP1-cTKO cells are cultured in the presence of 4OHT, Myc-hHP1β transgene can be removed by Cre-loxP recombination, thereby generating mESCs that lack HP1 completely.

B) Protein expression levels of H3K9 MTs and histone modification levels in HP1-cTKO cells were monitored by immunoblotting. The intensities were quantified using ImageJ software and summarized on the right. The thin gray line and bold colored line show individual data and means of the experimental replicates (n ≥ 3, biological replicates), respectively.

C) Comparison of H3K9me3 levels between HP1-deficient cells and Suv39h/Setdb1-deficient cells. The establishment of #33-6-SuvDKO is described in Figure S2. H3K9me levels were quantified using ImageJ software and are summarized at the bottom; the horizontal line represents the mean intensity of each cell. More than 75 nuclei were counted for each staining. **p < 0.01 (Tukey's honestly significant difference test).

D) Electron microscopy images of HP1-cTKO cells in the absence of 4OHT (left) and of cells treated with 4OHT for 4 days (right). Boxes represent enlarged images.
HP1 maintains protein stability of H3K9 methyltransferases and demethylases (2022)
Figure 4

Figure 4. HP1 ensures protein stability of H3K9 MTs by confining them to chromatin.

A) Schematic illustration of HP1 mutants used in this study. V23M and I161E mutants lose the ability to bind H3K9me3 and to form dimers, respectively.

B, C) Elucidation of protein-protein interaction between HP1 and H3K9 MTs by co-immunoprecipitation analysis. Whole-cell extracts of HEK293T cells co-expressing HA-HP1β mutants and epitope-tagged proteins of interest were subjected to immunoprecipitation with the anti-HA tag antibody. The immunocomplex was analyzed by immunoblotting with the indicated antibodies. The intensities were quantified using ImageJ software, normalized, and shown on the top.

D) Strategy to establish mESCs solely expressing mutated HP1 (left). We introduced expression constructs for 3xFlag-hHP1β (wild-type or mutants) into HP1-cTKO cells and then deleted the formerly introduced wild-type Myc-hHP1β transgene by 4OHT treatment.

E) Evaluation of HP1β mutants for maintaining H3K9me2/3 and ensuring protein stability of H3K9 MTs. H3K9me2/3 levels and protein levels of H3K9 MTs in HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).
HP1 maintains protein stability of H3K9 methyltransferases and demethylases (2022)
Figure 5

Figure 5. HP1 stabilizes H3K9 DMs, Jmjd1a and Jmjd1b, at the protein level.

A) Elucidation of protein-protein interaction between HP1 and Jmjd1a/b by co-immunoprecipitation analysis. Whole-cell extracts of wild-type mESCs and HP1β-deficient mESCs were subjected to immunoprecipitation with the anti-HP1β antibody. The immunocomplex was then analyzed by immunoblotting with the indicated antibodies.

B) Protein expression levels of Jmjd1a/b in HP1-cTKO cells were monitored by immunoblotting. The intensities were quantified using ImageJ software and summarized on the right. The thin gray line and bold colored line show individual data and the mean of experimental replicates, respectively.

C) Evaluation of HP1β mutants for ensuring protein stability of Jmjd1a/b. Protein levels of Jmjd1a/b in HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).
HP1 maintains protein stability of H3K9 methyltransferases and demethylases (2022)
Figure 6

Figure 6. H3K9me2/3 is required for maintaining protein stability of Jmjd1a/b

A) A model of HP1-mediated protein stability regulation of Jmjd1a/b. In wild-type cells, HP1 tethers Jmjd1a/b to chromatin through HP1-H3K9me2/3 interaction. We speculated that HP1 might be released from chromatin in Suv39h/Setdb1/G9a-deficient cells since H3K9me2/3 might be absent in these cells. In this situation, HP1 might no longer ensure protein stability of Jmjd1a/b.

B) Strategy to establish Suv39h/Setdb1/G9a-deficient mESCs. By disrupting G9a alleles in the #33-6-SuvDKO line by CRISPR/Cas, we established an mESC line, 3S1G, which carries the conditional knockout alleles for Setdb1 and conventionally knocked out alleles for Suv39h and G9a.

C) Impact of Suv39h/Setdb1/G9a depletion on HP1-mediated protein stability regulation of Jmjd1a/b. Jmjd1a/b were destabilized in mESCs lacking Suv39h/Setdb1/G9a, even though HP1 was present.

D) Functional evaluation of H3K9me2/3 for confining HP1 to chromatin. Subcellular localizations of HP1α, β and γ in TT2 and mESCs lacking Suv39h/Setdb1/G9a were compared. α-Tubulin, Ezh2, and histone H3 were used as representative markers for each fraction.

Expanded View Figure Legends
HOTAIR lncRNA promotes epithelial-mesenchymal transition by redistributing LSD1 at regulatory chromatin regions (2021)
Figure 1

Figure 1 - HOTAIR expression in epithelial Epi cells weakens tight junctions and promotes cell migration in LSD1-dependent manner.

A Stable Epi cell lines ectopically expressing CTR (GFP), full-length HOTAIR (HOT), HOTAIR lacking PRC2- or LSD1-interacting domains, HOTΔP and HOTΔL, respectively.

B-D Quantification of the wound area invaded in 24 hours (grey dots) for Epi cells expressing CTR, HOT, HOTΔP or HOTΔP (B), or Mes cells transfected with ASO CTR (control), ASO1 and ASO2 targeting HOTAIR (C), or Epi cells overexpressing CTR or HOT alone or together with LSD1 (LSD1-OE) (D). Bars correspond to Mean values and Confidence Interval (CI) of 95%. *** P-value < 10-4, ** P-value < 0.03 (Student t-test, N=48 for (B), N=6 for (C), N=30 for (D)).

E ZO-1 and GAPDH protein levels assessed in Epi cells by Western blot of whole protein extracts.

F Quantification of ZO-1 levels (grey dots) in four independent Western blot experiments by ImageJ. * P-value < 0.05 (Student's t-test), bars correspond to Mean ± Standard Deviation (N=4).

G ZO-1 quantification of IF images performed using the Fiji software and bar-plotted as normalized integrated densities per cell (grey dots) with bars corresponding to Mean ± Standard Error of the Mean (SEM) for at least 11 high-field units of at least 100 cells; * P-value < 0.05 (Student's t-test, N=11).