EMBO/soda-nel huggingface dataset:
MECP2 mutations affect ciliogenesis: a novel perspective for Rett syndrome and related disorders (2020)
Figure 3

Figure 3. Mecp2 deletion impairs Shh signaling pathway.

(A) Schematic representation of the Shh pathway in the cilium.

(B) MECP2-silenced or control hTERT-RPE-1 cells were starved for 48 h before treatment with 200 nM and 500 nM SAG for 24 h. Cells were stained with anti-Smo and anti-acetylated α-tubulin antibodies. The percentages of Smo-positive cilia were calculated. The graph shows the average of three independent experiments (mean ± S.E., *** p<0.001, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA demonstrated a significant silencing effect (F (1, 12) = 160; p<0.0001), a significant SAG treatment (F (2, 12) = 259.4; p<0.0001) and a significant interaction (F (2, 12) = 36.85; p<0.0001).

(C) WT or Mecp2 null MEF cells were treated with SAG (200 and 500 nM) for 24 h and the percentages of Smo-positive cilia were calculated. The graph shows the average of three independent experiments (mean ± S.E., *p<0.05, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA indicated a significant genotype effect (F (1, 12) = 11.22; p<0.01) and a significant SAG treatment (F (2, 12) = 11.7; p<0.0001).

(D) Gli1 protein levels were measured by Western blot in untreated (UT) and 100 nM SAG-treated WT and Mecp2 null MEF cells (n=5/6). Representative bands of Gli1, Mecp2 and α-tubulin are depicted above the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to the untreated WT samples (**p<0.01, ***p<0.001, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA reported a significant interaction (F (1, 18) = 6.652; p<0.05), a significant genotype effect (F (1,18) = 15.95; p<0.001) and a significant treatment (F (1, 18) = 20.15; p<0.001).

(E) Gli1 protein levels were measured by Western blot in WT and Mecp2 null neurons, following SAG treatment (200 and 400 nM). Representative bands of Gli1, Mecp2 and α-tubulin are depicted above the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to the untreated WT samples (*p<0.05, ***p<0.001, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA indicated a significant interaction (F (2, 20) = 3.788; p<0.05), a significant genotype effect (F (1, 20) = 4.362; p<0.05) and a significant treatment (F (2, 20) = 13.5; p<0.001).

(F, G) Gli1 mRNA expression levels upon SAG exposure in MEF cells (n=7 WT and n=9 null samples) (F; SAG 100 nM) and cortical neurons (n= 15 WT and n=12 null samples) (G; SAG 400 nM). The graphs show the mean ± S.E. of the fold change derived from at least three independent experiments (mean ± S.E., **p<0.01, by two-way ANOVA, followed by Bonferroni post hoc test).
MECP2 mutations affect ciliogenesis: a novel perspective for Rett syndrome and related disorders (2020)
Figure 4

Figure 4. Primary cilia defects are present in the cortex and cerebellum of Mecp2 mutant brains.

Primary cilium length was measured in the cortex of Mecp2 null mice and cerebellum of Mecp2 null and heterozygous mice.

(A) Representative immunostaining of Arl13b (red) and γ-tubulin (green) in the P14 mouse cortical layers. Nuclei were stained with DAPI (blue). Scale bar 10 µm.

(B, C) Average cilium length was measured in the cortex (mean ± S.E., n=4 WT and n=3 Mecp2 null) (B; *p<0.05, by Mann-Whitney test) and for each cortical layer (C; ***p<0.001, **p<0.01, *p<0.05, by two-way ANOVA followed by Bonferroni post hoc test). In C, cilium length is analysed in separate cortical layers while in B, the values indicate the average of cilium lengths for each layer.

(D) Representative immunostaining of Arl13b (green) in the internal (IGL) and external (EGL) granular layers of Mecp2 null (n=3) and heterozygous (het) cerebella (n=3) at P7, and the corresponding WT (n=3 males and n=3 females). Nuclei were stained with DAPI (blue). Scale bar 5 µm.

(E, F) Histograms show the mean ± S.E. of cilium length (n=3 males WT, n=3 females WT, n=3 Mecp2 null and Mecp2 het; **p<0.01, ***p<0.001, by Mann-Whitney test) in Mecp2 null (E) and heterozygous (F) mice, compared to the corresponding WT.

(G) Gli1 protein levels were measured by Western blot in WT and Mecp2 null cerebellum (n=7 WT and n=6 Mecp2 null). Representative bands of Gli1 and α-tubulin are depicted beside the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to WT animals (*p<0.05, by Student's t test).

(H) The mRNA expression levels of Gli1 and its downstream genes (Cyclin D1 and D2) were analyzed in WT and Mecp2 null cerebella (n=8 WT and n=7 Mecp2 null). The graph shows the fold change of transcripts compared to WT (mean ± S.E., *p<0.05, by Student's t test).
MECP2 mutations affect ciliogenesis: a novel perspective for Rett syndrome and related disorders (2020)
Figure 5

Figure 5. Tubacin rescues ciliogenesis in MeCP2 deficient cells

(A) WT and Mecp2 null MEF cells were starved for 48 h and then treated with the HDAC6 inhibitor tubacin (TUB) (1 μM) for 48 h or with DMSO (VEH) or left untreated (UT). Cells were fixed, stained for anti-acetylated α-tubulin and cilia were counted. The graph shows the percentage of ciliated cells out of three independent experiments (mean ± S.E., *p<0.05, **p<0.01, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA reported a significant genotype effect (F(1, 10) = 17.94; p<0.01), a significant tubacin treatment (F(2, 10) = 4.425; p<0.05) and a significant interaction (F(2, 10) = 7.266; p<0.05).

(B) Primary cortical neurons were treated at DIV5 with DMSO (VEH) or tubacin (TUB, 1 μM) for 48 h, or left untreated (UT), fixed at DIV7 and stained with anti-AC3 antibody. The graph shows the percentage of ciliated cells obtained from three independent experiments counting n=300 UT cells, n=216 VEH treated cells, n=267 TUB treated cells for each genotype (mean ± S.E., *p<0.05, **p<0.01, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA reported a significant genotype effect (F (1, 15) = 23.42; p<0.001), a significant treatment effect (F (2, 15) = 11.25; p<0.01) and a significant interaction (F (2, 15) = 4.853; p<0.05).

(C) MECP2 silenced or control hTERT-RPE-1 cells were starved for 48 h, treated with tubacin (TUB, 1 µM) for 24 h and then exposed to 200 nM SAG for 24 h in the presence of tubacin. Cells were stained with anti-Smo and anti-acetylated α-tubulin antibodies and the percentages of Smo-positive cilia were calculated. The graph shows the averages of three independent experiments (mean ± S.E., **p<0.01, by three-way ANOVA followed by Tukey's post hoc test). Statistical analysis reported a significant difference between genotype (F (1, 2) = 21.47; p<0.001), a significant SAG effect (F (2, 2) = 154.5; p<0.0001) and a significant tubacin effect (F (1, 2) = 25.18; p<0.001). In details, a significant interaction between genotype and SAG treatment (F (2, 2) = 5.413; p<0.05) and between SAG and tubacin treatment (F (2, 2) = 6.939; p<0.01) were reported, demonstrating respectively a difference between CTRL and MeCP2 depleted cells in response to SAG and a different SAG effect when cells were treated with tubacin.

(D) Representative immunostaining of Smo (green) and acetylated α-tubulin (red) in control (CTRL) and silenced (siMeCP2) hTERT-RPE-1 cells, after stimulation with 200 nM SAG in the presence or absence of the HDAC6 inhibitor tubacin (TUB). Scale bar 50 µm, and 20 µm in the enlarged image.
MECP2 mutations affect ciliogenesis: a novel perspective for Rett syndrome and related disorders (2020)
Figure 6

Figure 6. HDAC6 inhibition restores neuronal defects associated with the lack of Mecp2

(A) Representative images of MAP2 positive WT and Mecp2 null neurons (DIV7). At DIV5 neurons were treated with tubacin (TUB, 1µM for 48 h), Aurora A inhibitor TC-S 7010 (TC-S, 7 nM for 48 h) or left untreated and morphologically analyzed by Sholl analysis plug-in. Scale bar 20 µm.

(B) The graph reports the mean ± S.E. of the number of intersections measured 85 to 145 nm from the soma calculated by Sholl analysis for WT (n=39 neurons) and Mecp2 null neurons, when left untreated (n=57 neurons) or treated with tubacin (n=53 neurons) or TC-S 7010 (n=38 neurons). Data from WT and null neurons were compared by Mann-Whitney test (**p<0.01); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05, **p<0.01).

(C) Representative images of WT and Mecp2 null neurons (DIV14) immunostained for MAP2 (white), Synapsin1/2 (green) and Shank2 (red). Mecp2 null cells were treated at DIV12 with tubacin (TUB, 1µM for 48 h) or Aurora A inhibitor TC-S 7010 (TC-S, 7 nM for 48 h). Scale bar 15 µm, and 5 µm in the enlarged image.

(D, E) The graphs represent the mean ± S.E. of the Synapsin1/2 and the Shank2 puncta density in WT (n=67 neurons) and Mecp2 null neurons, when untreated (n=51 neurons) or treated with tubacin (n=59 neurons) or TC-S 7010 (n=55 neurons). Neurons derived from at least 6 different biological replicates. Data from WT and null neurons were compared by Mann-Whitney test (**p<0.01, ***p<0.001); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05, ***p<0.00­­1).

(F) The graph depicts the mean ± S.E. of the number of co-localized pre- and post-synaptic puncta (n=20 neurons deriving from 3 different biological samples). Data from WT and null neurons were compared by Mann-Whitney test (***p<0.001); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05).
MECP2 mutations affect ciliogenesis: a novel perspective for Rett syndrome and related disorders (2020)
Figure 7

Figure 7. RTT patients' fibroblasts show defective ciliogenesis which is rescued by tubacin treatment

(A) Schematic representation of the MeCP2 protein, with the methyl binding domain (MBD) highlighted in red and the transcriptional repression domain (TRD) in blue. Patients' mutations investigated in this study are indicated.

(B) Representative immunostaining of primary cilia and MeCP2 in cultured human fibroblasts from healthy controls (CTRL, n=3) and RTT patients. Fibroblasts were starved for 24 h and then stained for acetylated α-tubulin (green), MeCP2 (red) and DAPI (blue). Scale bar 50 µm.

(C-D) Histograms indicate (mean ± S.E.) the percentage of MeCP2 positive cells (C) and the percentage of ciliated cells in control and RTT fibroblasts (D) (*p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA, followed by Dunnett's post hoc test).

(E) The effect of tubacin (TUB) treatment (250 nM, 24 h) on primary cilium length of controls (CTRL) and RTT fibroblasts is reported. The graph indicates the mean ± S.E of the cilium length analyzed in untreated (n=130 CTRL; n=73 705delG; n=57 Q244X; n=68 R255X) and treated cells (n=25 CTRL; n=26 705delG; n=26 Q244X; n=49 R255X) (***p<0.001 vs untreated CTRL, §p<0.05 vs corresponding mutated untreated fibroblasts by two-way ANOVA, followed by Bonferroni post hoc test). Statistical analysis reported a significant treatment effect (F (1,429) = 26.56; p<0.0001) and a significant genotype (F(3,429) = 42.85; p<0.001).

Expended View Figure Legends
A NANOG-pERK reciprocal regulatory circuit regulates Nanog autoregulation and ERK signaling dynamics (2022)
Figure 1

Figure 1. Residual MEK1/2 activity in the ground state prevents complete derepression of Nanog.

(A) (left) FACS profiles of TβC44Cre6 cultured in indicated conditions for 3 passages. TβC44Cre6 is a Nanog null cell line, where β−geo cassette is inserted into one allele and GFP into another allele of the Nanog gene. The cells were cultured in Serum+LIF (SL) in presence of the 1 μM MEK1/2 inhibitor -PD0325901 (SLPD) or 3 μM GSK3β inhibitor -CHIR99021 (SLCHIR) or in serum-free media - N2B27 with PD0325901, CHIR99021, and LIF (2iL). The dotted line shows the FACS profile of unstained E14Tg2a cells used as negative control (-ve c). (right) Nanog:GFP population median of TβC44Cre6 (n=3).

(B) (left) FACS profile of NANOG-iRFP protein in NiRFP2A cells cultured in indicated conditions for 3 passages. The dotted line represents the FACS profile of unstained E14Tg2a cells used as negative control (-ve c). (right) NANOG-iRFP population median of NiRFP2A (n=4).

(C) RT-qPCR of pluripotency factors in indicated conditions (SL2i = SL+PD0325901+CHIR99021) (n=3).

(D) RT-qPCR analysis of pre- mRNA of Nanog and Oct4 (n=3).

(E) (left) Western blot of NANOG, OCT4, and SOX2. (right) Relative NANOG levels as estimated by densitometry (n=8). NANOG was nearly 7-fold more in PD, which is twice that of 2iL/SL2i.

(F) Western blot of pERK and ERK at 0, 1, 4, 8, 12, 16, and 24 hrs after media change in indicated treatments (n=3).

(G) Western blot of pERK and ERK in SLPD, SLCHIR, 2iL, and SL2i after 8, 12, 16, and 24 hrs of culture relative to SL, where the cells in SL were harvested 24 hrs after the media change (n>3).

(H) (left) Western blot of pERK and ERK in 1μM PD and increasing concentrations of CHIR in serum-free N2B27 media. (right) Relative pERK levels (n=3).

(I) (left) Western blot of pERK and ERK in 3μM CHIR and increasing concentrations of PD in serum-free N2B27 media. (right) Relative pERK levels (n=6).

Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in A-E, H, and I. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).
A NANOG-pERK reciprocal regulatory circuit regulates Nanog autoregulation and ERK signaling dynamics (2022)
Figure 2

Figure 2. FGF autocrine signaling pathway components are essential for Nanog autoregulation.

(A) (left) Schematic depiction of Tamoxifen (OHT) inducible TNERT cell line. TNERT and TDiN (Fig EV2A and B) are similar to NERTc3 and 44iN (Navarro et al., 2012), where the NANOG function is reinstated by 4-Hydroxytamoxifen (OHT) or Doxycycline respectively, and endogenous Nanog gene activity is reported by GFP. (Middle) FACS profile of TNERT treated with OHT (red) or no OHT (blue). The dotted line represents the FACS profile of unstained E14Tg2a cells used as negative control (-ve c). (Right) Nanog:GFP population median of TNERT (n=3).

(B) FACS profiles of TNERT, and TDiN treated with 2μM SU5402, with OHT/Doxycycline (red) or no OHT/Doxycycline (blue) (n=3). The dotted line represents the FACS profile of unstained E14Tg2a cells used as negative control (-ve c).

(C) Heat map representing transcript levels (FPKM) of Fgfbp1 from 8-cell to blastocyst stage analyzed from the single-cell sequencing data.

(D) Schematic depiction of TNERTFgfr1-/-, TNERTFgfr2-/, TNERTFgf4-/-, and TNERTFgfbp1-/- cell lines, which are derivatives of TNERT where Fgfr1, Fgfr2, Fgf4, and Fgfbp1 are knocked out respectively.

(E) FACS profiles of TNERT, TNERTFgfr1-/-, TNERTFgfr2-/-, TNERTFgf4-/-, and TNERTFgfbp1-/- cells, cultured in SL treated with OHT (red) or no OHT (blue) (n=3). The dotted line represents the E14Tg2a FACS profile used as negative control (-ve c).

(F) RT-qPCR of Nanog:GFP transcript from Nanog locus with primers complementary to 5'UTR of Nanog transcript.

(G) (left) Western blot of pERK in TNERT, TNERTFgfr1-/-, TNERTFgfr2-/-, TNERTFgf4-/-, and TNERTFgfbp1-/- with or no OHT. (Right) relative pERK levels estimated by densitometry (n=4).

(H) (top) FACS profiles of TNERT, TNERTFgfr1-/-, TNERTFgfr2-/-, TNERTFgf4-/-, TNERTFgfbp1-/- cultured in SL and TNERT+SLPD (n=3). The dotted line represents the FACS profile of unstained E14Tg2a cells used as negative control (-ve c). (Bottom) Nanog:GFP population median of indicated cell lines.

Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in A, F, G, and H. *P < 0.05, **P < 0.01, ***P < 0.001, and ns = not significant (paired two-tailed student's t-test).
A NANOG-pERK reciprocal regulatory circuit regulates Nanog autoregulation and ERK signaling dynamics (2022)
Figure 3

Figure 3. NANOG triggers autoregulation by inducing the expression of FGFR2, FGF4, and FGFBP1.

(A) RT-qPCR showing relative transcript levels after 0, 0.5, 1, 2, 4, 8, 12 and 18 hrs OHT treatment in TNERT (n=3). Esrrb, a known direct target of NANOG was used as the positive control.

(B) RT-qPCR of relative levels of pre- mRNA at the above indicated time points after OHT treatment in TNERT (n=3).

(C) ChIP analysis of NANOG on Fgf4, Fgfbp1, Fgfr1, Fgfr2, Nanog (validated target) and OFR (non-genic region (negative control)) loci in E14Tg2a cells cultured in SL or SLPD for 48 hrs (n=4).

(D) ChIP analysis of NANOG on promoters of above-indicated loci in EDiN cells cultured in Doxycycline (red) or no Doxycycline (blue) for 48 hrs (n=3).

(E) Western blot of FGFR1, FGFR2, and pERK in TNERT at one-hour intervals at indicated time points after OHT treatment (n>3).

(F) FACS analysis of FGFR2 on the cell surface of TNERT treated with (red) or no OHT (blue) (n=3). The dotted line represents the FACS profile of E14Tg2a cells stained with control IgG and secondary antibody used as negative control (-ve c).

(G-H) ELISA-based relative quantification of FGF4 (G) and FGFBP1 (H) in conditioned media from TNERT treated with or no OHT (n=3).

(I) Schematic of conditioned media experiment.

(J) FACS analysis of Tβc44Cre6 cell line in conditioned media collected from TNERT treated with OHT after 0, 18, 24, and 48 hrs. The dotted line represents the FACS profile of unstained E14Tg2a cells used as negative control (-ve c).

(K) (left) FACS analysis of TNERTZfp281-/- cells treated with (red) or with no OHT (blue) treatment. The dotted line represents the FACS profile of unstained E14Tg2a cells used as negative control (-ve c). (right) Nanog:GFP population median of TNERTZfp281-/- (n=3).

(L) FACS analysis of Tβc44Cre6 cell line in different conditioned media. Media from TNERT+OHT 0 hrs, TNERTFGF4-/-, Tβc44Cre6 48 hrs, (shades of blue color) fail to repress Nanog:GFP. Media from E14Tg2a- FGF4-OE (overexpression) 48 hrs, TNERT+OHT 48 hrs and 50ng/ml FGF4 (shades of maroon color) repress Nanog:GFP. The dotted line represents the FACS profile of unstained E14Tg2a cells used as negative control (-ve c).

Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in A-D, G, H, and K. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).
A NANOG-pERK reciprocal regulatory circuit regulates Nanog autoregulation and ERK signaling dynamics (2022)
Figure 5

Figure 5. ERK interacts and recruits NONO to repress Nanog transcription.

(A) Schematic of TNERTNono-/-cell line; a derivative of TNERT in which Nono is knocked-out.

(B) FACS profile of TNERTNono-/- treated with or no OHT (n=3). The dotted line represents the FACS profile of unstained E14Tg2a cells used as negative control (-ve c).

(C) Western blot of pERK and ERK in TNERT and TNERTNono-/- cells treated with or no OHT (n=3).

(D) Immunoprecipitation analysis showing interactions between ERK and NONO in the presence or absence of Nanog induction by Doxycycline in TDiN cells.

(E) (left) Western blot of pERK and ERK in E14Tg2a cells treated with PD or FGF4. (Middle and right) Relative levels of pERK and NONO in E14Tg2a cells treated with PD or FGF4 (n>4).

(F) Schematic representation of Nanog locus comprising the -6.0 to +2kb region. The vertical bars represent the relative positions of primer pairs used for ChiP-qPCR analysis. S1-S6 are located upstream of the TSS, the S7 primer pair is located around TSS, and S8 and S9 are located downstream in the first intron.

(G-K) ChIP-qPCR analysis of pERK (G), NONO (H), Pol2 (I), H3K4me3 (J) and H3K27me3 (K) on Nanog 5' region in E14Tg2a cells (blue), treated with FGF4 (green) and with PD (pink) (n=3).

(L) Cycloheximide chase assay of NANOG in SL, SLPD, and SLFGF4 in E14Tg2a cells.

(M) A cartoon illustrating the repression of Nanog by FGF signaling and derepression of Nanog in absence of FGF signaling. The FGF4 activates the FGF signaling cascade, resulting in the phosphorylation of ERK. pERK interacts and recruits NONO to the Nanog promoter and represses transcription of Nanog. pERK also affects the stability of the NANOG. In absence of FGF4, the pERK levels decrease resulting in enhanced stability of NANOG and transcription of Nanog locus by NANOG and other pluripotency factors resulting in derepression of Nanog locus.

Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in E and G-K. *P < 0.05, **P < 0.01, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).
A NANOG-pERK reciprocal regulatory circuit regulates Nanog autoregulation and ERK signaling dynamics (2022)
Figure 6

Figure 6. NANOG regulates ERK signaling dynamics and heterogeneity.

(A) Immunofluorescence of pERK (red) and NANOG (green) in the indicated ESCs. Scale bars 25 μM.

(B)The normalized fluorescence intensity of pERK was plotted against the normalized fluorescent intensity of NANOG.

(C) (left) Immunofluorescence of pERK and NANOG in 10% NANOG-high NiRFP2A cells at indicated time cultured in the same media collected from cells before FACS. (Right) The normalized fluorescence intensity of pERK was plotted against the normalized fluorescent intensity of NANOG at different time points. Scale bars 25 μM.

(D) (left) Western blot analysis of pERK, and NANOG in 10% NANOG-high NiRFP2A cells at indicated time cultured in the same media collected from cells before FAC sorting. (Right) Expression levels of NANOG and pERK in 10% NANOG-high NiRFP2A cells relative to 0 hrs during the time course of culture. The NANOG and pERK expression oscillate between high and low levels in the cells during culture.

(E) A working model of the NANOG-pERK reciprocal regulatory loop operating in ESCs. NANOG induces Fgfbp1 and Fgfr2 to enhance ERK signaling in Nanog-high cells. pERK along with NONO occupy the Nanog promoter to repress its transcription. The transcription repression results in reduced NANOG, which prevents induction of Fgfbp1and Fgfr2. The is reduces ERK activity relieving the repression on the Nanog promoter.

(F) A schematic depicting the progression of cells through different expression states of NANOG and pERK expression in the ESC population. The cells expressing high-NANOG induce Fgfbp1 and Fgfr2 to activate pERK by autocrine signaling to give rise to a high-NANOG:high-pERK state. The repression of Nanog transcription by pERK leads the cells through different intermediate levels of expression of NANOG and pERK resulting in a low-NANOG:low-pERK state. The low pERK permits transcription of Nanog and gradual induction of Fgfbp1 and Fgfr2 by NANOG culminating in a high-NANOG:high-pERK state. The cells will cycle through different levels of pERK and NANOG levels generating a heterogeneous population with a strong correlation between pERK and NANOG in an ESC cells culture.

Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in D. *P < 0.05, **P < 0.01 and ns = not significant (paired two-tailed student's t-test).

Expanded view figure legends