EMBO/SourceData huggingface dataset:
Figure 2A. IHC in DRG sections for pHDAC3 revealed that HDAC3 phosphorylation is decreased, primarily in the nucleus 24h after sciatic nerve axotomy (SNA) but not spinal dorsal column axotomy (DCA). Scale bars, 100 μm.B-C. Bar graphs show mean fold change levels (B) or % of pHDAC3+ cells (C) (pHDAC3+ threshold was set at the minimum intensity level of sham cells) ± s.e.m. DAPI counter staining was used to label nuclei N= 9-10 biological replicates. (***p<0.005) indicate significant difference versus sham or laminectomy (lam) (ANOVA followed by Bonferroni test).D. Western blot revealed that HDAC3 phosphorylation is decreased in DRG nuclear extracts but not in the cytoplasm 24h after SNA. E. Bar graphs show intensity measurement of nuclear vs cytoplasm immunoblot. Data is expressed as mean ± s.e.m. N= 3 biological replicates. (***p<0.005) indicate significant difference versus sham (Student´s t-test). F. HDAC3 activity (HDAC3 IP and activity assay) is strongly reduced after SNA but not DCA. Data is expressed as mean ± s.e.m. N= 3 biological replicates. (*p<0.05) indicate significant difference versus sham (ANOVA followed by Bonferroni test).
Figure 3PP4 activities are induced after SNA but not DCA, and are blocked by the calcium chelator EGTA. Data is expressed as mean ± s.e.m. N= 3-4 biological replicates. (***p<0.001) indicate significant difference versus sham or lam (Two-way ANOVA followed by Bonferroni test).PP2 activities are induced after SNA but not DCA, and are blocked by the calcium chelator EGTA. Data is expressed as mean ± s.e.m. N= 3-4 biological replicates. (***p<0.001) indicate significant difference versus sham or lam (Two-way ANOVA followed by Bonferroni test).C. SNA but not DCA significantly enhances calcium levels in DRG. Data is expressed as mean ± s.e.m. N= 4 biological replicates. (*p<0.05) indicate significant difference versus sham or lam (ANOVA followed by Bonferroni test).D-F. Immunoblotting of DRG homogenates shows that nerve injury-dependent nuclear HDAC3 dephosphorylation is reduced by the calcium scavenger EGTA upon in vivo delivery on the injured sciatic nerve (Axo=Axotomy). Cytoplasmic pERK levels are used as control of axotomy-dependent signalling. (E-F). Data is expressed as mean fold change of band intensity levels ± s.e.m. N= 3 biological replicates. (**p<0.01, ***p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test).Significant Pearson correlations between calcium levels (Fluo4AM) and H3K9ac, r2: 0.7802 (G) in cultured DRG neurons after KCl. The Pearson correlations were disrupted by administration of Fostriecin (calcium and H3K9ac, r2: 0.0007 Data is expressed as single cell fluorescence levels. N=50 cells per condition from 4 biological replicates.Significant Pearson correlations between calcium levels (Fluo4AM) ; or pHDAC3, r2: 0.6366 (H) in cultured DRG neurons after KCl. The Pearson correlations were disrupted by administration of Fostriecin calcium and pHDAC3, r2: 0.3025). Data is expressed as single cell fluorescence levels. N=50 cells per condition from 4 biological replicates.J-L. Immunofluorescence (J) in DRG tissue sections 24h after injury in vivo shows that the PP2a/PP4 inhibitor Fostriecin (F) inhibits the injury-induced decrease in pHDAC3 (K) and the increase in H3K9ac (L). Scale bar, 10 μm. (K-L). Data is expressed as mean fluorescence intensity in arbitrary units ± s.e.m. N= 3 (L) or 4 (K) biological replicates (*p<0.05; **p<0.01) indicate significant difference versus sham (ANOVA followed by Bonferroni test).M-N. Pharmacological inhibition of PP2a/PP4 impairs conditioning-induced DRG neurite outgrowth ex vivo (12h in culture). M. DRG neurite outgrowth (TUJ1 positive cells) 24h after sham or sciatic nerve axotomy (SNA) prior to i.t. administration of vehicle (V) or Fostriecin (F) (240µM). Scale bar, 100µm. (N) Data is expressed as average neurite length per neuron ± s.e.m. N= 3 biological replicates per condition, technical triplicate. *** p<0.005 indicate significant difference versus sham/vehicle (One way ANOVA followed by Bonferroni).
Figure 5A. The graph shows the number of differentially expressed (DE) genes 24h following SNA or DCA compared to their respective controls.B. RNAseq read counts show differential occupancy of H3K9ac on specific gene regions, including an increased occupancy close to the transcriptional start site (TSS) after SNA only, when compared to corresponding controls.C. Bar graph shows the number of genes that are upregulated or downregulated following SNA or DCA (RNAseq) with an increased or decreased H3K9ac occupancy (ChIPseq), respectively. D. Odds ratio analysis of the enrichment/depletion of genes with differential H3K9ac occupancy across each upregulated and downregulated gene cluster in (C); the numbers in red represent the p value given by Fisher's exact test (ns= not significant).
Figure 7A-B. AAV-HDAC3 mutant (Y298H; dominant negative deacetylase inactive) promoted DRG regenerative growth after spinal cord injury vs AAV-V5 control (injected 5 weeks prior to SCI in the sciatic nerve bilaterally) as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, (A-B) 200 μm.C-F. Immunofluorescence for H3K9ac. AAV-HDAC3 mutant induced upregulation of H3K9ac in vivo in DRG neurons, but not in surrounding nuclei of satellite cells. Scale bar, (C-F) 50 μm.G-J. New synaptic formations (VGlut1+) from regrowing axons are shown beyond the lesion site after injection of AAV-HDAC3mut. (G) Scale bar 200 μm, (H-J) High magnification insets depict co-localization of Dextran and vGlut1 staining. Scale bar 50 μm.K-L. Quantification of dextran positive axons shows that AAV-HDAC3mut promotes DRG regenerative growth across and beyond the spinal lesion site. Data is expressed as percentage of dextran+ axons at each distance vs dextran+ axons at -700μm from the lesion margin (K) or distance from the caudal margin of the lesion to the most rostral dextran+ axon tip for each animal (L). N= 10 animals per condition, ± s.e.m. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 indicate a significant difference (ANOVA followed by Bonferroni test).
Figure 8A-B. Intrathecally administered RGFP966 (966) through osmotic minipump for 14 days promoted DRG regenerative axonal growth after spinal cord injury as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, 200 μm.C-D. Quantification of axonal regeneration shows dextran axonal labelling across and beyond the spinal lesion site. Data is expressed as ratio of dextran+ axons vs dextran+ axons at -700 μm from the lesion site ± s.e.m (C) or distance from the caudal margin of the lesion to the last dextran+ axon tip (D). N= 6-10 animals per condition. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test).E. Immunofluorescence for H3K9ac and Neurofilament 200 (NF200) shows upregulated H3K9ac in vivo in DRG neurons after 966. Scale bar, 50 µm.F-G. Data is expressed as fold change of fluorescence intensity mean levels (F) or % of H3K9ac+ cells (G) (H3K9ac+ threshold was set at the mean intensity level of vehicle treated animals, DAPI counter staining was used to label nuclei from NF200+ or NF200- cells) vs veh± s.e.m. N= 11-12 biological replicates. (***p<0.005) indicate a significant difference vs respective Veh (ANOVA followed by Bonferroni test).
Figure 1(B) Western blot analysis of Atxn1 levels in cerebellar granule neurons (CGNs) after knockdown of Zbtb7a and/or Zbtb7b (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.(C) Western blot analysis of Atxn1 levels in the cerebella of wild-type (n = 3), Zbtb7a+/- (n = 4), Zbtb7b+/- (n = 3), Zbtb7a+/-; Zbtb7b+/- (n = 4) mice. Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.(D) Western blot analysis of Atxn1 levels in CGNs after overexpressing Zbtb7a and/or Zbtb7b (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.(E) Genetic interaction of wild-type human ATXN1(30Q) with either ZBTB7A or ZBTB7B expressed in the Drosophila eyes. Co-expression of ATXN1(30Q) with ZBTB7A or ZBTB7B severely disrupted the external Drosophila eye structure and increased ATXN1 levels. Scale bar: 100 μm
Figure 2(C) Western blot analysis of ATXN1 levels after overexpression of ZBTB7B wild-type (WT), zinc-finger domain mutant (ZFmut) or BTB domain mutants (BTBmut1 and BTBmut2) (n = 3). Data information: Mean ± SD, ***P<0.001 ****P<0.0001, one-way ANOVA.(D) qRT-PCR results of Atxn1 mRNA levels in the cerebella of wild-type (n = 3), Zbtb7b+/- (n = 4), and Zbtb7b-/- mice (n = 4). Data information: Mean ± SD, ***P<0.001 ****P<0.0001, one-way ANOVA.(E) MA plot (M: log ratio; A: mean average) of the differentially expressed genes (DEG) analysis from RNA-seq samples of the cells overexpressing either WT or ZFmut ZBTB7B. Four filters and the number of genes kept after applying each filter are described. Two genes that passed the 4th filter are labeled.(F) Expression heatmap of the 16 genes that passed the 3rd filter. Color scale: row z-score.(G) qRT-PCR results of RSK3 mRNA levels across different CRISPR knockout cell lines (n = 2). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(H) Epistatic relationship between ZBTB7B and RSK3 in terms of ATXN1 regulation. ATXN1 levels were measured after overexpression of ZBTB7B in the RSK3 knockout cell line (sgRSK3) and control cell lines (WT and sgControl) (n = 3). ZBTB7B+RSK3: co-overexpression of both ZBTB7B and RSK3. Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(I) qRT-PCR results of RSK3 mRNA levels after knockdown of ZBTB7B in Daoy cells (n = 2). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(J) qRT-PCR results of Rsk3 mRNA levels in the cerebella of Zbtb7b conditional knockout mice (n = 6). Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.(K) Position frequency matrix of ZBTB7B binding DNA sequence and a table of the predicted ZBTB7B binding sites on human RSK3 promoter. Start and End position numbers are described based on TSS. The seventy base pairs upstream RSK3 promotor includes top two potential binding sites and was used for luciferase assays. Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.(L) Luciferase assay result of RSK3 promoter in HEK 293T and HeLa cells transfected with either an empty or ZBTB7B expressing vector (n = 3). Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.
Figure 3(A) Western blot analysis of ATXN1 after knockdown of RSK3 in Daoy cells. Bottom left panel: qRT-PCR results of RSK3 mRNA. Bottom right panel: densitometry of the western blot image in the top panel (n = 3 except for shATXN1 where n = 1). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(B) Western blot analysis of ATXN1 after overexpression of RSK3, ZBTB7B, or eGFP in Daoy cells (n = 3). Data information: RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3. Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(C) Western blot analysis of ATXN1(82Q) after overexpression of either wild-type or inactive RSK3 in Daoy cells expressing ATXN1(82Q) (n = 3). Data information: RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3. Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(D) In vitro kinase assay of RSK3 with ATXN1. MSK1 was used as a positive control. Asterisk: auto-phosphorylated RSK3.(E) In vitro kinase assay of four different RSK members with ATXN1.(F) In vitro kinase assay of RSK3 with N-terminal (F1), middle (F2), and C-terminal fragment (F3) of ATXN1.(G) Mass spectrometry analysis of phosphorylated residues in ATXN1 F3 after in vitro kinase assay with RSK3.(H) Western blot analysis of ATXN1 after a treatment with RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.(I) Western blot analysis of total and phospho-S776 ATXN1 after a co-treatment with MSK1 (SB-747651A) and RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.
Figure 4(A) Images of Drosophila eyes expressing human ATXN1(82Q) with either knockdown or overexpression of the Drosophila RSK3 homolog Sk6II. Scale bar: 100 μm (top) and 20 μm (bottom).(B) Improvement of motor performance by the knockdown of Sk6II in Drosophila SCA1 model expressing ATXN1(82Q) in the central nervous system. Mean ± SEM, *P < 0.05, Linear mixed-effect model ANOVA, n = 10/genotype, four times of measurements.(C) A representative image of a mouse brain 4 weeks after AAV injection of shRsk3 into 12 weeks old SCA1 mice (Atxn1154Q/2Q) cerebellum. Scale bar: 2 mm.(D) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the cerebella of 16 weeks old SCA1 mice dissected 4 weeks after the stereotaxic injection of AAV9 harboring shControl (n = 3), shRsk3 (n = 3), or shAtxn1 (n = 2). Mean ± SD, *P<0.05, ***P<0.001 ****P<0.0001, one-way ANOVA.(E) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the brainstem (BS) and cerebella (CBM) of 8 weeks old Atxn1154Q/2Q (n = 6) and Atxn1154Q/2Q; Rsk3+/- mice (n = 7). Mean ± SD, *P<0.05, **P<0.01, two-tailed t-test.(F) Rotarod motor performance test result of Atxn1154Q/2Q; Rsk3+/- mice at 11-12 weeks old. Mean ± SEM, *P<0.05, two-way ANOVA, n = 35, 31, 34, 38 for WT, Rsk3+/-, Atxn1154Q/2Q, Atxn1154Q/2Q; Rsk3+/- genotype, respectively.
Figure 5(A) Fluorescent western blot analysis of Atxn1, Msk1, and Rsk3 in seven different brain regions of wild-type mice at 9 weeks old (n = 3). Each protein levels were represented relative to the protein levels in brainstem. Mean ± SD, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. Data information: BS: brainstem, CBM: cerebellum, MB: midbrain, ST: striatum, HC: hippocampus, CTX: cortex, OB: olfactory bulbs.(B) Representative BaseScope assay images of Rsk3 in the whole brain sagittal sections of wild-type (top) and Rsk3-/- mice (bottom) at 5-6 weeks old. The areas within the black squares are magnified in the right panels in each genotype. Scale bar = 2 mm in whole brain images; 50 μm in the magnified images. Data information: BS: brainstem, CBM: cerebellum(C) Representative IF images of Msk1 in the whole brain sagittal sections of wild-type (top) and Msk1-/- mice (bottom) at 6 months old. The areas within the white squares are magnified in the right panels in each genotype. Scale bar = 2 mm in whole brain images; 50 μm in the magnified images. Data information: BS: brainstem, CBM: cerebellum(D) Western blot analysis of total and active form of Rsk3 and Msk1 in the cerebella and brainstem of 12 weeks old WT and SCA1 mice (n = 4). Relative Rsk3 and Msk1 levels were represented into log2 scale. The protein levels of the two kinases are significantly different between the two brain regions. Mean ± SD, *P<0.05, **P<0.01, ****P <0.0001, two-way ANOVA. Data information: BS: brainstem, CBM: cerebellum(E) Western blot analysis of ATXN1, RSK3, pRSK3, MSK1, and pMSK1 in the postmortem cerebella, pons, and medulla oblongata of human controls and SCA1 patients (n = 3 per genotype). Total protein was used for normalization, and protein levels are represented relative to cerebellar protein levels in control group. C: Cerebellum, P: Pons, M: medulla oblongata. Values from P and M are combined and presented in BS. Mean ± SEM, *P<0.05, **P<0.01, two-way ANOVA, n = 3 (CBM) and 6 (BS). Data information: BS: brainstem, CBM: cerebellum
Figure 6(A) Western blot analysis of total and pS776 wild-type and mutant Atxn1 in the brainstem of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SD, *P<0.05, **P<0.01, ****P<0.0001, one-way ANOVA, n = 7, 6, 4, 7 following the order of genotypes in the western blot, respectively.(B) Plethysmography data measuring breathing ability (total ventilated gas volume per minute) of 34 weeks old mice with different genotypes. Mean ± SD, *P<0.05, two-tailed t-test, n = 12, 13, 15, 13, 15, 9, 15, 13 following the order of genotypes in the graph, respectively.(C) Representative immunohistochemistry images of Iba1+ reactive microglia in the coronally sectioned hypoglossal nucleus of 36 weeks old mice with different genotypes. Yellow arrows indicates Iba1+ microglia, and larger pyramidal shaped cells are motoneurons. scale bar = 50 μm.(D) Quantification of the number of Iba1+ cells shown in (C). Mean ± SD, *P<0.05, ***P<0.001, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from six sections per mouse.(E) Representative Hematoxylin and Eosin stained motoneurons in the coronally sectioned hypoglossal nucleus (white dashed line) of 36 weeks old mice with different genotypes. The motoneurons are dark purple with pyramidal shape within the nucleus. C: central canal. Scale bar = 100 μm.(F) Quantification of the number of motoneurons shown in (E). Mean ± SD, *P<0.05, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from six sections per mouse.(G) Kaplan-Meier survival graphs of SCA1 mice with heterozygous knockout of either one or both kinases. The data of Msk1+/-, Rsk3+/-, and Msk1+/-; Rsk3+/- mice overlap those of wild-type mice. *P<0.05, Gehan-Breslow-Wilcoxon test, n = 15, 15, 12, 13, 22, 11, 20, 19 following the order of genotypes in the legend on top of the figure, respectively.
Figure 7(A) Western blot analysis of total and pS776 wild-type and mutant Atxn1 in the cerebella of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SD, **P<0.01, ***P<0.001, one-way ANOVA, n = 7, 6, 4, 7 following the order of genotypes in the western blot, respectively.(B) Rotarod motor performance test result of 11-12 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SEM, *P<0.05, two-way ANOVA, n = 18, 12, 13, 11, 19, 13, 15, 13 following the order of genotypes in the legend on top of the figure, respectively.(C) Representative immunofluorescent (IF) images of cerebellar molecular layer between the cerebellum lobule V and VI across different genotypes at 36 weeks old, stained with anti-Calbindin 1 antibody. Scale bar = 100 μm.(D) Quantification of the thickness of top and bottom molecular layers shown in (C). Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from three sections per mouse.
Figure 1(E) Fold increase of Oct4-GFP+ colony number over control under forced expression of individual factors. n ≥ 5, where each dot indicates an independent experiment. Box-plots show median, 1st and 3rd quartile values. One-sample Wilcoxon test p-values are as indicated, with p values < 0.05 shown in red
Figure 2(B) Heatmap of average gene expression normalised to β-actin over an EpiSC resetting time course in 2i+LIF. Each row is coloured according to the unique minimum and maximum for that gene. The values shown are average expression of four independent experiments(C) Gene expression for Stat3, Klf2, Esrrb and Tfcp2l1 during EpiSC resetting relative to established mouse ESCs. β-actin serves as an internal control. Mean +/- SEM, n=4 independent experiments. * = p<0.05 Student"s t
Figure 3(E) Representative confocal images (top) and quantification (bottom) of Oct4-GFP reporter mean intensity. The indicated cell lines were treated with DOX for the first 2 days and imaged at Day 6. Mean +/- SD of 9 technical replicates. One representative experiment of two is shown. Scale bars=300µm(F) Representative alkaline phosphatase (AP) staining images (left) and quantification (right) of AP+ colonies after clonal replating, as described in panel
Figure 4(A) Top: Predictions of the number of regulation steps required for full activation of the indicated gene under control or dual expression of Esrrb and Tbx3 (+E/T). Bottom: Gene expression of EpiSCs harbouring empty vector (grey) or Esrrb/Tbx3 (orange), capture at D0 (F/A), D2 and D4 (as described in Fig 3D). Dashed black line: expression levels in ESCs maintained in 2i+LIF. Data normalised to empty vector cultures in F/A. Gapdh serves as an internal control. Mean +/- SEM, n=3 independent experiments. (B) As for panel A, comparison of control with dual expression of Esrrb and Klf4 (+E/K, green in bottom plot). Mean +/- SEM, n=3 independent experiments for bottom qRT-PCR panels(C) Left: Flow cytometry profiles of the resetting progression of EpiSCs stably transfected with the Esrrb-T2A-Klf4 construct and cultured in 2i+LIF with DOX for 2 and 4 days, with the indicated fraction of cells sorted for colony formation assay. Since the Venus reporter is under the control of a DOX responsive element, and the emission spectra of Venus and GFP fluorescence overlap, Oct4-GFP reporter could not be fully distinguished from Venus expression. Right: Number of AP+ colonies formed from 250 sorted cells from indicated fractions. Data points represent two technical replicates of one out of two independent experiments(D) Heatmap of single-cell expression measured by qRT-PCR of major ESC and EpiSC markers in un-induced EpiSCs (black), established ESCs (red), Day 2 High/Low (dark and light blue), and Day 4 High cells (green)(E) Scatterplots of the relative expression of pluripotency and transitional markers. Red bar corresponds to median gene expression, and each dot represents a single cell
Figure 5Left: 0.782 cABN predictions of the sequence of gene activation between gene pairs (white, OFF; blue, ON) along the resetting trajectory, compared to single cell gene expression measured by RT-qPCR. Each table summarises the percentage of single cells at the indicated stage of resetting (columns) that have the indicated expression state (rows). Right: Scatterplots showing single cell coordinates based on the expression of the gene pair
Figure 6(A) 0.782 cABN predictions of essential or dispensable factors for EpiSC resetting, compared with the experiment results shown in panel (B). (B) siRNA knockdown effects measured by Oct4-GFP+ colony formation at Day 6 of resetting. n=4 independent experiments(C) Left, resetting capacity of Klf2 and Klf4 KO EpiSCs measured by Oct4-GFP+ colony formation at Day 6 of resetting. Data points represent two independent experiments. Right, representative fluorescent and bright field images of wild type and Klf2 KO EpiSCs at Day 6 of resetting in 2i+LIF(D) Expression of naïve pluripotency, transition and somatic lineage markers in wild type and Klf2 KO EpiSCs during a resetting time course in 2i+LIF. Expression is normalised to wild type EpiSCs in A/F, and β-actin was used as internal control. The values shown correspond to the average expression of three technical replicates from one representative experiment out of two(E) Rescue of Klf2 KO EpiSC resetting by forced expression of individual network components measured by Oct4-GFP+ colony formation at Day 6 of resetting in 2i+LIF. Data points represent two independent experiments(F) Comparison between the effect of single factor knockdowns on ESC maintenance (n=4 independent experiments) and EpiSC resetting using experimental results(G) EpiSC resetting in 2i+LIF measured by Oct4-GFP+ colony formation after Stat3 siRNA in wild type EpiSCs (left), or Klf4 KO EpiSCs transfected with Tfcp2l1 and Gbx2 siRNAs. n=3 independent experiments in wild type cells (box plots indicate min, median, max), n=2 for Klf4-/- cells (box plots indicate min, mean, max)(H) EpiSC resetting in 2i+LIF measured by Oct4-GFP+ colony formation of Stat3 knockdown EpiSCs transiently transfected with Tfcp2l1, Gbx2 and Klf4. n=4 independent experiments: Student"s t-test, p-value indicated on plot. Box plots indicate min, median, max. See also Appendix Fi