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Files in this Data Supplement:
Fig. S1. Klf5 knockdown by RNA interference suppresses differentiation of ESCs. (A) ESC clone C7, stably transfected with EGFP under the control of neuron-specific α1-tubulin gene promoter, was grown in the presence of 200 µg/ml of G418 and induced to differentiate as described under Material and Methods. EGFP signal appears at day 4 and is maintained in fully differentiated neurons at day 10. (B) Differentiated ESC clone C7 was immunostained with anti-β3-tubulin antibody (red). Neuronal cells at day 13 of differentiation show co-expression of EGFP and β3-tubulin. (C) Klf5 knockdown by RNA interference results in the impairment of ESC differentiation into neurons. ESC clone C7 was used to screen a collection of shRNAs. The Klf5-targeting shRNA prevents the appearance of EGFP-positive cells 10 days after the induction of ESC differentiation. Scale bar: 100 µm. (D) Western blot analysis of protein extracts from CRL- or Klf5-shRNA-transfected ESCs shows that Klf5 silencing results in the decrease of the protein down to 20% of the basal levels.
Fig. S2. Klf5 expression in ESCs at day 3 of in vitro differentiation (A) and in blastocysts (B). (A) wt ESCs were grown for 3 days under differentiation conditions and stained with anti-Klf5 (red) and -Oct3/4 or -Nanog (green) antibodies. Klf5 immunostaining almost completely disappears. Scale bars: 50 µm. (B) Hatched blastocysts were fixed in 4% paraformaldehyde and stained with anti-Klf5 (red) and -Oct3/4 or -Nanog (green) antibodies. Confocal microscopy images show that Klf5 expression decreases at this stage in many cells of the inner cell mass, where Oct3/4 and Nanog are still expressed at high levels. Scale bars: 20 µm.
Fig. S3. (A) Klf5 knockdown results in morphological changes of ESCs. ESCs were transfected with either Klf5 or CRL shRNAs and grown for 3 days in culture medium with 2 µg/ml of puromycin. After selection, resistant cells were pooled and re-plated at 60% confluence to better observe cell morphology. The upper panel shows an example of groups of enlarged cells characteristic of Klf5 KD cells. Scale bar: 250 µm.
Fig. S4. Klf5 knockdown results in an aberrant differentiation of ESCs. ESCs transfected either with Klf5 shRNA (A) or CRL shRNA (B) were stained with anti-brachyury and -Cdx2 antibodies. 4 days after transfection, a significant number of cells positive for Cdx2 (red) or brachyury (green) specifically appear in Klf5-knockdown cells. Scale bars: 100 µm.
Fig. S5. Western blot analysis of Klf5 ectopic expression in ESCs. E15Tg2a cells were transfected with Klf5-3×FLAG under the control of β-actin promoter. Four G418 resistant clones were analysed by western blot with anti-Klf5 antibody; this analysis demonstrated that ectopic Klf5-3×FLAG and endogenous Klf5 are expressed at similar levels. 6 days after LIF withdrawal, endogenous Klf5 is undetectable, whereas Klf5-3×FLAG is still expressed.
Fig. S6. Klf5 does not interact with the Nanog promoter region containing the Oct4-Sox2 cis-element. EMSA were performed by using as a probe the ds oligonucleotide containing the Oct4-Sox2-binding site (underlined). Lane 1, free probe; lane 2, wt ESC nuclear extract was prepared as described previously (Bevilacqua et al., 2005) and incubated with 32P-labelled ds oligonucleotide; lane 3, as in lane 2, but the extract was preincubated with a 50-fold molar excess of unlabelled oligonucleotide probe; lane 4, as in lane 2, but the extract was preincubated with a 50-fold molar excess of unlabelled oligonucleotide designed on the basis of CR4 region of the Oct3/4 promoter (Chew et al., 2005), which contains the binding site for the Oct4-Sox2 dimer; lane 5, as in lane 2, but the extract was preincubated with a 50-fold molar excess of unlabelled oligonucleotide probe containing a mutation in the Oct4-Sox2 binding site; lane 6, as in lane 2, but the extract was preincubated with Klf5 antibody; lane 7, nuclear extract from ESCs transfected with Klf5-Flag was incubated with 32P-labelled ds oligonucleotide as in lane 2; lane 8, as in lane 7, but the extract was preincubated with anti-FLAG antibody. The results indicate that the region containing the Oct4-Sox2 cis-element does not interact with Klf5 and that Klf5 overexpression does not modify the Oct4-Sox2 binding to the promoter. The lower panel reports the sequence of the Nanog promoter; the sequence of the oligonucleotide used in this experiment is underlined; the Oct4-Sox2-binding site is included in the grey box. The arrow indicates the band shift of the Oct3/4-Sox2 complex.
Fig. S7. Klf5 promoter region contains a conserved putative Nanog cis-element. Alignment of sequences in the region upstream of the Klf5 putative transcription start site is shown. The mouse sequence starts at nucleotide 98,179,221 of chromosome 14. Uppercase letters indicate conserved nucleotides in the Nanog cis-element. The lower panel shows a schematic representation of Klf5 and reports the position of the putative Nanog-binding site (arrowhead) and those of the two regions (K1, K2) amplified in the ChIP experiments of Fig. 4D.
Fig. S8. Klf5 overexpression or knockdown do not change ESC proliferation. ESCs transfected with Klf5 siRNA, CRL siRNA, Klf5 expression vector or control DNA were labelled with 10 µM BrdU for 45 minutes. The cells were stained with anti-BrdU antibody (green) and counted in at least ten non-overlapping fields. The percentages of BrdU-positive cells were: CRL-siRNA transfected, 50±4; Klf5-siRNA transfected, 52±7; mock transfected, 53±5; Klf5 overexpressing (Klf5 ov.), 58±5. Scale bars: 250 µm.
Fig. S9. Anti-Klf5-antibody specificity. (A) ESCs, NIH3T3, MEFs and MS5 were fixed and stained with the anti-Klf5 antibody. No signal is detectable in the cells (NIH3T3, MEFs and MS5) not expressing Klf5. (B) NIH3T3 cells were transfected with Klf5 or with empty vector. The western blot of the total extracts prepared from these cells is shown, indicating that the antibody recognises only one band in the transfected-cell extract. (C) Western blot analysis of ESC extract with anti-Klf5 antibody, which stains one band of the expected size.
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