7 research outputs found
Ruthenium- and Rhodium-Catalyzed Chemodivergent Couplings of Ketene Dithioacetals and α‑Diazo Ketones via C–H Activation/Functionalization
Chemodivergent
coupling of α-acylketene dithioacetals with
diazo compounds has been realized under catalyst control. The RuÂ(II)-catalyzed
C–H activation occurred at the olefinic position, and 1:2 coupling
with α-diazoketoesters leads to furfurylation. In contrast,
the RhÂ(III)-catalyzed C–H functionalization occurred at both
the olefinic and the <i>ortho</i> CÂ(aryl)–H positions,
and [4 + 2] annulation afforded naphthalenones. Synthetic applications
have been performed to demonstrate the usefulness of the coupling
system
Sp5 induces the expression of Nanog to maintain mouse embryonic stem cell self-renewal
<div><p>Activation of signal transducer and activator of transcription 3 (STAT3) by leukemia inhibitory factor (LIF) maintains mouse embryonic stem cell (mESC) self-renewal. Our previous study showed that trans-acting transcription factor 5 (Sp5), an LIF/STAT3 downstream target, supports mESC self-renewal. However, the mechanism by which Sp5 exerts these effects remains elusive. Here, we found that Nanog is a direct target of Sp5 and mediates the self-renewal-promoting effect of Sp5 in mESCs. Overexpression of <i>Sp5</i> induced <i>Nanog</i> expression, while knockdown or knockout of <i>Sp5</i> decreased the <i>Nanog</i> level. Moreover, chromatin immunoprecipitation (ChIP) assays showed that Sp5 directly bound to the Nanog promoter. Functional studies revealed that knockdown of <i>Nanog</i> eliminated the mESC self-renewal-promoting ability of Sp5. Finally, we demonstrated that the self-renewal-promoting function of Sp5 was largely dependent on its zinc finger domains. Taken together, our study provides unrecognized functions of Sp5 in mESCs and will expand our current understanding of the regulation of mESC pluripotency.</p></div
I<sub>2</sub>‑Mediated Intramolecular C–H Amidation for the Synthesis of N‑Substituted Benzimidazoles
A practical
intramolecular C–H amidation methodology has been developed
using molecular iodine under basic conditions. The required imine
substrates were readily obtained by condensation of simple <i>o</i>-phenylenediamine derivatives and aldehydes. The transition-metal-free
cyclization reaction described here works well with crude imines and
allows for the sequential synthesis of N-protected benzimidazoles
without purification of the less stable condensation intermediates.
This operationally simple synthetic approach is broadly applicable
to a variety of aromatic, aliphatic, and cinnamic aldehydes to produce
diverse 1,2-disubstituted benzimidazole derivatives in an efficient
and scalable fashion
Screening the downstream pluripotency genes regulated by <i>Sp5</i>.
<p>(A) HA-tagged <i>Sp5</i> was introduced into 46C mESCs and the protein level of HA-tagged <i>Sp5</i> was determined by Western blot. α-Tubulin was used as a loading control. (B) Phase-contrast and alkaline phosphatase (AP) staining images of PB and PB-<i>Sp5</i> mESCs cultured under serum-containing conditions in the absence of LIF for eight days. Scale bar, 100 μm. (C) Immunofluorescence of PB and PB-<i>Sp5</i> mESCs cultured under basal media conditions in the absence of LIF. Scale bar, 100 μm. (D) qRT–PCR analysis of the expression levels of mESC pluripotency markers (<i>Oct4</i>, <i>Sox2</i>, and <i>Nanog</i>) and differentiation-associated genes (<i>Gata4</i>, <i>Gata6</i>, and <i>T</i>) in PB and PB-<i>Sp5</i> 46C mESCs cultured in the absence of LIF. Data represent the mean±s.d of three biological replicates. **p < 0.01 vs PB. (E) qRT-PCR analysis of <i>Klf2/4/5</i>, <i>Nanog</i>, <i>Esrrb</i>, <i>Gbx2</i>, <i>Myc</i>, <i>Tfcp2l1</i>, <i>Tbx3</i> and <i>Pim1/3</i> expression in PB and PB-<i>Sp5</i> 46C mESCs cultured under LIF/serum-containing conditions. Data represent the mean±s.d. of three biological replicates. **p < 0.01 vs PB. (F) qRT–PCR analysis of <i>Sp5</i>, <i>Nanog</i> and <i>Klf2</i> transcripts in <i>scramble</i> and <i>Sp5</i> shRNA mESCs cultured under LIF/serum-containing conditions. Data represent the mean±s.d. of three biological replicates. *p < 0.05, **p < 0.01 vs <i>scramble</i> shRNA control.</p
Sp5 directly regulates the transcription of <i>Nanog</i>.
<p>(A) Independent validation of Nanog as an Sp5-bound target by ChIP-qPCR with fifteen primers set to scan different fragments of the Nanog promoter. Primers set at sites 1, 3, 5, 8, 9 and 15 represent significant enrichment. Data represent the mean±s.d. of three biological replicates. *p < 0.05, **p < 0.01 vs IgG. (B) Schematic illustration of luciferase reporter plasmids and <i>Sp5</i> expression activates the P<sub>Nanog</sub>-luciferase reporter. Data represent the mean±s.d. of three biological replicates. **p < 0.01 vs PB.</p
Delineation of the zinc finger domains of Sp5 impairs the self-renewal-promoting effect of Sp5.
<p>(A) Schematic illustration of the <i>Sp5</i> deletion (Δ) mutants. Zinc finger domains are shown as green boxes. (B) Western blot analysis of Flag-tagged <i>Sp5</i> and <i>Sp5</i> deletion mutants with anti-FLAG antibody. (C) AP staining images of PB, PB-<i>Sp5</i> and <i>Sp5</i> deletion mutant mESCs cultured under serum-containing conditions in the absence of LIF for eight days. Scale bar, 100 μm. (D) Quantification of AP-positive colonies in Fig. 4C. (E) qRT–PCR analysis of the expression of <i>Nanog</i> in PB, PB-<i>Sp5</i> and <i>Sp5</i> deletion mutant mESCs cultured in LIF/serum-containing media. Data represent the mean±s.d. of three biological replicates. **p < 0.01 vs PB.</p
Sp5 relies on Nanog to promote mESC self-renewal.
<p>(A) qRT–PCR analysis of <i>Nanog</i> expression in PB-<i>Sp5</i> mESCs infected with <i>Nanog</i> knockdown lentiviruses and cultured in LIF/serum-containing media. The transcript level was normalized to the <i>scramble</i> shRNA control. Data represent the mean±s.d of three biological replicates. **p < 0.01 vs <i>scramble</i> shRNA control. (B) Immunofluorescence of PB-<i>Sp5</i> cells infected with <i>scramble</i> control and <i>Nanog</i> shRNA lentiviruses cultured under serum-containing conditions in the absence of LIF for eight days. Scale bar, 100 μm. (C) AP staining images of <i>scramble</i> control and <i>Nanog</i> shRNA mESCs overexpressing PB-<i>Sp5</i> cultured under serum-containing conditions. Scale bar, 100 μm. (D) qRT–PCR analysis of the expression of mESC pluripotency markers (<i>Oct4</i>, <i>Sox2</i>, <i>Klf4</i>, <i>Esrrb</i> and <i>Tfcp2l1</i>) and differentiation-associated genes (<i>Gata4</i>, <i>Gata6</i>, <i>Mixl1</i>, <i>Sox1</i> and <i>Cdx2</i>) in <i>scramble</i> and <i>Nanog</i>-knockdown mESCs transfected with PB-<i>Sp5</i> cultured in the absence of LIF. Data represent the mean±s.d of three biological replicates. *p < 0.05, **p < 0.01 vs scramble control. (E) qRT–PCR analysis of the expression of <i>Gata4</i> and <i>Gata6</i> in PB and PB-<i>Sp5</i> 46C mESC-derived embryoid bodies harvested on day 6. Data represent the mean±s.d. of three biological replicates. **p < 0.01 vs PB EBs. (F) qRT–PCR analysis of <i>Gata4</i> and <i>Gata6</i> transcript levels in wild type and S<i>p5</i>KO mESC-derived embryoid bodies. Data represent the mean±s.d. of three biological replicates. **p < 0.01 vs 46C EBs.</p