Rad4 mainly functions in Chk1-mediated DNA damage checkpoint pathway.

<p><b>A</b>, wild type cells or cells with the integrated mutations were treated with (+) or without (−) MMS at 30°C for 1 hour. Phosphorylation of Chk1 was monitored by the mobility shift assay as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092936#s2" target="_blank">Materials and Methods</a>. A section of the Ponceau S stained membrane is shown for the loading (bottom). <b>B</b>, Rad3-dependent phosphorylation of Cds1-Thr¹¹ was examined by phosphor-specific antibody (top panel). Loading of Cds1 is shown in the lower panel. <b>C</b>, sensitivity of the cells to acute HU (left) or MMS (right) treatment. Cells were treated with the drugs in YE6S medium at 30°C. At each time point, an aliquot of the culture was removed and spread on YE6S plates for the cells to recover. Colonies were counted and viability was presented as percentages of the untreated cells. Each data point represents an average of three independent experiments for each mutant. <b>D</b>, synthetic effects of the double mutants containing Δ<i>cds1</i> or Δ<i>chk1</i> with the indicated <i>rad4</i> mutations were examined by spot assay.</p

The C13Y and K56R mutations abolish the scaffolding function of Rad4 <i>in vivo</i>.

<p><b>A</b>, Rad9 was IPed from the lysates of MMS-treated cells with the indicated mutation (bottom panel). Wild type or mutant Rad4 (top two panels) Co-IPed with Rad9 was detected by Western blotting using anti-Rad4 antibodies. Phosphorylated Rad9-Thr⁴¹² was shown in the third panel from the top. <b>B</b>, Co-IP of Rad4 containing the indicated mutations with phosphorylated Rad9 from HU-treated cells. <b>C</b>, Co-IP of Rad4 and Rad9 with Crb2 from MMS-treated cells.</p

Genetic screen of novel <i>rad4</i> mutants with enhanced sensitivities to HU and MMS in the <i>rad4⁺</i> shut-off strain.

<p><b>A</b>, thiamine-controlled cell growth of the shut-off strain (nmt-rad4) in which the endogenous promoter of <i>rad4⁺</i> was replaced with a thiamine repressive <i>nmt81</i> promoter. Logarithmically growing cells were diluted in fivefold steps and spotted on EMM6S plates with (+) or without (-) thiamine. The plates were incubated at 30°C for 3 days before being photographed. <b>B</b>, thiamine-regulated expression of Rad4 was examined by Western blotting. Untagged Rad4, Rad4 with the deletion of whole C-terminus (ΔC) and HA-tagged Rad4 were expressed on a vector in the shut-off strain. Expression of Rad4 in the presence (+) or absence (-) of thiamine was examined by using anti-Rad4 antibodies (Top). The same membrane was stripped and blotted with anti-HA antibody (bottom). Asterisks indicate the cross-reacting materials. <b>C</b>, molecular architecture of Rad4 with relative locations of the newly isolated (solid circles) and previously reported (open circles) mutations and the AAD domain. The four BRCT repeats are marked by roman numerals. The regions covered by mutational PCRs in the genetic screening are also shown. <b>D</b>, sensitivities of the <i>rad4</i> mutants to HU and MMS were assessed by spot assay in the shut-off strain. The newly screened (top part) and the previously reported (lower part) mutations are marked on the right. Wild type cells, Δ<i>rad3</i> mutant, and the shut-off strain carrying an empty vector or the vector expressing wild-type Rad4 were used as controls.</p

The C13Y and K56R mutations abolish the scaffolding function of Rad4 <i>in vitro</i>.

<p><b>A</b>, schematic diagram of the Rad4 fragments used in this experiment. Each fragment was fused with a GST tag for protein purification and detection in the binding assay using anti-GST antibodies. <b>B</b>, preferential binding of the N- and C-terminal pair of BRCT repeats of Rad4 to Crb2 and Rad9, respectively. Recombinant GST or GST-tagged Rad4 fragments were incubated with immunopurified Crb2 (middle panels) or Rad9 (right panels) bound to the anti-HA antibody beads. The bound proteins were released from the beads in gel loading buffer and analyzed by Western blotting. The membrane was striped and reprobed with anti-HA antibody for Crb2 and Rad9 (bottom panels). Aliquots of the binding reaction were analyzed separately by SDS PAGE as the input (left panel). <b>C</b>, equal amount of the Rad4 fragment b with or without the C13Y-K56R mutation was incubated with Crb2 bound on beads. The input and the Rad4 protein bound to Crb2 were analyzed as in B. <b>D</b>, fragment e with or without the E368K mutation was similarly tested for binding to phosphorylated Rad9. Phosphorylation of Rad9-Thr⁴¹² was detected by blotting of the same membrane with the phospho-specific antibody.</p

<i>In vitro</i> kinase assay using purified Rad3-Rad26 and Cds1(D312E) as the substrate.

<p><b>A</b>, time course of the <i>in vitro</i> kinase reaction. Rad3-Rad26 was affinity-purified from <i>S. pombe</i> using anti-Flag antibody resin, eluted with Flag peptide, and incubated with the kinase-inactive Cds1(D312E) as the substrate in the standard kinase buffer containing 200 μM ATP. At each time point, a small aliquot of the reaction was taken out and analyzed by SDS PAGE followed by Western blotting to examine the phosphorylation of Cds1-Thr¹¹. Rad3 and Rad26 in the same samples were detected by using anti-HA and anti-flag antibody, respectively. Loading of the substrate was shown by Ponceau S staining. The kinase dead Rad3(D2249E)-Rad26 was similarly prepared and used as the control (last lane on the right). <b>B</b>, phosphorylation of Cds1-Thr¹¹ was examined in the presence of increasing concentrations of Rad3-Rad26. The reaction was carried out at 30°C for 30 min. <b>C</b>, full-length Rad4 was purified from <i>S. pombe</i>. Removal of the N-terminal GST tag was analyzed by SDS PAGE (left) and confirmed by Western blotting (right) using anti-Rad4 antibodies. Asterisks indicate the degradation products of Rad4. <b>D</b>, the Rad3 kinase reactions were carried out at 30°C for 20 min in the absence or presence of increasing amount of purified Rad4 and analyzed as in A.</p

Drug sensitivities of <i>S. pombe</i> with the integrated <i>rad4</i> mutations.

<p><b>A</b>, the two-step marker switching method used for integration of the mutations. The genomic locus of <i>rad4⁺</i> is shown on top. <b>B</b>, sensitivities of the cells with integrated <i>rad4</i> mutations to HU or MMS were examined by spot assay. The newly identified (upper part) and the previously reported (lower part) mutants are marked on the right. <b>C</b>, expression of Rad4 in the cells with the integrated <i>rad4</i> mutations was detected in whole cell lysates by Western blotting using anti-Rad4 antibodies. The asterisk denotes the cross-reactive material. Note: like the T45M mutant, A60P is a <i>ts</i> mutant.</p

Enhancing Visible-Light Absorption of 2D Carbon Nitride by Constructing 2D/2D van der Waals Heterojunctions of Carbon Nitride/Nitrogen-Superdoped Graphene

Carbon nitride sheets (CNs) down to the two-dimensional (2D) limit have been widely used in photoelectric conversion due to their inherent band gap and extremely short charge-carrier diffusion distance. However, the utilization of visible light remains low due to the rapid recombination of photogenerated electron–hole pairs and enlarged band gap. Here, atomically thin 2D/2D van der Waals heterojunctions (vdWHs) of N-superdoped graphene (NG) and CNs (CNs/NG) are fabricated via a facile electrostatic self-assembly method. Our results revealed that the vdWHs can increase the visible-light absorption of CNs by extending the absorption edge from 455 to up to 490 nm. The recombination of photogenerated electron–hole pairs is inhibited because superdoped N in CNs/NG facilitates the transmission of photogenerated carriers in the melon chain. This study opens a new avenue for narrowing the band gap and promoting photoexcited carrier separation in carbon-nitride-based materials

Light-Promoted Arylsilylation of Alkenes with Hydrosilanes

Herein, we report light-promoted photo/hydrogen atom transfer dual catalysis for arylsilylation of alkenes via the radical–radical cross-coupling with diverse hydrosilanes, which provides a simple and efficient method to prepare various organosilicon compounds with a wide range of substrate scope and good functional group tolerance under transition-metal- and chemical-oxidant-free conditions. Furthermore, the arylsilylation of alkenes can also proceed via the possible electron donor–acceptor complex under exogenous photocatalyst-free conditions

Table4_Assessment of CircRNA Expression Profiles and Potential Functions in Brown Adipogenesis.XLSX

Brown adipose tissue (BAT) is specialized for energy expenditure, thus a better understanding of the regulators influencing BAT development could provide novel strategies to defense obesity. Many protein-coding genes, miRNAs, and lncRNAs have been investigated in BAT development, however, the expression patterns and functions of circRNA in brown adipogenesis have not been reported yet. This study determined the circRNA expression profiles across brown adipogenesis (proliferation, early differentiated, and fully differentiated stages) by RNA-seq. We identified 3,869 circRNAs and 36.9% of them were novel. We found the biogenesis of circRNA was significantly related to linear mRNA transcription, meanwhile, almost 70% of circRNAs were generated by alternative back-splicing. Next, we examined the cell-specific and differentiation stage-specific expression of circRNAs. Compared to white adipocytes, nearly 30% of them were specifically expressed in brown adipocytes. Further, time-series expression analysis showed circRNAs were dynamically expressed, and 117 differential expression circRNAs (DECs) in brown adipogenesis were identified, with 77 upregulated and 40 downregulated. Experimental validation showed the identified circRNAs could be successfully amplified and the expression levels detected by RNA-seq were reliable. For the potential functions of the circRNAs, GO analysis suggested that the decreased circRNAs were enriched in cell proliferation terms, while the increased circRNAs were enriched in development and thermogenic terms. Bioinformatics predictions showed that DECs contained numerous binding sites of functional miRNAs. More interestingly, most of the circRNAs contained multiple binding sites for the same miRNA, indicating that they may facilitate functions by acting as microRNA sponges. Collectively, we characterized the circRNA expression profiles during brown adipogenesis and provide numerous novel circRNAs candidates for future brown adipogenesis regulating studies.</p

Table6_Assessment of CircRNA Expression Profiles and Potential Functions in Brown Adipogenesis.XLSX

Brown adipose tissue (BAT) is specialized for energy expenditure, thus a better understanding of the regulators influencing BAT development could provide novel strategies to defense obesity. Many protein-coding genes, miRNAs, and lncRNAs have been investigated in BAT development, however, the expression patterns and functions of circRNA in brown adipogenesis have not been reported yet. This study determined the circRNA expression profiles across brown adipogenesis (proliferation, early differentiated, and fully differentiated stages) by RNA-seq. We identified 3,869 circRNAs and 36.9% of them were novel. We found the biogenesis of circRNA was significantly related to linear mRNA transcription, meanwhile, almost 70% of circRNAs were generated by alternative back-splicing. Next, we examined the cell-specific and differentiation stage-specific expression of circRNAs. Compared to white adipocytes, nearly 30% of them were specifically expressed in brown adipocytes. Further, time-series expression analysis showed circRNAs were dynamically expressed, and 117 differential expression circRNAs (DECs) in brown adipogenesis were identified, with 77 upregulated and 40 downregulated. Experimental validation showed the identified circRNAs could be successfully amplified and the expression levels detected by RNA-seq were reliable. For the potential functions of the circRNAs, GO analysis suggested that the decreased circRNAs were enriched in cell proliferation terms, while the increased circRNAs were enriched in development and thermogenic terms. Bioinformatics predictions showed that DECs contained numerous binding sites of functional miRNAs. More interestingly, most of the circRNAs contained multiple binding sites for the same miRNA, indicating that they may facilitate functions by acting as microRNA sponges. Collectively, we characterized the circRNA expression profiles during brown adipogenesis and provide numerous novel circRNAs candidates for future brown adipogenesis regulating studies.</p