Journal: Plant Physiology

Article Title: The role of Arabidopsis Splicing Factor 30 in floral transition and the implications of its alternative splicing

doi: 10.1093/plphys/kiaf335

Figure Lengend Snippet: SPF30 mutation caused reduced transcript levels and increased intron retention of FLC . A) Schematic view of FLC gene structure. Exons are represented by boxes and introns are indicated by lines. Dark boxes indicate coding regions (CDS) and light boxes indicate untranslated regions (UTR). Arrows indicate the primers used for RT-qPCR analysis. B) RT-qPCR analysis of FLC intron retention in 12-d-old spf30 and wild-type (WT) seedlings. Intron retention was calculated as the level of unspliced transcripts normalized to the level of spliced transcripts for each detected intron. Specifically, primer pairs F1/R1 and F1/R1s were used for intron 1, F5/R5 and F5 s/R5s for intron 5, and F6/RT and F6s/RT for intron 6. Error bars represent SE from three biological replicates, and asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; Student's t -test). C) RT-qPCR analysis of total and spliced FLC transcripts in spf30 and WT. Total transcripts were detected with primer pair FT/RT; spliced transcripts were detected with F1/R1s. Error bars represent SE from three biological replicates, and asterisks indicate significant differences (*, P < 0.05; Student's t -test). D and E) RT-qPCR analysis of FLC intron 1 retention (D) and total transcript levels (E) in WT, spf30 and spf30 complemented with gSPF30 . Error bars represent SE from two biological replicates. F) Rosette leaf number at bolting in WT, spf30 , flc and flc spf30 under long-day conditions. Numbers are shown as means ± SE ( n ≥ 36). Different letters indicate significant differences ( P < 0.05; Tukey's post hoc test).

Article Snippet: A 60-nt 3′ fragment of the first intron of FLC was synthesized with a 3′ CY5 label by Sangon Biotech Company (China).

Techniques: Mutagenesis, Quantitative RT-PCR

Journal: Plant Physiology

Article Title: The role of Arabidopsis Splicing Factor 30 in floral transition and the implications of its alternative splicing

doi: 10.1093/plphys/kiaf335

Figure Lengend Snippet: Complementation effects of the CDS of individual SPF30 transcript isoforms. A) Schematic representation of the constructs used for spf30 complementation. 35S stands for a double CaMV 35S promoter. c stands for coding sequence (CDS). B and C) RT-qPCR analysis of FLC intron 1 retention (B), and total transcript levels (C) in 12-d-old wild-type (WT), spf30 and spf30 complemented with individual SPF30 isoforms. Intron retention was calculated as the ratio of unspliced to spliced FLC transcripts. Error bars represent SE from three biological replicates. Asterisks indicate significant differences in comparison to spf30 (*, P < 0.05; Student's t -test). D) RT-qPCR analysis of SPF30 expression in spf30 complementation lines. Error bars represent SE from three biological replicates. For cSPF30.1 , cSPF30.2 and cSPF30.4, a primer pair spanning the T-DNA insertion site in exon 8 was used; for c SPF30.3 , isoform-specific primers were used. E) Flowering time of spf30 complemented with c SPF30.1 (left) or other SPF30 isoforms (right). Rosette leaf number at bolting is shown as means ± SE ( n ≥ 16). Asterisks indicate significant differences in comparison to spf30 (*, P < 0.05; Student's t -test).

Article Snippet: A 60-nt 3′ fragment of the first intron of FLC was synthesized with a 3′ CY5 label by Sangon Biotech Company (China).

Techniques: Construct, Sequencing, Quantitative RT-PCR, Comparison, Expressing

Journal: Plant Physiology

Article Title: The role of Arabidopsis Splicing Factor 30 in floral transition and the implications of its alternative splicing

doi: 10.1093/plphys/kiaf335

Figure Lengend Snippet: The second intron in the full-length SPF30.3 transcript ( SPF30.3FL ) can be further spliced in Arabidopsis. A) Schematic view of GFP -tagged SPF30.1 and SPF30.3FL . 35S indicates a double CaMV 35S promoter. The two boxes flanking intron 2 indicate coding regions (CDS). Arrows indicate primers used in B. Asterisk indicates the position of a stop codon. B) RT-PCR analysis of GFP -tagged SPF30.1 and SPF30.3FL transcripts expressed in wild-type (WT) Arabidopsis protoplasts. Plasmids carrying SPF30.1 and SPF30.3FL serve as controls. IR, PS and CS represents fully retained, partially spliced and fully spliced second intron, respectively. The band corresponding to partially spliced second intron is indicated by a white triangle and was analyzed by Sanger sequencing. In addition to the isoforms identified in , an additional product (#C) was detected and illustrated bellow, with a partial SPF30 genome structure shown for reference. Bar colors are described in . C) Subcellular localization of GFP-tagged SPF30.1 and SPF30.3FL in WT Arabidopsis protoplasts. Bars = 10 μ m. D) RT-qPCR analysis of GFP -tagged SPF30.1 and SPF30.3FL transcripts expressed in protoplast made of spf30 . The characteristic alternative splicing (AS) patterns (primers indicated by arrows) were analyzed in comparison to the total amount of exogenously expressed SPF30 transcripts. Error bars represent SE from three biological replicates. Asterisks indicate significant differences (**, P < 0.01; Student's t -test). E) RT-qPCR analysis of FLC intron 1 retention, spliced FLC transcript levels and exogenous SPF30 expression levels in WT, spf30 and spf30 protoplasts transfected with GFP -tagged SPF30.1 or SPF30.3FL transcripts. Error bars represent SE from three biological replicates. Asterisks indicate significant differences in comparison to spf30 (*, P < 0.05; Student's t -test).

Article Snippet: A 60-nt 3′ fragment of the first intron of FLC was synthesized with a 3′ CY5 label by Sangon Biotech Company (China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Quantitative RT-PCR, Alternative Splicing, Comparison, Expressing, Transfection

Journal: Plant Physiology

Article Title: The role of Arabidopsis Splicing Factor 30 in floral transition and the implications of its alternative splicing

doi: 10.1093/plphys/kiaf335

Figure Lengend Snippet: The second intron negatively impacts the function of SPF30 . A) Schematic representation of SPF30 genomic deletion constructs. gSPF30Δintron2 lacks the second intron, while gSPF30Δ7introns retains only intron 2, with the rest seven introns removed. B to D) RT-qPCR analysis of FLC intron 1 retention (B), spliced FLC transcript levels (C), and exogenous SPF30 expression (D) in 12-d-old wild-type (WT), spf30 and spf30 complemented with different SPF30 genomic constructs. # indicates independent transgenic lines. Error bars represent SE from two technical replicates. E) Flowering time of spf30 lines complemented with SPF30 genomic constructs. Rosette leaf number at bolting is shown as means ± SE ( n ≥ 16). Different letters indicate significant differences ( P < 0.05; Tukey's post hoc test).

Article Snippet: A 60-nt 3′ fragment of the first intron of FLC was synthesized with a 3′ CY5 label by Sangon Biotech Company (China).

Techniques: Construct, Quantitative RT-PCR, Expressing, Transgenic Assay

Journal: Plant Physiology

Article Title: The role of Arabidopsis Splicing Factor 30 in floral transition and the implications of its alternative splicing

doi: 10.1093/plphys/kiaf335

Figure Lengend Snippet: Working model of SPF30 . SPF30 is alternatively spliced into various transcript isoforms. The fully spliced SPF30.1 encodes the full-length SPF30.1 protein, which is associated with spliceosomal components and is required in floral transition by affecting FLC expression and splicing. Alternative isoforms with partially retained intron 2 carry a premature termination codon (PTC) and are targeted for nonsense-mediated mRNA decay (NMD), enabling a negative regulation. Isoforms with intron 2 fully retained are immune to NMD and can serve as intermediate transcripts for further splicing to produce SPF30.1 or NMD-sensitive transcripts. By generating these different isoforms, the effects of the functional SPF30 protein can be fine-tuned in the cells. Dotted lines indicate hypothetical pathways.

Article Snippet: A 60-nt 3′ fragment of the first intron of FLC was synthesized with a 3′ CY5 label by Sangon Biotech Company (China).

Techniques: Expressing, Functional Assay

Journal:

Article Title: Strong Antifungal Activity of SS750, a New Triazole Derivative, Is Based on Its Selective Binding Affinity to Cytochrome P450 of Fungi

doi: 10.1128/AAC.46.2.308-314.2002

Figure Lengend Snippet: Chemical structures of three triazole derivatives (SS750, FLC, and ITC).

Article Snippet: As a solvent for SS750 and FLC, 0.5% carboxymethyl cellulose-sodium was used, and for ITC, 40% (wt/vol) hydroxypropyl-β-cyclodextrin (Funakoshi Co., Ltd., Tokyo, Japan) was used by a method slightly modified from that reported previously ( 13 ).

Techniques:

Journal:

Article Title: Strong Antifungal Activity of SS750, a New Triazole Derivative, Is Based on Its Selective Binding Affinity to Cytochrome P450 of Fungi

doi: 10.1128/AAC.46.2.308-314.2002

Figure Lengend Snippet: MICs of SS750, FLC, ITC, and AMB for Candida species and C. neoformans a

Article Snippet: As a solvent for SS750 and FLC, 0.5% carboxymethyl cellulose-sodium was used, and for ITC, 40% (wt/vol) hydroxypropyl-β-cyclodextrin (Funakoshi Co., Ltd., Tokyo, Japan) was used by a method slightly modified from that reported previously ( 13 ).

Techniques:

Journal:

Article Title: Strong Antifungal Activity of SS750, a New Triazole Derivative, Is Based on Its Selective Binding Affinity to Cytochrome P450 of Fungi

doi: 10.1128/AAC.46.2.308-314.2002

Figure Lengend Snippet: Therapeutic effects of SS750, FLC, and ITC against systemic (A) and pulmonary (B) candidiasis caused by C. albicans IFM 40009 in immunosuppressed mice. The MICs for the infectious strain were 0.063 μg/ml for SS750, 0.025 μg/ml for FLC, and 0.031 μg/ml for ITC. SS750 and the reference drugs were orally administered to 10 animals per group once daily for 4 consecutive days, starting at 1 h after infection. Symbols: □, 64 mg/kg; ▪, 16 mg/kg; ○, 4 mg/kg; •, 1 mg/kg; ▵, 0.25 mg/kg; ▴, 0.063 mg/kg; ◊, untreated control; *, significantly different from the survival of untreated controls (P < 0.01 by the Kaplan-Meier technique and the log rank test).

Article Snippet: As a solvent for SS750 and FLC, 0.5% carboxymethyl cellulose-sodium was used, and for ITC, 40% (wt/vol) hydroxypropyl-β-cyclodextrin (Funakoshi Co., Ltd., Tokyo, Japan) was used by a method slightly modified from that reported previously ( 13 ).

Techniques: Infection, Control

Journal:

Article Title: Strong Antifungal Activity of SS750, a New Triazole Derivative, Is Based on Its Selective Binding Affinity to Cytochrome P450 of Fungi

doi: 10.1128/AAC.46.2.308-314.2002

Figure Lengend Snippet: IC 50 s and MICs of SS750, FLC, and ITC for C. albicans and C. krusei

Article Snippet: As a solvent for SS750 and FLC, 0.5% carboxymethyl cellulose-sodium was used, and for ITC, 40% (wt/vol) hydroxypropyl-β-cyclodextrin (Funakoshi Co., Ltd., Tokyo, Japan) was used by a method slightly modified from that reported previously ( 13 ).

Techniques:

Journal:

Article Title: Strong Antifungal Activity of SS750, a New Triazole Derivative, Is Based on Its Selective Binding Affinity to Cytochrome P450 of Fungi

doi: 10.1128/AAC.46.2.308-314.2002

Figure Lengend Snippet: Affinities of binding of SS750 (○), FLC (▴), ITC (□), and KTC (•) to the microsomal cytochrome P450 of C. albicans ATCC 90028, C. krusei ATCC 6258, rat liver, and human liver on the basis of the carbon monoxide displacement spectrum. ITC was not tested at a concentration of >100 μM because of its insolubility. The differences in absorbance between 448 nm (C. albicans and C. krusei) or 450 nm (rat liver and human liver) and 490 nm are shown as a percentage of that observed in controls. Results are the means ± standard errors of three experiments. Symbols: * and **, P < 0.05, and P < 0.01, respectively, versus the results for SS750, according to Tukey’s multiple comparison.

Article Snippet: As a solvent for SS750 and FLC, 0.5% carboxymethyl cellulose-sodium was used, and for ITC, 40% (wt/vol) hydroxypropyl-β-cyclodextrin (Funakoshi Co., Ltd., Tokyo, Japan) was used by a method slightly modified from that reported previously ( 13 ).

Techniques: Binding Assay, Concentration Assay, Comparison