MPK6-mediated phosphorylation regulates MASS localization and function.

(A) In vitro kinase assay showing MKK5DD-activated MPK6 phosphorylation of MASS2.2. (B) Upper panel showing the localization of YFP-tagged MASS1 and MASS1 phosphor-variants (green). Lower panel shows the YFP intensity profiling along the lines drawn in the above images. Red arrows indicate YFP signals at the plasma membrane. Scale bars represent 10 μm. (C) Quantification of YFP intensity partition in designated subcellular regions shown in (B). (D) Upper panel, confocal images showing localization of YFP-tagged MASS2.2 and phosphor-variants. Lower panel, intensity profiling of the corresponding YFP signals along the lines drawn in the above images. Red arrows indicate YFP signals at the plasma membrane. Scale bars represent 10 μm. (E, F) Quantification of clustered stomata index in 5-dpg adaxial cotyledons expressing YFP-tagged MASS2.2 and phosphor-variants (E) and myristoylated GFP-tagged proteins as designated (F). * in (E), significantly different compared with the WT (Col) values. ** in (F) significantly different between the two samples being compared (bars). (Student’s t-test, *P < 0.05, **P < 0.01). n.s: not significant.</p

Fine-scale analysis of the MASS2 subdomains.

(A) Diagram of MASS2.3 subdomains with proposed functions. N76, the N-terminal domain containing 76-aa; C72, the C-terminal domain containing 72-aa; Δ29N, the N-terminal 29-aa deleted; Δ13C, the C-terminal 13-aa delete; Δ25C, the C-terminal 25-aa deleted. (B-G) Confocal images of 3-dpg adaxial cotyledon epidermis showing the localization of GFP-fused MASS2.3 variants (green). (H-J) Confocal images of 3-dpg adaxial cotyledon epidermis showing stomatal phenotypes of expressing GFP-MASS2.3 full-length and truncated versions (green). Brackets indicate stomatal clusters and abnormal cell divisions. Cell outlines in (H-J) were stained with PI. Scale bar represents 20 μm in (B-G) and 50 μm in (H-J).</p

Table3_Gonadal transcriptome sequencing reveals sexual dimorphism in expression profiling of sex-related genes in Asian arowana (Scleropages formosus).XLS

Asia arowana (Scleropages formosus) is an ornamental fish with high economic value, while its sex determination mechanism is still poorly understood. By far, no morphological evidence or molecular marker has been developed for effective distinguishment of genders, which poses a critical challenge to our captive breeding efforts. In this study, we sequenced gonadal transcriptomes of adult Asian arowanas and revealed differential expression profiling of sex-related genes. Based on the comparative transcriptomics analysis of testes (n = 3) and ovaries (n = 3), we identified a total of 8,872 differentially expressed genes (DEGs) and 18,490 differentially expressed transposable elements (TEs) between male and female individuals. Interestingly, the expression of TEs usually has been more significantly testis-biased than related coding genes. As expected, several genes related to females (such as foxl2 and cyp19a1a) are significantly transcribed in the ovary, and some genes related to male gonad development (such as dmrt1, gsdf and amh) are highly expressed in the testis. This sexual dimorphism is valuable for ascertaining the differential expression patterns of sex-related genes and enriching the genetic resources of this economically important species. These valuable genetic materials thereby provide instructive references for gender identification and one-to-one breeding practices so as to expand fish numbers for a rapid elevation of economic value.</p

Involvement of CX3CR1 in spinal LTP.

<p>(A & B) When an anti-CX3CR1 neutralizing antibody (CX3CR1 AB, 30 μg/30 μl) was administrated 2 h before TSS, 10-trains TSS-induced spinal LTP was evidently suppressed (A). Simultaneously, the expression of CX3CR1 in spinal dorsal horn was also decreased by CX3CR1 AB (B). * p<0.05 vs. IgG control. (C) TSS stably induced spinal LTP of C-fiber-evoked field potentials in wild type mice, but failed to induce in CX3CL1 knockout mice. (D & E) TSS-induced mechanical allodynia (D) and thermal hyperalgesia (E) only occurred in wild type mice but not in CX3CL1 knockout mice. ** p>0.01 vs. Baseline (before TSS).</p

Table2_Gonadal transcriptome sequencing reveals sexual dimorphism in expression profiling of sex-related genes in Asian arowana (Scleropages formosus).XLS

Asia arowana (Scleropages formosus) is an ornamental fish with high economic value, while its sex determination mechanism is still poorly understood. By far, no morphological evidence or molecular marker has been developed for effective distinguishment of genders, which poses a critical challenge to our captive breeding efforts. In this study, we sequenced gonadal transcriptomes of adult Asian arowanas and revealed differential expression profiling of sex-related genes. Based on the comparative transcriptomics analysis of testes (n = 3) and ovaries (n = 3), we identified a total of 8,872 differentially expressed genes (DEGs) and 18,490 differentially expressed transposable elements (TEs) between male and female individuals. Interestingly, the expression of TEs usually has been more significantly testis-biased than related coding genes. As expected, several genes related to females (such as foxl2 and cyp19a1a) are significantly transcribed in the ovary, and some genes related to male gonad development (such as dmrt1, gsdf and amh) are highly expressed in the testis. This sexual dimorphism is valuable for ascertaining the differential expression patterns of sex-related genes and enriching the genetic resources of this economically important species. These valuable genetic materials thereby provide instructive references for gender identification and one-to-one breeding practices so as to expand fish numbers for a rapid elevation of economic value.</p

Table1_Gonadal transcriptome sequencing reveals sexual dimorphism in expression profiling of sex-related genes in Asian arowana (Scleropages formosus).DOCX

Asia arowana (Scleropages formosus) is an ornamental fish with high economic value, while its sex determination mechanism is still poorly understood. By far, no morphological evidence or molecular marker has been developed for effective distinguishment of genders, which poses a critical challenge to our captive breeding efforts. In this study, we sequenced gonadal transcriptomes of adult Asian arowanas and revealed differential expression profiling of sex-related genes. Based on the comparative transcriptomics analysis of testes (n = 3) and ovaries (n = 3), we identified a total of 8,872 differentially expressed genes (DEGs) and 18,490 differentially expressed transposable elements (TEs) between male and female individuals. Interestingly, the expression of TEs usually has been more significantly testis-biased than related coding genes. As expected, several genes related to females (such as foxl2 and cyp19a1a) are significantly transcribed in the ovary, and some genes related to male gonad development (such as dmrt1, gsdf and amh) are highly expressed in the testis. This sexual dimorphism is valuable for ascertaining the differential expression patterns of sex-related genes and enriching the genetic resources of this economically important species. These valuable genetic materials thereby provide instructive references for gender identification and one-to-one breeding practices so as to expand fish numbers for a rapid elevation of economic value.</p

Table4_Gonadal transcriptome sequencing reveals sexual dimorphism in expression profiling of sex-related genes in Asian arowana (Scleropages formosus).XLS

Asia arowana (Scleropages formosus) is an ornamental fish with high economic value, while its sex determination mechanism is still poorly understood. By far, no morphological evidence or molecular marker has been developed for effective distinguishment of genders, which poses a critical challenge to our captive breeding efforts. In this study, we sequenced gonadal transcriptomes of adult Asian arowanas and revealed differential expression profiling of sex-related genes. Based on the comparative transcriptomics analysis of testes (n = 3) and ovaries (n = 3), we identified a total of 8,872 differentially expressed genes (DEGs) and 18,490 differentially expressed transposable elements (TEs) between male and female individuals. Interestingly, the expression of TEs usually has been more significantly testis-biased than related coding genes. As expected, several genes related to females (such as foxl2 and cyp19a1a) are significantly transcribed in the ovary, and some genes related to male gonad development (such as dmrt1, gsdf and amh) are highly expressed in the testis. This sexual dimorphism is valuable for ascertaining the differential expression patterns of sex-related genes and enriching the genetic resources of this economically important species. These valuable genetic materials thereby provide instructive references for gender identification and one-to-one breeding practices so as to expand fish numbers for a rapid elevation of economic value.</p

Contribution of IL-18 and IL-23 to spinal LTP.

<p>(A & B) In the spinal cord, IL-18 was mainly produced in Iba1-labled microglia and co-localized with CX3CR1 (A); both IL-18R and IL-23 were expressed in astrocytes (B). (C) As compared with control (PBS, 0.01M 30 μl), the spinal LTP was obviously suppressed by administrating IL-18BP (7 μg/30 μl) 20 min before 10-trains TSS. (D) Similarly, the spinal LTP was also suppressed by an anti-IL-23 neutralizing antibody (IL-23 AB, 6 μg/30 μl), which was administrated 40 min before 10-trains TSS.</p

Involvement of CX3CL1 in spinal LTP.

<p>(A) As compared with 10-trains TSS-induced LTP, 3-trains TSS induced a LTP with smaller potentiated extent. While exogenous CX3CL1 (0.75 μg/30 μl) was applied 30 min before TSS, 3-trains TSS-induced LTP was robustly potentiated. (B) The facilitative effect of exogenous CX3CL1 (0.75 μg/30 μl) on 3 trains TSS-induced LTP was completely blocked by CX3CR1 AB (30 μg/30 μl), which was applied 2 h before TSS (1.5 h before delivering CX3CL1). (C) There was a delayed facilitative effect of 3.75 μg/30 μl exogenous CX3CL1 on baseline C-response, as compared with control PBS, and no influence of CX3CL1 was observed on baseline C-response at the dose of 0.75 μg/30 μl. (D) Western blot showed 30 min after 10-trains TSS, the expression of membrane-bound CX3CL1 was evidently reduced in the spinal dorsal horn, whereas soluble CX3CL1 level was upregulated in spinal CSF. Inset: the membrane-bound CX3CL1 and soluble CX3CL1 were detected at the 95 kDa and 72 kDa band respectively in the spinal dorsal horn (SDH) and CSF by an anti-CX3CL1 antibody. (E) ELASA assay showed that soluble CX3CL1 in the CSF was significantly upregulated at 30 min after TSS. (F) Western blot showed that Cathepsin S level was upregulated in the CSF at 30 min after TSS. * p<0.05 vs. Sham control.</p

MASS2 interacts with YDA.

(A) Yeast two-hybrid assay for MASS2.2 interaction with YDA. The BD and AD empty vectors were used as negative controls. Interaction tests were shown on the medium supplemented with -Leu-Trp-His. (B) In vitro pull-down assays using recombinant proteins, MBP-YDA and His-MASS2.3. MBP alone was used as negative control. Immunoblots were visualized by anti-His and anti-MBP. (C) BiFC assays to test the interaction between YDAKI and MASS2 in tobacco leaf epidermis. The expression of half YFPs (YFPN and YFPC) were used as negative controls. Scale bars represent 50 μm. (D) Co-IP assay to test the interaction between YDAKI and MASS2. 35S::4xMyc-MASS2g was transiently co-expressed with 35S::YDAKI-YFP or 35S::YFP in tobacco leaves.</p