Rbm46 downregulates the mRNA level of <i>β-Catenin</i>.

(A) pPE-E14 cells (Control) and pPy-Rbm46 cells were lysed and subjected to western blot with the indicated antibodies. (B) RT-PCR analysis of the indicated mRNA levels in pPE-E14 cells (Control) and pPy-Rbm46 cells. Experiment was performed in triplicate. Error bars showed the standard deviation. **, pRbm46 shRNA cells were lysed and subjected to western blot with the indicated antibodies. (D) RT-PCR analysis of the indicated mRNA levels in Non-silence cells and Rbm46 shRNA cells. Experiment was performed in triplicate. Error bars showed the standard deviation. ***, pRbm46 RNAi. Luciferase activities were measured after 48 h. The Luciferase activity was normalized to the control reporter (SV40 Poly A) activity. Experiments were performed in triplicate. Error bars showed the standard deviation. ***, p<0.001; t-test for (G) and one-way ANOVA with Tukey’s post hoc test for (H).</p

Rbm46 regulates mouse embryonic stem cell differentiation by targeting <i>β-Catenin</i> mRNA for degradation

<div><p>Embryonic stem cells (ESCs) are pluripotent cells and have the capability for differentiation into any of the three embryonic germ layers. The Wnt/<i>β-Catenin</i> pathway has been shown to play an essential role in ESC differentiation regulation. Activation of <i>β-Catenin</i> by post-translational modification has been extensively studied. However, mechanism(s) of post-transcriptional regulation of <i>β-Catenin</i> are not well defined. In this study, we report an RNA recognition motif-containing protein (RNA binding motif protein 46, RBM46) which regulates the degradation of <i>β-Catenin</i> mRNA. Our results show that Rbm46 is distributed primarily in the cytoplasm of mouse ESCs (mESCs) and is elevated during the process of ESC differentiation. In addition, overexpression of Rbm46 results in differentiation of mESCs into trophectoderm, while knock-down of <i>Rbm46</i> leads to mESC differentiation into endoderm. β-Catenin, a key effector in the Wnt pathway which has been reported to play a significant role in the regulation of ESC differentiation, is post-transcriptionally regulated by Rbm46. Our study reveals Rbm46 plays a novel role in the regulation of ESC differentiation.</p></div

Downregulation of Rbm46 promotes differentiation of mESC into endoderm.

(A) RT-PCR and Western Blot analysis of the Rbm46 shRNA-E14 stable cell lines. (B) Left panel shows the morphology of the Non-silence cells and Rbm46 shRNA cells. Middle panel shows the AKP staining and immunostaining with SSEA-1 antibody of Non-silence cells and Rbm46 shRNA cells. (C) FACs analysis of the SSEA-1 positive cells in Non-silence cells and Rbm46 shRNA cells. (D) and (E) Representative images and tumor weight analysis of the teratocarcinoma formed by Non-silence cells and Rbm46 shRNA cells. (F) RT-PCR analysis of mRNA level of the marker from different layers in Non-silence cells and Rbm46 shRNA cells. The columns show the quantification of the indicated gene relative to Gapdh. The average value in the Non-silence group is normalized as 1. Experiment was performed in triplicate. Error bars showed the standard deviation. *, pRbm46 shRNA cells. The columns show the quantification of the indicated gene relative to Gapdh. The average value in the Non-silence group is normalized as 1. Experiment was performed in triplicate. Error bars showed the standard deviation. *, p<0.05; ***, p<0.001; t-test.</p

Rbm46 interacts with the P-bodies.

<p>(A) pPy-Rbm26 cells which can stably express Flag-Rbm46 were lysed and subjected to FLAG immunoprecipitation, followed by MS analysis. (B) Flag-Rbm46 was transfected into E14 cells for 24 hours and were lysed and subjected to FLAG immunoprecipitation. Total RNA extracted from the eluate was subjected to RT-PCR with primers specific to <i>β-Catenin</i> and <i>Gapdh</i> (negative control). (C) Double immunofluorescence staining using antibodies against Rbm46 (red) and Pabpc1 (green) shows a clear co-localization in E14/T mESCs.</p

Overexpression of Rbm46 results in differentiation of mESC into trophectoderm.

<p>(A) Left panel shows the morphology of the pPE-E14 cells and pPy-Rbm46 cells. Middle panel shows the AKP staining and immunostaining with SSEA-1 antibody of the pPE-E14 cells and pPy-Rbm46 cells. (B) FACs analysis of the SSEA-1 positive cells in pPE-E14 cells and pPy-Rbm46 cells. (C) and (D) Representative images and tumor weight analysis of the teratocarcinoma formed by pPE-E14 cells and pPRbm46 cells. (E) RT-PCR analysis of mRNA level of the marker from different layers in pPE-E14 cells and pPy-Rbm46 cells. The columns show the quantification of the indicated <i>gene</i> relative to <i>Gapdh</i>. The average value in the control group is normalized as 1. Experiment was performed in triplicate. Error bars showed the standard deviation. *, p<0.05; **, p<0.01, t-test. (F) RT-PCR analysis of the trophectoderm markers expressed in the teratocarcinoma formed by pPy-Rbm46 cells. The columns show the quantification of the indicated <i>gene</i> relative to <i>Gapdh</i>. The average value in the control group is normalized as 1. Experiment was performed in triplicate. Error bars showed the standard deviation. *, p<0.05, t-test.</p

Rbm46 inhibits mESC proliferation.

<p>(A) RT-PCR and Western Blot analysis of the pPy-Rbm46-E14 stable cell lines. (B) CCK-8 analysis of the proliferation rate of pPE-E14 cells and pPy-Rbm46 cells within 72 hours after seeding. Experiment was performed in triplicate. Error bars showed the standard deviation. (C) Statistics analysis of the average diameter of the colonies formed by pP-E14 cells and pPy-Rbm46 cells, respectively. Experiment was performed in triplicate. Error bars showed the standard deviation. ***, p<0.001; t-test. (D) Representative profiles of the pPE-E14 cells and pPy-Rbm46 cells by Annexin V/PI staining. (E) and (F) RT-PCR analysis of <i>Oct4</i> and <i>Rbm46</i> mRNA level at the indicated time points in E14 cells induced to undergo differentiation by removal of LIF. Experiments were performed in triplicate. Error bars showed the standard deviation.</p

Down-regulation of <i>Pcsk1n</i> advanced C-terminal cleavage and activity of PC1/3 in MIN6 cells.

<p><b>A</b>, MIN6 cells were transfected with <i>Pcsk1n</i> siRNA for 48 h, and protein was collected to analyze the PC1/3 C-terminal cleavage with western blots. <b>B</b>, ratio of densitometry scanning of 87 kDa and 66 kDa PC1/3 bands was analyzed using MIN6 cells transfected with <i>Pcsk1n</i> siRNA (black bar) or scrambled siRNA (white bar) (n = 3). <b>C</b>, enzyme activity was analyzed using MIN6 cells transfected with <i>Pcsk1n</i> siRNA (black circles) or scrambled siRNA (white circles) with equivalent protein (n = 3). <b>D</b>, the specificity of enzyme activity measured was tested by PC1/3 specific inhibitor Ac-LLRVKR-NH₂ using MIN6 cells. Black squares, <i>Pcsk1n</i> siRNA; Black circles, scrambled siRNA; White squares, <i>Pcsk1n</i> siRNA + inhibitor; White circles, scrambled siRNA + inhibitor. A representative experiment is shown; the experiment was repeated three times. Each value represents the mean ± SE. *<i>p<</i>0.05.</p

Additional file 1: of Differentiation of adult human retinal pigment epithelial cells into dopaminergic-like cells in vitro and in the recipient monkey brain

Figure S1. Chemical compounds reported to induce neural cell differentiation and reprogramming. Figure S2. Chemically induced reprogramming of RPE cells into dopamine-producing cells. (A) Immunofluorescent staining of chemically induced RPE cells for FOXA2 (scale bar: 50 μm).(B) Immunofluorescent staining of chemically induced RPE cells for TH (scale bar: 50 μm). (Empty, without any chemical small molecules; S, SB431542; C, CHIR99021; Com SC, combination of SB431542 and CHIR99021; Com LSC, combination of LDN193189, SB431542 and CHIR99021.). Figure S3. Characteristics of chemically induced dopamine-producing cells. (A) Immunofluorescent staining of chemically induced RPE cells for VMAT2 or DAT (scale bar: 50 μm). (B) Immunofluorescent staining of chemically induced RPE cells for GRIK2 or Calbindin along with TH (scale bar: 50 μm). (DOCX 9774 kb

<i>Pax6</i> deficiency led to decrease of PC1/3 C-terminal cleavage and activity in mice.

<p><b>A</b>, expression of 87 kDa and 66 kDa PC1/3 in isolated islets in three operations as measured by western blot, each blot show the islets protein from several mice in wild type (from left to right, <i>n</i> = 7, 3, 3) versus <i>Pax6</i> mutant mice (<i>n</i> = 9, 3, 3). 60 µg islets protein of wild-type and <i>Pax6</i> mutant mice was used. <b>B</b>, ratio of densitometry scanning of 87 kDa and 66 kDa PC1/3 bands is shown (n = 3). <b>C</b>, <i>Pax6</i> mutant mice (<i>Pax6^m/+</i>) exhibited lowered activity of PC1/3. Islets protein of wild-type (<i>n</i> = 6) and <i>Pax6</i> mutant mice (<i>n</i> = 6) with equivalent PC1/3 normalized by western blot (the upper row) were used to determine PC1/3 activity in the presence of 400 µM fluorogenic substrate. Wild type: white bars, <i>Pax6^m/+</i>: black bars. A representative experiment is shown; the experiment was repeated three times. Each value represents the mean ± SE. *<i>p</i><0.05, **<i>p</i><0.01.</p

Down-regulationof <i>Pcsk1n</i> rescued the effect of Pax6 knock-down on PC1/3 and proinsulin processing.

<p><b>A</b>, MIN6 cells were transfected with <i>Pax6</i>, <i>Pax6</i> and <i>Pcsk1n</i> or scrambled siRNA in triplicate, and 48 hours later protein was analyzed by western blot. <b>B</b>, ratio of densitometry scanning of 87 kDa and 66 kDa PC1/3 bands is shown (n = 3). <b>C</b>, enzyme activity of PC1/3 was examined in <i>Pax6</i> RNAi or <i>Pax6</i> and <i>Pcsk1n</i> double RNAi treated MIN6 cells with equivalent protein. Scrambled siRNA was used as negative control (n = 3). Black circles, values for transfected with Pax6 siRNA; Grey circles, values for transfected with both <i>Pax6</i> and <i>Pcsk1n</i> siRNA; White circles, scrambled siRNA. <b>D</b>, the true insulin and total insulin concentrations in conditional medium of MIN6 cells were measured by ELISA. The cells were transfected with scrambled siRNA, Pax6 siRNA or <i>Pax6</i> and <i>Pcsk1n</i> siRNA, and aliquots of conditional medium were applied to ELISA Kits for total insulin or true insulin detection (n = 3). True insulin, the mature form of insulin, which was derived from proinsulin cleaved by PC1/3 and PC2; total insulin, the sum of true insulin and proinsulin. White bars, value for transfected with scrambled siRNA; Black bars, value for transfected with Pax6 siRNA; Grey bars, value for transfected with both of <i>Pax6</i> and <i>Pcsk1n</i> siRNA. A representative experiment is shown; the experiment was repeated three times. Each value represents the mean ± SE. *<i>p</i><0.05, **<i>p</i><0.01.</p