Journal: PLoS ONE

Article Title: Specification of Region-Specific Neurons Including Forebrain Glutamatergic Neurons from Human Induced Pluripotent Stem Cells

doi: 10.1371/journal.pone.0011853

Figure Lengend Snippet: ( A & B ) Immunostaining for the forebrain functional markers TBR1 and MAP2 ( A ), and CTIP2 and VGLUT1 ( B ) on cells differentiated for 5 ( A ) or 6 ( B ) weeks from H9 hESC and TZ1 and YZ1 hiPSC lines. ( C ) Immunostaining for the dopaminergic neuron marker TH on cells differentiated from hESC/hiPSC-derived and FGF8/SHH-treated neural progenitors. ( D ) Immunostaining for the spinal motor neuronal marker HB9 (with βIII-tubulin as a neuronal control marker) on cells differentiated from hESC/hiPSC-derived and RA/SHH-treated neural progenitors. ( E ) Some cells were positive for S100β (an astrocyte marker) or Synapsin at two months after differentiation from H9 or TZ1 cells. Cell nuclei were counterstained with Hoechst 33342. Bar, 50 µm. ( F ) Percentage of cells immunostained positive for TBR1, HB9, and TH counted for A , C , and D , respectively.

Article Snippet: Mouse anti-PAX6 (final dilution 1∶5000), rat anti-HOXB4 (1∶20), mouse anti-MNR2 (HB9, 1∶50), and rabbit anti-βIII-tubulin (1∶5000) antibodies were from Developmental Studies Hybridoma Bank (Iowa City, IA).

Techniques: Immunostaining, Functional Assay, Marker, Derivative Assay

Journal: Cell Death & Disease

Article Title: LncRNA MNX1-AS1 promotes progression of intrahepatic cholangiocarcinoma through the MNX1/Hippo axis

doi: 10.1038/s41419-020-03029-0

Figure Lengend Snippet: a Heatmap and volcano map of DEGs (FDR < 0.05, |log2FC| > 2) based on the TCGA-CHOL data sets. The locations of MNX1-AS1 and MNX1 were labeled (Supplementary Table : 5842 up and 1115 down). b Heatmap and volcano map of DEGs (FDR < 0.05, |log2FC| > 2) based on the GSE107943 data sets. The locations of MNX1-AS1 and MNX1 were labeled (Supplementary Table : 6842 up and 1149 down). c Venn diagram of upregulated genes in TCGA-CHOL and GSE107943 data sets, 3544 common upregulated genes were found (all FDR < 0.05, log2FC > 2), and both MNX1-AS1 and MNX1 were included. d Schematic representation of the MNX1-AS1 and MNX1. MNX1-AS1 and MNX1 were located at two chains of the same chromosome, and the coding region of MNX1-AS1 overlaps with the promoter region of MNX1 to a certain extent. e The expression of MNX1-AS1 and MNX1 was significantly higher in ICC tissues compared with adjacent normal tissues in the GSE107943 data set. Their expression was highly and positively correlated (Pearson: P < 0.001, R 2 = 0.875, *** P < 0.001 by Wilcoxon test). f MNX1-AS1 and MNX1 expression levels were quantified in 33 pairs of ICC tissues and adjacent normal tissues using qRT-PCR, and their expression was highly and positively correlated (Pearson: P < 0.001, R 2 = 0.88, *** P < 0.001 by Wilcoxon test) in ICC tissues. g The MNX1-AS1 expression levels in three cholangiocarcinoma cell lines (RBE, QBC939, and FRH0201) and one normal bile duct epithelial cell line (HiBEpiC) were quantified by qRT-PCR, the experiment was repeated three times for each cell line (** P < 0.01, *** P < 0.001 by ANOVA). h FISH assay was used to show the localization of MNX1-AS1 in FRH0201 and RBE cell lines, which indicated that MNX1-AS1 was mainly located in the nucleus. i Immunohistochemical staining showed that MNX1 protein expression was significantly higher in ICC tissues than that in normal tissues.

Article Snippet: The slices were then incubated overnight with the mouse anti-MNX1 (1:200, Sigma-Aldrich) at 4 °C.

Techniques: Labeling, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Cell Death & Disease

Article Title: LncRNA MNX1-AS1 promotes progression of intrahepatic cholangiocarcinoma through the MNX1/Hippo axis

doi: 10.1038/s41419-020-03029-0

Figure Lengend Snippet: a MNX1 protein was markedly increased in pcDNA-MNX1-AS1 or pcDNA-MNX1 transfected RBE cells and significantly decreased in si-MNX1-AS1 or si-MNX1 transfected FRH0201 cells. The experiment was repeated three times for each group (*** P < 0.001 by t -test). b The GeneCards and PROMO databases were used to predict TFs which may bind to the promoter region of MNX1. Only five TFs were shared between the two databases: YY1, c-Myc, MAZ, USF1, WT1. c ChIP assay showed the enrichment of c-Myc and MAZ over the MNX1 promoter region in FRH0201 and RBE cells. d The expression level of MNX1 protein was significantly decreased in si-c-Myc or si-MAZ transfected FRH0201 cells. e RegRNA database was used to predict c-Myc and MAZ binding sites on the nucleotide sequence of MNX1-AS1, which indicated that the binding sites did exist. RIP assay in FRH0201 cells further determined the previous predicting results. The experiment was repeated three times for each group. (*** P < 0.001 by t -test).

Article Snippet: The slices were then incubated overnight with the mouse anti-MNX1 (1:200, Sigma-Aldrich) at 4 °C.

Techniques: Transfection, Expressing, Binding Assay, Sequencing

Journal: Cell Death & Disease

Article Title: LncRNA MNX1-AS1 promotes progression of intrahepatic cholangiocarcinoma through the MNX1/Hippo axis

doi: 10.1038/s41419-020-03029-0

Figure Lengend Snippet: a CCK-8 and colony formation assays indicated that the overexpressed MNX1-AS1 enhanced the proliferation ability of RBE cells, whereas knockdown of MNX1-AS1 elicited the opposite results in FRH0201 cells. The experiment was repeated three times for each group. (* P < 0.05, *** P < 0.001 by t -test). b Cell migration and invasion assays showed that the overexpressed MNX1-AS1 in RBE cells enhanced cell migration and invasion abilities, whereas knockdown of MNX1-AS1 elicited the opposite trends in FRH0201 cells. The experiment was repeated three times for each group. (* P < 0.05, *** P < 0.001 by t -test). c Endothelial tube formation assay demonstrated that the upregulated MNX1-AS1 enhanced cell angiogenic ability, whereas downregulation of MNX1-AS1 expression repressed cell angiogenic ability. The experiment was repeated three times for each group. (* P < 0.05, ** P < 0.01 by t -test).

Article Snippet: The slices were then incubated overnight with the mouse anti-MNX1 (1:200, Sigma-Aldrich) at 4 °C.

Techniques: CCK-8 Assay, Migration, Endothelial Tube Formation Assay, Expressing

Journal: Cell Death & Disease

Article Title: LncRNA MNX1-AS1 promotes progression of intrahepatic cholangiocarcinoma through the MNX1/Hippo axis

doi: 10.1038/s41419-020-03029-0

Figure Lengend Snippet: a The effect of MNX1-AS1 on tumor formation after subcutaneous transplantation in vivo. The left image showed the dissected tumors from FRH0201 cells stably transfected with scrambled (NC-MNX1-AS1 group) or sh-MNX1-AS1 (SH-MNX1-AS1 group); the middle image showed the tumor volume growth curves of SH-MNX1-AS1 and NC-MNX1-AS1 groups between 0 and 4 weeks; the right image showed the scatter plots of tumor weight with horizontal lines. The results clarified that downregulated MNX1-AS1 repressed the growth of ICC in vivo. (*** P < 0.001 by t -test). b The effect of MNX1-AS1 on tumor metastasis in vivo. The left image showed the dissected lungs from FRH0201 cells stably transfected with scrambled (NC-MNX1-AS1 group) or sh-MNX1-AS1 (SH-MNX1-AS1 group); the middle image showed Hematoxylin–Eosin (HE) staining and Ki-67 IHC staining between the two groups; the right image showed Ki-67 IHC score between the two groups. (** P < 0.01 by t -test). The IHC staining was repeated three times for each group. The results indicated that downregulated MNX1-AS1 repressed the metastasis of ICC in vivo.

Article Snippet: The slices were then incubated overnight with the mouse anti-MNX1 (1:200, Sigma-Aldrich) at 4 °C.

Techniques: Transplantation Assay, In Vivo, Stable Transfection, Transfection, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: LncRNA MNX1-AS1 promotes progression of intrahepatic cholangiocarcinoma through the MNX1/Hippo axis

doi: 10.1038/s41419-020-03029-0

Figure Lengend Snippet: a Pie diagram illustrated the distribution of MNX1-related elements. b Heatmap of ChIP-seq results. c GO enrichment analysis of ChIP-seq of MNX1. Top 10 terms of each class were performed. BP biological processes, CC cellular components, MF molecular functions. d KEGG pathway analysis of ChIP-seq of MNX1 showed that MNX1 was involved in diverse cancer-related pathways (Supplementary Table ).

Article Snippet: The slices were then incubated overnight with the mouse anti-MNX1 (1:200, Sigma-Aldrich) at 4 °C.

Techniques: ChIP-sequencing

Journal: Cell Death & Disease

Article Title: LncRNA MNX1-AS1 promotes progression of intrahepatic cholangiocarcinoma through the MNX1/Hippo axis

doi: 10.1038/s41419-020-03029-0

Figure Lengend Snippet: a The motif analysis was conducted to ChIP-seq of MNX1. b ChIP assay was performed to determine whether MNX1 bound to the promoter of Ajuba. The result indicated that MNX1 bound to the promoter of Ajuba (P2 and P3). c The expression of essential molecules in the Hippo signaling pathway after upregulating or downregulating the expression of MNX1-AS1 or MNX1 by western blot assay. The results indicated that overexpression of MNX1 facilitated the expression of Ajuba protein, and subsequently repressed the activity of the Hippo pathway, the western blot assay was repeated three times for each group. (*** P < 0.001 by t -test).

Article Snippet: The slices were then incubated overnight with the mouse anti-MNX1 (1:200, Sigma-Aldrich) at 4 °C.

Techniques: ChIP-sequencing, Expressing, Western Blot, Over Expression, Activity Assay

Journal: Cell Death & Disease

Article Title: LncRNA MNX1-AS1 promotes progression of intrahepatic cholangiocarcinoma through the MNX1/Hippo axis

doi: 10.1038/s41419-020-03029-0

Figure Lengend Snippet: LncRNA MNX1-AS1 facilitates the expression of MNX1 via recruiting transcription factors c-Myc and MAZ in the nucleus. Then, the MNX1 protein prompts the expression of Ajuba protein via binding to its promoter region. The increasing Ajuba protein in the cytoplasm subsequently suppresses the Hippo pathway by inhibits Mst1/2 and Lats1. These processes eventually lead to the increasing of YAP1 in the nucleus. The elevated YPA1 in the nucleus facilitates the tumorigenesis of ICC.

Article Snippet: The slices were then incubated overnight with the mouse anti-MNX1 (1:200, Sigma-Aldrich) at 4 °C.

Techniques: Expressing, Binding Assay

Journal: bioRxiv

Article Title: A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

doi: 10.1101/2023.12.12.571328

Figure Lengend Snippet: (a) RfxCas13d domain organization and RfxCas13d-N2V7 and RfxCas13d-N2V8 mutations. (b, c) Relative all-V and V3 mRNA in (b) HEK293T and (c) SH-SY5Y cells transfected with RfxCas13d, RfxCas13d-N2V7 and RfxCas13d-N2V8 with crRNA-13 or a non-targeted (NTG) crRNA. All values normalized to untreated cells ( n = 3). (d) Volcano plot of the RNA-seq analysis comparing HEK293T cells transfected with (left) RfxCas13d or (right) RfxCas13d-N2V8 with crRNA-13 to each variant with NTG crRNA ( n = 3). Lines denote a >1.25-fold change (FC) and an FDR-adjusted P < 0.01. (e) Number of differentially expressed genes (DEGs) [>1.25-FC, FDR-adjusted P < 0.01] from (d). (f) Gene ontology (GO) and biological process (BP) term analysis for the DEGs in (d). Line denotes FDR-adjusted P < 0.05. (g) Venn diagram of overlapping DEGs. (h) Immunostaining of neurospheres. Cells positive for Hb9 and choline acetyltransferase (ChAT). Scale bar, 75 µm. (i) Brightfield and fluorescent images of neurospheres seven days after treatment with AAV-PHP.eB-EGFP-KASH. (j, k) Relative (j) V3 to all-V mRNA and (k) CBLN1 and CBLN2 mRNA in neurospheres treated with AAV-PHP.eB-RfxCas13d-N2V8-crRNA-13 or -NTG ( n = 4). Values indicate means and error bars indicate SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; (b, c) one-tailed unpaired t-test comparing crRNA-13 to NTG crRNA for each variant; (j, k) one-tailed unpaired t-test comparing crRNA-13 to NTG crRNA (b, c, j, k). All data points are biologically independent samples.

Article Snippet: Primary antibodies were mouse anti-HB9 (1:200; Thermo Fisher Scientific, PA5-23407), and goat anti-ChAT (1:50; EMD Millipore, AB144P.

Techniques: Transfection, RNA Sequencing Assay, Variant Assay, Immunostaining, One-tailed Test