Journal: Cell reports

Article Title: Variation of Human Neural Stem Cells Generating Organizer States In Vitro before Committing to Cortical Excitatory or Inhibitory Neuronal Fates

doi: 10.1016/j.celrep.2020.107599

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were used at the concentration indicated by manufacturer’s protocol: CaM Kinase II alpha (6G9) (NB100–1983), LMX1A (NBP1–81303) Novusbio; SYNAPSIN (106 001), HOMER (160 003) Synaptic System; EGFR (Ab231), FGFR1 phosphoY654 (Ab59194), TBR1 (Ab31940), REELIN (Ab18570), CYCLIN D1 (Ab10540), FGFR2 (Ab10648), BMPR1A (Ab38560) Abcam; HES1 (11988), p-SMAD1/5 (9516), CYCLIN D1 (2926), pERK1/2 (4370), FGFR1 (9740) Cell Signaling Technology; PAX6 (PRB-278P) BioLegend; NESTIN (MAB1259), OTX2 (AF1979), PDGFR alpha (AF1062; AF307), SOX2 (AF2018; MAB2018), SOX21 (AF3538), TuJ1 (MAB1195), EGFR (AF1280), O4 (MAB 1326) R&D Systems; GFAP (Z 0334) DAKO; HES5 (sc-13859), CUX1 (sc-13024), TLE4 (sc-9125), FGFR3 (sc-9007), LHX2 (sc-19344) Santa Cruz Biotechnology; FOXP2 (ABE73), TBR1 (AB2261), REELIN (MAB5366) Millipore; FOXG1 (M227) Takara/Clontech; anti GAD65/67 was kindly gifted by Dr. Christian Geis, Hans Berger Department of Neurology, Jena University Hospital, Germany ( ).

Techniques: Virus, Plasmid Preparation, Recombinant, Transfection, Antibody Labeling, In Vitro, Microarray, Gene Expression, Derivative Assay, Software

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A Workflow for comprehensive identification of transcriptionally active lncRNAs in the gastric mucosa of patients with GC. B Pie chart depicting gene loci harboring H3K4me3 peaks at transcription start sites (TSSs). These loci were categorized as protein-coding genes, lncRNA genes, or other genes. Peaks were categorized as patient-specific, healthy individual-specific, and common. C H3K4me3 peaks of representative genes in each category. D Summarized qRT-PCR results for 15 selected lncRNAs from eight GC cell lines. E qRT-PCR analysis of TCONS_00006264 (TM4SF1-AS1) and TM4SF1 in GC cell lines and normal stomach tissue. The locations of these two genes are shown at the top. ( n = 3). F Expression of TM4SF1-AS1 in primary GCs ( n = 374) and normal stomach tissues ( n = 32) from The Cancer Genome Atlas (TCGA) dataset. * P < 0.05.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Expressing

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A qRT-PCR analysis of TM4SF1-AS1 in the indicated GC cell lines transfected with siRNAs targeting TM4SF1-AS1 (si-1 and 2) or a control siRNA (si-Ctrl). ( n = 3). B Results of cell viability assays with GC cell lines transfected with the indicated siRNAs. ( n = 8). C qRT-PCR analysis of TM4SF1-AS1 in HSC-45 cells with inducible shRNAs targeting TM4SF1-AS1 (sh-1 and 2) or a control shRNA (sh-Ctrl). Cells were incubated for 8 days with or without doxycycline (Dox). ( n = 3). D Colony formation assays using HSC-45 cells with inducible shRNAs. Cells were incubated for 8 days with or without Dox. Summarized results are shown on the right; error bars represent SDs. ( n = 3). E Tumor growth in mice injected with HSC-45 cells inducibly expressing the indicated shRNAs. Mice were treated with or without Dox. Growth curves (left), resected tumors (middle), and tumor weights (right) are shown. F qRT-PCR analysis of TM4SF1-AS1 in the resected tumors in E . ( n = 4). G Tumor growth in mice injected with SNU638 cells stably expressing GFP or TM4SF1-AS1. Growth curves are shown on the left, and resected tumors are shown on the right. ( n = 4). H qRT-PCR analysis of TM4SF1-AS1 in the resected tumors in G . ( n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001, NS not significant.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Transfection, Control, shRNA, Incubation, Injection, Expressing, Stable Transfection

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A qRT-PCR analysis of TM4SF1-AS1 in the indicated cellular fractions from HSC-45 cells. U6 snRNA and ACTB served as endogenous controls. ( n = 3). B Association between TM4SF1-AS1 and Pur-α in the indicated subcellular fractions from HSC-45 cells. Pur-α was detected by western blotting in proteins pulled-down with BrU-labeled TM4SF1-AS1, antisense of TM4SF1-AS1 or beads. C Association between Pur-α and YB-1. Immunoprecipitated YB-1 or Pur-α from HSC-45 cell extracts were probed for co-precipitating proteins. D YB-1 was immunoprecipitated from HSC-45 cells with or without RNase A treatment, after which Pur-α was detected by western blotting. E YB-1 or Pur-α in the indicated subcellular fractions from HSC-45 cells were immunoprecipitated with or without RNase A treatment and then probed for co-precipitating proteins. F Results of RIP assays. Pur-α (upper) or YB-1 (lower) was immunoprecipitated from HSC-45 cells, after which co-precipitated TM4SF1-AS1 was detected with qRT-PCR. IgG served as a negative control. ( n = 3). G qRT-PCR analysis of Pur-α (upper) and YB-1 (lower) in HSC-45 cells transfected with the indicated siRNAs. ( n = 3). H Results of cell viability assays with HSC-45 cells transfected with the indicated siRNAs. ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Labeling, Immunoprecipitation, Negative Control, Transfection

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A Heatmap showing expression of genes suppressed by TM4SF1-AS1 knockdown in HSC-45 cells. B GO analysis using the genes in A . C qRT-PCR analysis of representative IRDS genes in HSC-45 cells transfected with the indicated siRNAs. ( n = 3). D GSEA analysis using the microarray data. Gene sets with an FDR < 0.01 are shown on the left. Enrichment plots of indicated gene sets are shown on the right. E Western blot analysis of total and phosphorylated STAT1 in HSC-45 cells transfected with the indicated siRNAs. (F, G) qRT-PCR analysis of interferon genes in HSC-45 cells transfected with the indicated siRNAs ( F ) or in SNU638 cells stably transfected with the indicated genes ( G ). ( n = 3). H Western blot analysis of total and phosphorylated STAT1 in SNU638, SNU638-GFP, and SNU638-TM4SF1-AS1 cells. I qRT-PCR analysis of IRDS genes in SNU638-GFP and SNU638-TM4SF1-AS1 cells. ( n = 3). J Western blot analysis of STAT1 and Pur-α in SNU638-TM4SF1-AS1 cells transfected with the indicated siRNAs targeting Pur-α. K qRT-PCR analysis of IRDS genes in SNU638-TM4SF1-AS1 cells transfected with the indicated siRNAs. ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Transfection, Microarray, Western Blot, Stable Transfection

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A Workflow of ChIRP coupled with mass spectrometry (ChIRP-MS) or RNA-seq (ChIRP-RNA-seq) to identify molecules associated with TM4SF1-AS1. B qRT-PCR confirming enrichment of TM4SF1-AS1 in ChIRP products derived from SNU638-TM4SF1-AS1 cells. ( n = 3). C Protein-protein interaction (PPI) network among the proteins identified by ChIRP-MS. Functional categories of the proteins are indicated by node colors. D RIP-qPCR assays validating the ChIRP-MS results. The indicated proteins in HSC-45 cells were immunoprecipitated, and co-precipitated TM4SF1-AS1 was detected by qRT-PCR. IgG served as a negative control. ( n = 3). E Immunofluorescence images showing staining of the SG marker G3PB2 in SNU638, SNU638-GFP, and SNU638-TM4SF1-AS1 cells treated with or without sodium arsenite (SA). Summarized results are shown on the right ( n = 5). Scale bars = 10 μm. F Immunofluorescence indicating G3BP2 (green), TIA1 (white), and inducible MS2-tagged TM4SF1-AS1 (red) in SNU638 cells. Cells were transfected with a MS2 coat protein (MCP)-RFP plasmid and incubated for 8 days with or without Dox. Magnified views of the respective markers are shown on the right. Scale bars = 10 μm. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Mass Spectrometry, RNA Sequencing Assay, Quantitative RT-PCR, Derivative Assay, Functional Assay, Immunoprecipitation, Negative Control, Immunofluorescence, Staining, Marker, Transfection, Plasmid Preparation, Incubation

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A Immunofluorescence indicating RACK1 (green), G3BP1 (white), and inducible MS2-tagged TM4SF1-AS1 (red) in SNU638 cells. The cells were transfected with a MS2 coat protein (MCP)-RFP plasmid and incubated for 8 days with or without Dox. Magnified views of the respective markers are shown on the right. Scale bars = 10 μm. B Immunofluorescence images of G3PB2 in HSC-45 cells incubated for 8 days with or without Dox and expressing the indicated inducible shRNAs. Representative images are shown on the left. Summarized results are on the right. ( n = 5). C , D Apoptosis ( C ) and cell cycle ( D ) assays in HSC-45 cells incubated with or without Dox for 8 days and expressing the indicated inducible shRNAs. Representative results are shown on the left. Summarized results are shown on the right. ( n = 3). E , F Western blot analysis of PARP, caspase-3 ( E ) and p38 ( F ) in HSC-45 cells incubated with or without Dox for 8 days and expressing the indicated inducible shRNAs. Cleaved PARP and cleaved caspase-3 are indicated by arrows ( E ). Phosphorylated p38 is indicated by an arrow ( F ). ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Incubation, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A , B Immunofluorescence images showing Pur-α (A) or YB-1 (B), TIA1 and inducible MS2-tagged TM4SF1-AS1 in SNU638 cells incubated for 8 days with or without Dox. Scale bars = 10 μm. C Immunofluorescent staining of G3PB2 in SNU638-TM4SF1-AS1 cells transfected with the indicated siRNAs. Representative results are shown on the left. Summarized results are shown on the right. ( n = 5). Scale bars = 10 μm. D Localization of GFP-tagged Pur-α or YB-1, G3BP1 and TIA1 immunofluorescence in SNU638 cells. Cells were transfected with vectors encoding GFP (upper), GFP-tagged Pur-α (middle) or GFP-tagged YB1 (bottom). Magnified views of the respective markers are shown on the right. Scale bars = 10 μm. E Cell cycle analysis of HSC-45 cells expressing the indicated siRNAs. ( n = 3). F , G Western blot analysis of PARP and caspase-3 ( F ) and total and phosphorylated p38 ( G ) in HSC-45 cells transfected with the indicated siRNAs. Cleaved PARP, cleaved caspase-3 and phosphorylated p38 are indicated by arrows. ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Immunofluorescence, Incubation, Staining, Transfection, Cell Cycle Assay, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: In the early steps of gastric tumorigenesis, TM4SF1-AS1 is transcriptionally activated. TM4SF1-AS1 sequesters RACK1 within SGs, which leads to suppression of stress-responsive MAPK signaling and inhibition of apoptosis (created with BioRender.com).

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Inhibition