Journal: Neural Regeneration Research

Article Title: Salsolinol as an RNA m 6 A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy

doi: 10.4103/NRR.NRR-D-23-01592

Figure Lengend Snippet: Changes in the gene expression profiles and signaling pathways in PC12 cells treated with Sal. (A) Heatmaps of the relative expression levels of total DEGs between PC12 cells treated with Sal and controls. (B) The results identified 740 upregulated genes and 1665 downregulated genes after Sal treatment. (C) The mRNA levels of m6A-related genes, as determined by RNA sequencing. (D) KEGG analysis of downregulated DEGs in the Sal treatment group. (E) The mRNA levels of genes involved in the Hippo signaling pathway, as determined by RNA sequencing. Data in C and E were normalized by the control group. Data are expressed as the mean ± SD ( n = 3/group). * P < 0.05, *** P < 0.001, vs. Ctrl group (Student’s t -test). ALKBH5: alkB homolog 5; Ctrl: control; DEG: differentially expression gene; FTO: fat mass and obesity-associated protein; LATS1/2: large tumor suppressor homolog 1/2; m 6 A: N 6 -methyladenosine; METTL14: methyltransferase-like 14; METTL3: methyltransferase-like 3; MST1/2: STE20-like serine/threonine kinases1/2; Sal: salsolinol; TAZ: transcriptional coactivator with PDZ-binding motif; WTAP: Wilms tumor 1-associated protein; YAP1: Yes-associated protein 1.

Article Snippet: The membrane was blocked with 5% skimmed milk and incubated with mouse anti-FTO monoclonal antibody (1:1000, Abcam, Cat# ab92821, RRID: AB_10565042), rabbit anti-ALKBH5 monoclonal antibody (1:1000, Abcam, Cat# ab195377, RRID: AB_2827986), rabbit anti-METTL3 monoclonal antibody (1:1000, Abcam, Cat# ab195352, RRID: AB_2721254), mouse anti-YAP1 monoclonal antibody (1:8000, Proteintech, Chicago, IL, USA, Cat# 66900-1-Ig, RRID: AB_2882229), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:100,000, Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), rabbit anti-YTH domain-containing family protein 1 (YTHDF1) polyclonal antibody (1:2000, Proteintech, Cat# 17479-1-AP, RRID: AB_2217473), rabbit anti-YTH domain-containing family protein 2 (YTHDF2) polyclonal antibody (1:5000, Proteintech, Cat# 24744-1-AP, RRID: AB_2687435), mouse anti-YTH domain-containing family protein 3 (YTHDF3) monoclonal antibody (1:1000, Santa Cruz Biotechnology, Texas, CA, USA, Cat# sc-377119, RRID: AB_2687436), rabbit anti-p62 polyclonal antibody (1:1000, MBL, Tokyo, Japan, Cat# PM045, RRID: AB_1279301), rabbit anti-light chain 3 beta (LC3B) polyclonal antibody (1:1000, Cell Signaling Technology, Boston, MA, USA, Cat# 2775, RRID:AB_915950), and rabbit anti-phospho-YAP1 (Ser127) (1:1000, Cell Signaling Technology, Cat# 13008, RRID: AB_2650553) at 4°C overnight.

Techniques: Expressing, RNA Sequencing Assay, Control, Binding Assay, Wilms Tumor Assay

Journal: Neural Regeneration Research

Article Title: Salsolinol as an RNA m 6 A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy

doi: 10.4103/NRR.NRR-D-23-01592

Figure Lengend Snippet: The effect of Sal on YAP1 expression and m 6 A modification of Yap1 mRNA. (A) YAP1 and p-YAP1 expression in PC12 cells treated with different concentrations of Sal. (B) Yap1 mRNA levels in cells treated with Sal. (C) SRAMP website prediction of the m 6 A modification sites in rat Yap1 mRNA. Red indicates very high reliability, and blue indicates high reliability. (D) The 1510 and 1520 sites in rat Yap1 mRNA underwent m 6 A modification; IgG was used as the negative control. (E) The 2156 site in rat Yap1 mRNA underwent m 6 A modification; IgG was used as the negative control. (F) The 2795 site in rat Yap1 mRNA underwent m 6 A modification; IgG was used as the negative control. (G) The m 6 A levels at the 1510 and 1520 sites in rat Yap1 mRNA increased significantly in cells treated with Sal. (H) The m 6 A levels at the 2156 site in the rat Yap1 mRNA increased significantly after Sal induction. (I) The m 6 A levels at the 2795 site in the rat Yap1 mRNA increased significantly after induction by Sal. Data are expressed as the mean ± SD ( n = 3/group). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . Ctrl group (Student’s t -test). Ctrl: Control; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; IP: immunoprecipitation; m 6 A: N 6 -methyladenosine; p-YAP1: phospho-YAP1; Sal: salsolinol; YAP1: Yes-associated protein 1.

Article Snippet: The membrane was blocked with 5% skimmed milk and incubated with mouse anti-FTO monoclonal antibody (1:1000, Abcam, Cat# ab92821, RRID: AB_10565042), rabbit anti-ALKBH5 monoclonal antibody (1:1000, Abcam, Cat# ab195377, RRID: AB_2827986), rabbit anti-METTL3 monoclonal antibody (1:1000, Abcam, Cat# ab195352, RRID: AB_2721254), mouse anti-YAP1 monoclonal antibody (1:8000, Proteintech, Chicago, IL, USA, Cat# 66900-1-Ig, RRID: AB_2882229), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:100,000, Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), rabbit anti-YTH domain-containing family protein 1 (YTHDF1) polyclonal antibody (1:2000, Proteintech, Cat# 17479-1-AP, RRID: AB_2217473), rabbit anti-YTH domain-containing family protein 2 (YTHDF2) polyclonal antibody (1:5000, Proteintech, Cat# 24744-1-AP, RRID: AB_2687435), mouse anti-YTH domain-containing family protein 3 (YTHDF3) monoclonal antibody (1:1000, Santa Cruz Biotechnology, Texas, CA, USA, Cat# sc-377119, RRID: AB_2687436), rabbit anti-p62 polyclonal antibody (1:1000, MBL, Tokyo, Japan, Cat# PM045, RRID: AB_1279301), rabbit anti-light chain 3 beta (LC3B) polyclonal antibody (1:1000, Cell Signaling Technology, Boston, MA, USA, Cat# 2775, RRID:AB_915950), and rabbit anti-phospho-YAP1 (Ser127) (1:1000, Cell Signaling Technology, Cat# 13008, RRID: AB_2650553) at 4°C overnight.

Techniques: Expressing, Modification, Negative Control, Control, Immunoprecipitation

Journal: Neural Regeneration Research

Article Title: Salsolinol as an RNA m 6 A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy

doi: 10.4103/NRR.NRR-D-23-01592

Figure Lengend Snippet: m 6 A RNA methylation regulates YAP1 expression in vitro and in vivo . (A) m 6 A modification at the 1510 and 1520 sites of rat Yap1 mRNA after FTO or ALKBH5 was knocked down. (B) m 6 A modification at the 2156 site of the rat Yap1 mRNA after FTO or ALKBH5 was knocked down. (C) m 6 A modification at the 2795 site of the rat Yap1 mRNA after FTO or ALKBH5 was knocked down. (D) YAP1 protein expression did not change in PC12 cells after knocking down FTO. (E) YAP1 protein expression decreased in PC12 cells after knocking down ALKBH5. (F) YAP1 protein expression remained unchanged In the striatum of the rat brain after FTO was knocked down and decreased after ALKBH5 was knocked down. (G) YAP1 protein expression remained unchanged in the midbrain of the rat after FTO was knocked down and decreased after ALKBH5 was knocked down. Data were normalized by the control group. Data are expressed as the mean ± SD ( n = 3/group). * P < 0.05, ** P < 0.01, **** P < 0.0001, vs. Scr group (Student’s t -test). ALKBH5: alkB homolog 5; FTO: fat mass and obesity-associated protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; IP: immunoprecipitation; m 6 A: N 6 -methyladenosine; Scr: scramble; shALKBH5: short hairpin RNA of alkB homolog 5; shFTO: short hairpin RNA of fat mass and obesity-associated protein; YAP1: Yes-associated protein 1.

Article Snippet: The membrane was blocked with 5% skimmed milk and incubated with mouse anti-FTO monoclonal antibody (1:1000, Abcam, Cat# ab92821, RRID: AB_10565042), rabbit anti-ALKBH5 monoclonal antibody (1:1000, Abcam, Cat# ab195377, RRID: AB_2827986), rabbit anti-METTL3 monoclonal antibody (1:1000, Abcam, Cat# ab195352, RRID: AB_2721254), mouse anti-YAP1 monoclonal antibody (1:8000, Proteintech, Chicago, IL, USA, Cat# 66900-1-Ig, RRID: AB_2882229), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:100,000, Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), rabbit anti-YTH domain-containing family protein 1 (YTHDF1) polyclonal antibody (1:2000, Proteintech, Cat# 17479-1-AP, RRID: AB_2217473), rabbit anti-YTH domain-containing family protein 2 (YTHDF2) polyclonal antibody (1:5000, Proteintech, Cat# 24744-1-AP, RRID: AB_2687435), mouse anti-YTH domain-containing family protein 3 (YTHDF3) monoclonal antibody (1:1000, Santa Cruz Biotechnology, Texas, CA, USA, Cat# sc-377119, RRID: AB_2687436), rabbit anti-p62 polyclonal antibody (1:1000, MBL, Tokyo, Japan, Cat# PM045, RRID: AB_1279301), rabbit anti-light chain 3 beta (LC3B) polyclonal antibody (1:1000, Cell Signaling Technology, Boston, MA, USA, Cat# 2775, RRID:AB_915950), and rabbit anti-phospho-YAP1 (Ser127) (1:1000, Cell Signaling Technology, Cat# 13008, RRID: AB_2650553) at 4°C overnight.

Techniques: Methylation, Expressing, In Vitro, In Vivo, Modification, Control, Immunoprecipitation, shRNA

Journal: Neural Regeneration Research

Article Title: Salsolinol as an RNA m 6 A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy

doi: 10.4103/NRR.NRR-D-23-01592

Figure Lengend Snippet: YTHDF2 induces degradation of Yap1 mRNA in dopaminergic neurons. (A) The effect of different concentrations of Sal on the expression of YTHDF1, YTHDF2, and YTHDF3. (B) Confirmation of YTHDF2 overexpression in PC12 cells. (C) YAP1 protein expression decreased after YTHDF2 was overexpressed. (D) Confirmation of YTHDF2 knockdown in PC12 cells. (E) YAP1 expression was not changed after YTHDF2 was knocked down. (F) NC and siYTHDF2 cells were treated with actinomycin D for 90 minutes, and Yap1 mRNA was analyzed after pre-determined durations. The half-life of Yap1 mRNA increased after YTHDF2 was knocked down. Data were normalized by the NC group. Data are expressed as the mean ± SD ( n = 3/group). ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . NC or ctrl group (Student’s t -test). Ctrl: Control; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; NC: normal control; oe-YTHDF2: overexpression of YTHDF2; Sal: salsolinol; siYTHDF2: small interfering RNA of YTHDF2; YAP1: Yes-associated protein 1; YTHDF1/2/3: YTH domain-containing family protein 1/2/3.

Article Snippet: The membrane was blocked with 5% skimmed milk and incubated with mouse anti-FTO monoclonal antibody (1:1000, Abcam, Cat# ab92821, RRID: AB_10565042), rabbit anti-ALKBH5 monoclonal antibody (1:1000, Abcam, Cat# ab195377, RRID: AB_2827986), rabbit anti-METTL3 monoclonal antibody (1:1000, Abcam, Cat# ab195352, RRID: AB_2721254), mouse anti-YAP1 monoclonal antibody (1:8000, Proteintech, Chicago, IL, USA, Cat# 66900-1-Ig, RRID: AB_2882229), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:100,000, Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), rabbit anti-YTH domain-containing family protein 1 (YTHDF1) polyclonal antibody (1:2000, Proteintech, Cat# 17479-1-AP, RRID: AB_2217473), rabbit anti-YTH domain-containing family protein 2 (YTHDF2) polyclonal antibody (1:5000, Proteintech, Cat# 24744-1-AP, RRID: AB_2687435), mouse anti-YTH domain-containing family protein 3 (YTHDF3) monoclonal antibody (1:1000, Santa Cruz Biotechnology, Texas, CA, USA, Cat# sc-377119, RRID: AB_2687436), rabbit anti-p62 polyclonal antibody (1:1000, MBL, Tokyo, Japan, Cat# PM045, RRID: AB_1279301), rabbit anti-light chain 3 beta (LC3B) polyclonal antibody (1:1000, Cell Signaling Technology, Boston, MA, USA, Cat# 2775, RRID:AB_915950), and rabbit anti-phospho-YAP1 (Ser127) (1:1000, Cell Signaling Technology, Cat# 13008, RRID: AB_2650553) at 4°C overnight.

Techniques: Expressing, Over Expression, Knockdown, Control, Small Interfering RNA

Journal: Neural Regeneration Research

Article Title: Salsolinol as an RNA m 6 A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy

doi: 10.4103/NRR.NRR-D-23-01592

Figure Lengend Snippet: Autophagy regulated by Sal is partly related to the inhibition of YAP1. (A) Sal treatment decreased the expression of p62 and increased LC3. (B) Autophagy-related proteins were examined in cells treated with Sal alone or in combination with BafA1 treatment. (C) In PC12 cells with YAP1 knockdown, the levels of p62 decreased and LC3 increased. Data are expressed as the mean ± SD ( n = 3/group). * P < 0.05, *** P < 0.001, vs. NC group (Student’s t -test). (D) In PC12 cells treated with Verteporfin, the levels of p62 decreased and LC3 increased. (E) A schematic model in which Sal acts as an RNA m 6 A methylation inducer, mediating dopaminergic neuronal death by regulating YAP1 and autophagy. ALKBH5: alkB homolog 5; BafA1: Bafilomycin A1; FTO: fat mass and obesity-associated protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; LC3: light chain 3; m 6 A: N 6 -methyladenosine; METTL3: methyltransferase-like 3; NC: normal control; p62: Sequestosome 1; Sal: salsolinol; siYAP1: small interfering RNA of YAP1; YAP1: Yes-associated protein 1; YTHDF2: YTH domain-containing family protein 2.

Article Snippet: The membrane was blocked with 5% skimmed milk and incubated with mouse anti-FTO monoclonal antibody (1:1000, Abcam, Cat# ab92821, RRID: AB_10565042), rabbit anti-ALKBH5 monoclonal antibody (1:1000, Abcam, Cat# ab195377, RRID: AB_2827986), rabbit anti-METTL3 monoclonal antibody (1:1000, Abcam, Cat# ab195352, RRID: AB_2721254), mouse anti-YAP1 monoclonal antibody (1:8000, Proteintech, Chicago, IL, USA, Cat# 66900-1-Ig, RRID: AB_2882229), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:100,000, Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), rabbit anti-YTH domain-containing family protein 1 (YTHDF1) polyclonal antibody (1:2000, Proteintech, Cat# 17479-1-AP, RRID: AB_2217473), rabbit anti-YTH domain-containing family protein 2 (YTHDF2) polyclonal antibody (1:5000, Proteintech, Cat# 24744-1-AP, RRID: AB_2687435), mouse anti-YTH domain-containing family protein 3 (YTHDF3) monoclonal antibody (1:1000, Santa Cruz Biotechnology, Texas, CA, USA, Cat# sc-377119, RRID: AB_2687436), rabbit anti-p62 polyclonal antibody (1:1000, MBL, Tokyo, Japan, Cat# PM045, RRID: AB_1279301), rabbit anti-light chain 3 beta (LC3B) polyclonal antibody (1:1000, Cell Signaling Technology, Boston, MA, USA, Cat# 2775, RRID:AB_915950), and rabbit anti-phospho-YAP1 (Ser127) (1:1000, Cell Signaling Technology, Cat# 13008, RRID: AB_2650553) at 4°C overnight.

Techniques: Inhibition, Expressing, Knockdown, Methylation, Control, Small Interfering RNA

Journal: iScience

Article Title: Highly efficient generation of self-renewing trophoblast from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110874

Figure Lengend Snippet:

Article Snippet: Monoclonal Anti-YAP1 antibody produced in mouse , Millipore-Sigma , WH0010413M1-100UG.

Techniques: Purification, Produced, Virus, Control, Recombinant, Lysis, Extraction, Cell Culture, Expressing, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Western Blot, Multiplex Assay, Sequencing, Software, Modification