Journal: Annals of Translational Medicine

Article Title: YTHDF3 facilitates triple-negative breast cancer progression and metastasis by stabilizing ZEB1 mRNA in an m 6 A-dependent manner

doi: 10.21037/atm-21-6857

Figure Lengend Snippet: YTHDF3 expression is correlated with poor prognosis in TNBC patients. (A,B) Kaplan-Meier analysis of the association between DFS and YTHDF3 mRNA expression in TNBC patients using TCGA database and BC-GenExMiner. (C) Representative IHC images of weak, moderate, and strong staining of YTHDF3 in TNBC tumor tissues (×200 magnification, scale bar: 50 μm). (D,E) Kaplan-Meier analysis of the association between DFS and OS with YTHDF3 protein expression in TNBC patients using the IHC method. YTHDF3, the YTH domain family 3; TNBC, triple-negative breast cancer; DFS, disease-free survival; TCGA, The Cancer Genome Atlas; IHC, immunohistochemistry; OS, overall survival.

Article Snippet: Samples were transferred to polyvinylidene fluoride (PVDF) membranes and incubated with the following primary antibodies: anti-YTHDF3 (ab220161, Abcam, Cambridge, UK), anti-ZEB1 (21544-1-AP, Proteintech, Wuhan, China), anti-E-cadherin (20874-1-AP, Proteintech, Wuhan, China), anti-N-cadherin (22018-1-AP, Proteintech, Wuhan, China), anti-vimentin (10366-1-AP, Proteintech, Wuhan, China), and anti-GAPDH (ab181602, Abcam, Cambridge, UK).

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Annals of Translational Medicine

Article Title: YTHDF3 facilitates triple-negative breast cancer progression and metastasis by stabilizing ZEB1 mRNA in an m 6 A-dependent manner

doi: 10.21037/atm-21-6857

Figure Lengend Snippet: Associations of YTHDF3 expression with clinicopathological characteristics for triple-negative breast cancer patients

Article Snippet: Samples were transferred to polyvinylidene fluoride (PVDF) membranes and incubated with the following primary antibodies: anti-YTHDF3 (ab220161, Abcam, Cambridge, UK), anti-ZEB1 (21544-1-AP, Proteintech, Wuhan, China), anti-E-cadherin (20874-1-AP, Proteintech, Wuhan, China), anti-N-cadherin (22018-1-AP, Proteintech, Wuhan, China), anti-vimentin (10366-1-AP, Proteintech, Wuhan, China), and anti-GAPDH (ab181602, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Annals of Translational Medicine

Article Title: YTHDF3 facilitates triple-negative breast cancer progression and metastasis by stabilizing ZEB1 mRNA in an m 6 A-dependent manner

doi: 10.21037/atm-21-6857

Figure Lengend Snippet: Univariate and multivariate cox proportional hazard model for DFS and OS in TNBC patients

Article Snippet: Samples were transferred to polyvinylidene fluoride (PVDF) membranes and incubated with the following primary antibodies: anti-YTHDF3 (ab220161, Abcam, Cambridge, UK), anti-ZEB1 (21544-1-AP, Proteintech, Wuhan, China), anti-E-cadherin (20874-1-AP, Proteintech, Wuhan, China), anti-N-cadherin (22018-1-AP, Proteintech, Wuhan, China), anti-vimentin (10366-1-AP, Proteintech, Wuhan, China), and anti-GAPDH (ab181602, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Annals of Translational Medicine

Article Title: YTHDF3 facilitates triple-negative breast cancer progression and metastasis by stabilizing ZEB1 mRNA in an m 6 A-dependent manner

doi: 10.21037/atm-21-6857

Figure Lengend Snippet: Knockdown of YTHDF3 suppresses cell migration, invasion, and EMT in MDA-MB-231 and BT-549 TNBC cells. (A) The protein expression of YTHDF3 in the MDA-MB-231, BT-549, HCC1937, and HS578T TNBC cell lines was detected by Western blot. (B,C) The knockdown efficiency of shYTHDF3 was analyzed by qRT-PCR and Western blot in MDA-MB-231 and BT-549 cells. (D,E) Cell migration and invasion ability were measured using the wound healing assay and transwell assay with YTHDF3 inhibition in MDA-MB-231 and BT-549 cells (transwell assay with crystal violet staining, ×200 magnification). (F) The protein levels of the EMT markers (E-cadherin, N-cadherin, and vimentin) were detected by Western blot with YTHDF3 knockdown in MDA-MB-231 and BT-549 cells. **, P<0.01; ***, P<0.001. YTHDF3, the YTH domain family 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; EMT, endothelial mesenchymal transition; TNBC, triple-negative breast cancer; qRT-PCR, quantitative real time-polymerase chain reaction.

Article Snippet: Samples were transferred to polyvinylidene fluoride (PVDF) membranes and incubated with the following primary antibodies: anti-YTHDF3 (ab220161, Abcam, Cambridge, UK), anti-ZEB1 (21544-1-AP, Proteintech, Wuhan, China), anti-E-cadherin (20874-1-AP, Proteintech, Wuhan, China), anti-N-cadherin (22018-1-AP, Proteintech, Wuhan, China), anti-vimentin (10366-1-AP, Proteintech, Wuhan, China), and anti-GAPDH (ab181602, Abcam, Cambridge, UK).

Techniques: Knockdown, Migration, Expressing, Western Blot, Quantitative RT-PCR, Wound Healing Assay, Transwell Assay, Inhibition, Staining, Real-time Polymerase Chain Reaction

Journal: Annals of Translational Medicine

Article Title: YTHDF3 facilitates triple-negative breast cancer progression and metastasis by stabilizing ZEB1 mRNA in an m 6 A-dependent manner

doi: 10.21037/atm-21-6857

Figure Lengend Snippet: ZEB1 is a key downstream target for YTHDF3 in triple-negative breast cancer. (A-F) The correlation between YTHDF3 and EMT-TFs, SNAIL1, SNAIL2, TWIST1, TWIST2, ZEB1, and ZEB2, was evaluated using Spearman’s correlation analysis based on TCGA database. (G) The mRNA expression of ZEB1 was identified by qRT-PCR after YTHDF3 knockdown in MDA-MB-231 and BT-549 cells. (H) The protein expression of ZEB1 was identified by Western blot after YTHDF3 knockdown in MDA-MB-231 and BT-549 cells. **, P<0.01. YTHDF3, the YTH domain family 3; SNAIL1/2, snail family transcriptional repressor 1/2; TWIST1/2, twist family transcription factor 1/2; ZEB1/2, zinc finger E-box-binding homeobox 1/2; EMT-TF, endothelial mesenchymal transition transcription factor. TCGA, The Cancer Genome Atlas; qRT-PCR, quantitative real time-polymerase chain reaction.

Article Snippet: Samples were transferred to polyvinylidene fluoride (PVDF) membranes and incubated with the following primary antibodies: anti-YTHDF3 (ab220161, Abcam, Cambridge, UK), anti-ZEB1 (21544-1-AP, Proteintech, Wuhan, China), anti-E-cadherin (20874-1-AP, Proteintech, Wuhan, China), anti-N-cadherin (22018-1-AP, Proteintech, Wuhan, China), anti-vimentin (10366-1-AP, Proteintech, Wuhan, China), and anti-GAPDH (ab181602, Abcam, Cambridge, UK).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Western Blot, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Annals of Translational Medicine

Article Title: YTHDF3 facilitates triple-negative breast cancer progression and metastasis by stabilizing ZEB1 mRNA in an m 6 A-dependent manner

doi: 10.21037/atm-21-6857

Figure Lengend Snippet: YTHDF3 enhances ZEB1 mRNA stability in an m6A-dependent manner. (A) RNA-binding protein immunoprecipitation (RIP) assay confirmed that YTHDF3 can bind to ZEB1 mRNA in MDA-MB-231 and BT-549 cells. (B) RIP assay validated that ZEB1 mRNA was modified with m6A modification in MDA-MB-231 and BT-549 cells. (C,D) The mRNA stability analysis verified that ZEB1 mRNA was drastically decreased after YTHDF3 knockdown in MDA-MB-231 and BT-549 cells. *, P<0.05; **, P<0.01; ***, P<0.001. YTHDF3, the YTH domain family 3; ZEB1, zinc finger E-box-binding homeobox 1; m6A, N6-methyladenosine.

Article Snippet: Samples were transferred to polyvinylidene fluoride (PVDF) membranes and incubated with the following primary antibodies: anti-YTHDF3 (ab220161, Abcam, Cambridge, UK), anti-ZEB1 (21544-1-AP, Proteintech, Wuhan, China), anti-E-cadherin (20874-1-AP, Proteintech, Wuhan, China), anti-N-cadherin (22018-1-AP, Proteintech, Wuhan, China), anti-vimentin (10366-1-AP, Proteintech, Wuhan, China), and anti-GAPDH (ab181602, Abcam, Cambridge, UK).

Techniques: RNA Binding Assay, Immunoprecipitation, Modification, Knockdown, Binding Assay

Journal: Annals of Translational Medicine

Article Title: YTHDF3 facilitates triple-negative breast cancer progression and metastasis by stabilizing ZEB1 mRNA in an m 6 A-dependent manner

doi: 10.21037/atm-21-6857

Figure Lengend Snippet: The YBX1/CTPS1 axis is involved in the progression of TNBC. (A,B) Cell migration and invasion ability were determined by wound healing and transwell assays after downregulation of YTHDF3 and overexpression of ZEB1 (transwell assay with crystal violet staining, ×200 magnification). (C) The protein levels of the EMT markers (E-cadherin, N-cadherin, and vimentin) after downregulation of YTHDF3 and overexpression of ZEB1were detected by Western blot. (D) The correlation between YTHDF3 and ZEB1 protein expression was evaluated by Spearman’s correlation analysis based on immunohistochemistry analysis. (E) Representative staining images of YTHDF3 and ZEB1 in TNBC tumor tissues (×200 magnification). (F) Kaplan-Meier analysis of the disease-free survival and overall survival with ZEB1 protein expression in TNBC patients. (G) Kaplan-Meier analysis of the disease-free survival and overall survival in TNBC patients expressing both YTHDF3 and ZEB1. Scale bar: 50 μm. **, P<0.01; ***, P<0.001 (shCtrl vs. shYTHDF3); and #, P<0.05, ##, P<0.01 (shYTHDF3 + Vector vs. shYTHDF3 + ZEB1). YTHDF3, the YTH domain family 3; ZEB1, zinc finger E-box-binding homeobox 1; TNBC, triple-negative breast cancer; EMT, epithelial-mesenchymal transition.

Article Snippet: Samples were transferred to polyvinylidene fluoride (PVDF) membranes and incubated with the following primary antibodies: anti-YTHDF3 (ab220161, Abcam, Cambridge, UK), anti-ZEB1 (21544-1-AP, Proteintech, Wuhan, China), anti-E-cadherin (20874-1-AP, Proteintech, Wuhan, China), anti-N-cadherin (22018-1-AP, Proteintech, Wuhan, China), anti-vimentin (10366-1-AP, Proteintech, Wuhan, China), and anti-GAPDH (ab181602, Abcam, Cambridge, UK).

Techniques: Migration, Over Expression, Transwell Assay, Staining, Western Blot, Expressing, Immunohistochemistry, Plasmid Preparation, Binding Assay

Journal: Molecular cell

Article Title: Altered m 6 A Modification of Specific Cellular Transcripts Affects Flaviviridae Infection

doi: 10.1016/j.molcel.2019.11.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following antibodies were used for immunoblot: anti-METTL3 (Novus Biologicals, 1:1000), anti-METTL14 (Sigma-Aldrich, 1:5000), anti-FTO (Abcam, 1:1000), anti-YTHDF1 (Proteintech, 1:1000), anti-YTHDF2 (Proteintech, 1:1000), anti-YTHDF3 (Sigma-Aldrich, 1:1000), anti-ALKBH5 (Sigma-Aldrich, 1:1000), anti-WTAP (Proteintech, 1:1000) anti-FLAG M2 (Sigma-Aldrich, 1:5000), anti-tubulin (Sigma-Aldrich, 1:5000), anti-HCV NS5A (clone 9E10, gift of Charles Rice, Rockefeller University ( Lindenbach et al., 2005 ), 1:1000), anti-RIOK3 (Proteintech, 1:1000), anti-CIRBP (Proteintech 1:1000), anti-DENV NS3 (GeneTex, 1:1000), anti-ZIKV NS3 (GeneTex, 1:1000), anti-HCV NS4A (Genscript custom ( Horner et al., 2011 )), 1:1000), anti-eIF2α (Cell Signaling, 1:1000), anti-phospho-eIF2α (Cell Signaling, 1:1000), anti-GADD34 (Proteintech, 1:1000), anti-HSPA5 (Cell Signaling, 1:1000), anti-H2A.X (Cell Signaling, 1:1000), anti-U170K serum (gift of Dr. Jack Keene, Duke University, ( Query and Keene, 1987 ), 1:1000)

Techniques: Recombinant, Protease Inhibitor, Purification, SYBR Green Assay, Luciferase, Reporter Assay, Cell Viability Assay, Clone Assay, Transfection, Plasmid Preparation, Infection, Functional Assay, Software