Journal: Frontiers in Oncology

Article Title: TFAP2C-Mediated lncRNA PCAT1 Inhibits Ferroptosis in Docetaxel-Resistant Prostate Cancer Through c-Myc/miR-25-3p/SLC7A11 Signaling

doi: 10.3389/fonc.2022.862015

Figure Lengend Snippet: TFAP2C transcriptionally activates PCAT1 expression in DTX-resistant cells. (A) Venn diagram showing the possible transcription factors within the promoter regions of PCAT1 by online bioinformatics analysis (HUMAN TFDB and ALGGEN). (B-D) The mRNA and protein levels of TFAP2C were detected by qRT-PCR and Western blot in DTX-resistant PCa cells and their sensitive cells. (E) The mRNA levels of TFAP2C, PCAT1 and SLC7A11 in PC3/DR cells were detected after transfected with siTFAP2C and control siRNA. (F) qRT-PCR assay was used to assess the expression of TFAP2C, PCAT1 and SLC7A11 in PC3 cells after TFAP2C overexpression. (G) Schematic diagram showing location of TFAP2C binding sites on the promoter regions of PCAT1. (H) ChIP-qPCR was used to detect the binding efficiency of TFAP2C on the PCAT1 promoter. (I) The luciferase activities were measured in TFAP2C overexpressed PC3 cells after co-transfected with PCAT1 wild-type and mutant reporter plasmids. (J) Cell viability was assessed by CCK-8 assay in TFAP2C-knockdown PC3 cells after transfected with PCAT1 and vector plasmids. (K) The relative expression of TFAP2C was measured by qRT-PCR in 30 pairs of clinical PCa samples. (L) The positive relation between TFAP2C and PCAT1 in clinical samples (n=30) by qRT-PCR. (M) The schematic diagram showing that TFAP2C-induced PCAT1 inhibits ferroptosis in DTX-resistant PCa cells through c-Myc/miR-25-3p/SLC7A11 signaling. The data are presented as the mean ± SD, * P < 0.05.

Article Snippet: The primary antibodies were as follows: SLC7A11 (1:1000, Abcam, USA, Cat. ab175186), GPX4 (1:1000, Abcam, USA, Cat. ab125066), TFAP2C (1:1000, Proteintech, China, Cat. 14572-1-AP), c-Myc (1:1000, Proteintech, Cat. 67447-1-Ig) and β-actin (1:1000, Proteintech, China, Cat. 66009-1-Ig).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Control, Over Expression, Binding Assay, ChIP-qPCR, Luciferase, Mutagenesis, CCK-8 Assay, Knockdown, Plasmid Preparation

Journal: eLife

Article Title: Defects in translation-dependent quality control pathways lead to convergent molecular and neurodevelopmental pathology

doi: 10.7554/eLife.66904

Figure Lengend Snippet:

Article Snippet: After blocking in 5% nonfat dry milk (Cell Signaling, #9999S), blots were probed with primary antibodies at 4°C overnight: rabbit anti-phospho-eIF2α S51 (Cell Signaling, #9721, 1:1000), rabbit anti-eIF2α (Cell Signaling, #9722, 1:2000), rabbit anti-Hbs1l (Proteintech, 10359–1-AP, 1;1000), rabbit anti-Pelo (Proteintech, 10582–1-AP, 1:2000), rabbit anti-GAPDH (Cell Signaling, #2118, 1:10,000), rabbit anti-phospho-p70S6K T389 (Cell Signaling, #9234, 1:1000), rabbit anti-phospho-p70S6K (Cell Signaling, #2708, 1:1000), mouse anti-vinculin (Sigma, V-9131, 1:20,000).

Techniques: RNAscope, Multiplex Assay, SYBR Green Assay, Sequencing, Quantitative RT-PCR, Software

Journal: Molecular Therapy. Nucleic Acids

Article Title: The miR-93-3p/ZFP36L1/ZFX axis regulates keratinocyte proliferation and migration during skin wound healing

doi: 10.1016/j.omtn.2020.11.017

Figure Lengend Snippet: ZFP36L1 is targeted by miR-93-3p in HaCaT cells (A) Alignment of miR-93-3p and its corresponding complementary binding sequence in the ZFP36L1 3′ UTR, as determined by the TargetScan bioinformatics algorithm. (B) HaCaT cells were transfected with 200 ng mL −1 luciferase reporter plasmid containing wild-type (WT) or mutant (Mut 1, Mut 2, and Mut all) ZFP36L1 3′ UTR or empty vector (Vector) together with 20 nM pre-miR-93-3p or pre-miR-Ctrl, and luciferase activity was measured 48 h later. (C and D) HaCaT cells were transfected with 20 nM pre-miR-93-3p/anti-miR-93-3p for 48 h, and ZFP36L1 expression was analyzed by qRT-PCR and western blot. (E) ZFP36L1 expression was analyzed by qRT-PCR in wound biopsies at the indicated time points. (F) Correlation analysis of ZFP36L1 and miR-93-3p in mouse wound biopsies by Pearson’s correlation coefficient (n = 6). (G) qRT-PCR for ZFP36L1 in the keratinocytes (PAM212 and HaCaT cell). (H) Immunofluorescence staining of ZFP36L1 (green) in wound sections, which were counterstained using 4′,6-diamidino-2-phenylindole (DAPI; blue, nucleus). Scale bar, 50 μm. Data of one representative experiment out of four independent experiments are shown with mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Sections were further incubated overnight with the primary antibodies toward ZFP36L1 (Proteintech; 12306-1-AP, 1:200) and Ki-67 (Servicebio; GB13030-2, 1:200) at 4°C, followed by goat anti-rabbit secondary antibody labeled with Alexa Fluor 488 at room temperature for 1 h. With the use of a fluorescence microscope (Olympus, Tokyo, Japan), signals were detected, and images were captured and analyzed using cellSens image analysis software (Olympus)

Techniques: Binding Assay, Sequencing, Transfection, Luciferase, Plasmid Preparation, Mutagenesis, Activity Assay, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: The miR-93-3p/ZFP36L1/ZFX axis regulates keratinocyte proliferation and migration during skin wound healing

doi: 10.1016/j.omtn.2020.11.017

Figure Lengend Snippet: Silencing of ZFP36L1 promotes HaCaT cell proliferation and migration HaCaT cells were transfected with 20 nM shRNA control or shRNA ZFP36L1 for 48 h. (A and B) The silencing efficiency was analyzed by measuring ZFP36L1 levels with qRT-PCR and western blot. (C) The expression of proliferation marker Ki-67 was analyzed in the transfected HaCaT cells using qRT-PCR. (D) Cell proliferation was measured by the EdU assay. Scale bar, 50 μm. The percentages of EdU+ cells are shown. (E) Cell-cycle analysis by flow cytometry. The percentages of cells in the G1, S, and G2/M phases of the cell cycle are shown. (F) Colony formation of the transfected HaCaT cells. (G) Scratch assays were performed to assess the migration of the transfected HaCaT cells. Photographs were taken at the indicated time points after scratch injury. (H) Representative photographs of the Transwell migration assay for the transfected HaCaT cells. The number of cells passing through the membrane was counted. Data of one representative experiment out of four independent experiments are shown with mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Sections were further incubated overnight with the primary antibodies toward ZFP36L1 (Proteintech; 12306-1-AP, 1:200) and Ki-67 (Servicebio; GB13030-2, 1:200) at 4°C, followed by goat anti-rabbit secondary antibody labeled with Alexa Fluor 488 at room temperature for 1 h. With the use of a fluorescence microscope (Olympus, Tokyo, Japan), signals were detected, and images were captured and analyzed using cellSens image analysis software (Olympus)

Techniques: Migration, Transfection, shRNA, Control, Quantitative RT-PCR, Western Blot, Expressing, Marker, EdU Assay, Cell Cycle Assay, Flow Cytometry, Transwell Migration Assay, Membrane

Journal: Molecular Therapy. Nucleic Acids

Article Title: The miR-93-3p/ZFP36L1/ZFX axis regulates keratinocyte proliferation and migration during skin wound healing

doi: 10.1016/j.omtn.2020.11.017

Figure Lengend Snippet: ZFP36L1 affects ZFX expression in HaCaT cells (A) Six conserved AU-rich elements located in the 3ʹ UTR of ZFX transcripts of humans and mice. The sequence and location of the AU-rich elements are presented as a graphic. (B) Schematic representation of the UTR1–UTR6 regions. (C) RT-PCR quantification of RNA immunoprecipitations (IPs). Association of ZFP36L1 with ZFX 3′ UTR regions (UTR1, UTR2, UTR3, UTR4, UTR5, and UTR6). Whole-cell lysates from HaCaT cells were used for immunoprecipitation using anti-ZFP36L1 antibody or nonspecific IgG. Total RNA was extracted from immunoprecipitated complexes, reverse transcribed, and subjected to PCR analysis using primers to amplify the UTR1–UTR6 regions of ZFX 3′ UTR. The negative control was IgG (mouse). Glyceraldehyde 3-phosphate dehydrogenase expression was determined (input) to ensure that equal amounts of samples were taken during the RNA immunoprecipitation experiment. (D) HaCaT cells were transiently transfected for 48 h with ZFP36L1 plasmid (control, 0.5, 1.0, or 1.5 μg) and were then immunoblotted for ZFP36L1 and ZFX protein expression. (E) HaCaT cells were transiently transfected for 48 h with ZFP36L1 plasmid (control, 0.5, 1.0, or 1.5 μg), and RNA immunoprecipitation was performed using an anti-ZFP36L1 antibody. Total RNA was extracted from the immunoprecipitated complexes, reverse transcribed, and subjected to PCR analysis using ZFX 3′ UTR2 primers and densitometric analysis. (F) HaCaT cells were transiently transfected with shRNA ZFP36L1 (0, 24, and 48 h) and were then immunoblotted for ZFP36L1 and ZFX protein expression. (G) HaCaT cells were transiently transfected with shRNA ZFP36L1 (0, 24, and 48 h), and RNA immunoprecipitation was performed using anti-ZFP36L1 antibody. Total RNA was extracted from the immunoprecipitated complexes, reverse transcribed, and subjected to PCR analysis using ZFX 3′ UTR2 primers and densitometric analysis. Data of one representative experiment out of four independent experiments are shown with mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Sections were further incubated overnight with the primary antibodies toward ZFP36L1 (Proteintech; 12306-1-AP, 1:200) and Ki-67 (Servicebio; GB13030-2, 1:200) at 4°C, followed by goat anti-rabbit secondary antibody labeled with Alexa Fluor 488 at room temperature for 1 h. With the use of a fluorescence microscope (Olympus, Tokyo, Japan), signals were detected, and images were captured and analyzed using cellSens image analysis software (Olympus)

Techniques: Expressing, Sequencing, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Reverse Transcription, Negative Control, RNA Immunoprecipitation, Transfection, Plasmid Preparation, Control, shRNA

Journal: Molecular Therapy. Nucleic Acids

Article Title: The miR-93-3p/ZFP36L1/ZFX axis regulates keratinocyte proliferation and migration during skin wound healing

doi: 10.1016/j.omtn.2020.11.017

Figure Lengend Snippet: miR-93-3p promotes HaCaT cell proliferation and migration by upregulating ZFX expression HaCaT cells were transfected with 20 nM pre-miR-93-3p, ZFP36L1, or pre-miR-93-3p + ZFP36L1 for 48 h. (A and B) The expression of ZFX was determined by qRT-PCR and western blot. (C) The expression of proliferation marker Ki-67 was analyzed in the transfected HaCaT cells using qRT-PCR. (D) Cell proliferation was measured by the EdU assay. Scale bar, 50 μm. The percentages of EdU+ cells are shown. (E) Cell-cycle analysis was performed by flow cytometry. The percentages of cells in the G1, S, and G2/M phases of the cell cycle are shown. (F) Colony formation of the transfected HaCaT cells. (G) Scratch assays were performed to assess the migration of HaCaT cells. Photographs were taken at the indicated time points after scratch injury. (H) Representative photographs of the Transwell migration assay for HaCaT cells. The number of HaCaT cells passing through the membrane was counted. Data of one representative experiment out of four independent experiments are shown with mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Sections were further incubated overnight with the primary antibodies toward ZFP36L1 (Proteintech; 12306-1-AP, 1:200) and Ki-67 (Servicebio; GB13030-2, 1:200) at 4°C, followed by goat anti-rabbit secondary antibody labeled with Alexa Fluor 488 at room temperature for 1 h. With the use of a fluorescence microscope (Olympus, Tokyo, Japan), signals were detected, and images were captured and analyzed using cellSens image analysis software (Olympus)

Techniques: Migration, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Marker, EdU Assay, Cell Cycle Assay, Flow Cytometry, Transwell Migration Assay, Membrane

Journal: Molecular Therapy. Nucleic Acids

Article Title: The miR-93-3p/ZFP36L1/ZFX axis regulates keratinocyte proliferation and migration during skin wound healing

doi: 10.1016/j.omtn.2020.11.017

Figure Lengend Snippet: Inhibition of miR-93-3p delays skin wound healing in vivo Skin wounds were treated with anti-miR-Ctrl or anti-miR-93-3p and treated with or without control shRNA (Ad-shC) or Ad-shZFP36L1 for 14 days. (A) Left: representative images of treated wounds at 0 h, 24 h, 7 days, and 14 days postwound-injury. Right: level of wound closure is expressed as a percentage of the wound area from the initial wound area. (B−D) The expression of miR-93-3p, ZFP36L1, and ZFX, respectively, was analyzed by qRT-PCR in the wound-edge skin 7 days after injury. (E) Immunofluorescence staining for Ki-67 (green) in the wound-edge skin 7 days after injury, which was counterstained using DAPI (blue, nucleus). Scale bar, 50 μm; n = 6. Data are shown with mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus anti-miR-Ctrl #p < 0.05, ##p < 0.01, ###p < 0.001 versus Ad-shC.

Article Snippet: Sections were further incubated overnight with the primary antibodies toward ZFP36L1 (Proteintech; 12306-1-AP, 1:200) and Ki-67 (Servicebio; GB13030-2, 1:200) at 4°C, followed by goat anti-rabbit secondary antibody labeled with Alexa Fluor 488 at room temperature for 1 h. With the use of a fluorescence microscope (Olympus, Tokyo, Japan), signals were detected, and images were captured and analyzed using cellSens image analysis software (Olympus)

Techniques: Inhibition, In Vivo, Control, shRNA, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining