Journal: MedComm

Article Title: Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5‐Hydroxymethylcytosine‐Mediated Sp1 Transcription Factor Expression

doi: 10.1002/mco2.70261

Figure Lengend Snippet: 5‐hmC levels in senescent HUVECs and PQ‐induced aging mice. (A) β‐galactosidase staining and senescent cell ratio in replicative senescent HUVECs ( n = 4 for each group, unpaired two‐sided t ‐test, scale bars = 100 µm). (B) Protein levels of p16, p21, and p53 in replicative senescent HUVECs ( n = 3 for each group, unpaired two‐sided t ‐test). (C) 5‐hmC levels in replicative senescent HUVECs assayed by using a dot blot; methylene blue staining used as an internal control of the DNA content of samples ( n = 3 for each group, unpaired two‐sided t ‐test). (D) Levels of p‐γH2AX and 5‐hmC in replicative senescent HUVECs ( n = 6 for each group, unpaired two‐sided t ‐test, scale bars = 100 µm). Levels of p‐γH2AX and 5‐hmC in the heart (E, scale bars = 100 µm) and aorta tissue (F, scale bars = 400 µm) of PQ‐treated mice ( n = 3 for each group, unpaired two‐sided t ‐test). (G) 5‐mC and 5‐hmC landscape changes of the p21 in the senescent cells compared with the young cells. (H) 5‐mC and 5‐hmC landscape changes in the p53 in the senescent cells compared with the young cells. All data are expressed as the mean ± standard error of mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: HEK 293T cells were transfected with p53 shRNA plasmid (sc‐29435‐SH, Santa Cruz) or human p53 overexpression plasmid for 24 h. Then, the 293T cells were cotransfected with a mixture of the TET3 promoter plasmid and the pRL‐SV40 plasmid containing renilla luciferase gene.

Techniques: Staining, Dot Blot, Control

Journal: MedComm

Article Title: Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5‐Hydroxymethylcytosine‐Mediated Sp1 Transcription Factor Expression

doi: 10.1002/mco2.70261

Figure Lengend Snippet: Role of TET3 in regulating cellular senescence. (A) Protein levels of TET2 and TET3 in replicative senescent HUVECs ( n = 3 for each group, unpaired two‐sided t ‐test). (B) Protein levels of TET2 and TET3 in HUVECs treated with PQ ( n = 4 for each group, unpaired two‐sided t ‐test). (C) Protein levels of p16, p21, and p53, and (D) Immunofluorescent imaging of p‐γH2AX after TET2 or TET3 silencing in HUVECs treated with PQ ( n = 3 for each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test, scale bars = 100 µm). (E, F) Protein and mRNA levels of p16, p21, and p53 after TET2 or TET3 overexpression ( n = 3 for each group, unpaired two‐sided t ‐test). (G) Immunofluorescent imaging of p‐γH2AX in HUVECs after TET2 or TET3 overexpression ( n = 3 for each group, unpaired two‐sided t ‐test, scale bars = 50 µm). (H) Cell proliferation after TET3 overexpression in HUVECs ( n = 9 for each group, unpaired two‐sided t ‐test). All the data are expressed as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: HEK 293T cells were transfected with p53 shRNA plasmid (sc‐29435‐SH, Santa Cruz) or human p53 overexpression plasmid for 24 h. Then, the 293T cells were cotransfected with a mixture of the TET3 promoter plasmid and the pRL‐SV40 plasmid containing renilla luciferase gene.

Techniques: Imaging, Over Expression

Journal: MedComm

Article Title: Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5‐Hydroxymethylcytosine‐Mediated Sp1 Transcription Factor Expression

doi: 10.1002/mco2.70261

Figure Lengend Snippet: Role of TET3 knockout in regulating cellular senescence. (A, B) Protein and mRNA levels of p16, p21, and p53 in HUVECs with TET3 knockout ( n = 3 for each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test). (C) β‐galactosidase staining and senescent cell ratio in HUVECs with TET3 knockout ( n = 3 for each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test, scale bars = 100 µm). (D) Cell proliferation ratio in HUVECs with TET3 knockout ( n = 12 for each group, unpaired two‐sided t ‐test). (E, F) MRNA levels of SASP in HUVECs with TET3 knockout ( n = 3 for each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test). (G) Immunofluorescence imaging of p‐γH2AX and 5‐hmC in HUVECs with TET3 knockout ( n = 9 for each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test, scale bars = 100 µm). All data are expressed as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: HEK 293T cells were transfected with p53 shRNA plasmid (sc‐29435‐SH, Santa Cruz) or human p53 overexpression plasmid for 24 h. Then, the 293T cells were cotransfected with a mixture of the TET3 promoter plasmid and the pRL‐SV40 plasmid containing renilla luciferase gene.

Techniques: Knock-Out, Staining, Immunofluorescence, Imaging

Journal: MedComm

Article Title: Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5‐Hydroxymethylcytosine‐Mediated Sp1 Transcription Factor Expression

doi: 10.1002/mco2.70261

Figure Lengend Snippet: Role of TET3 in regulating aging in mice. (A, B) Protein level of TET3 in the heart and aorta tissues of PQ‐treated mice ( n = 3 for each group, unpaired two‐sided t ‐test, scale bars = 100 µm). (C, D) Protein levels of p21 and p53 in the senescent heart and aorta tissues of TET3‐heterozygous mice treated with PQ ( n = 3 for each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test, scale bars = 100 µm). Levels of p‐γH2AX and 5‐hmC in the senescent heart (E, scale bars = 100 µm) and aorta tissues (F, scale bars = 400 µm) of TET3‐heterozygous mice treated with PQ ( n = 6 for E, and 3 for F each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test). All data are expressed as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: HEK 293T cells were transfected with p53 shRNA plasmid (sc‐29435‐SH, Santa Cruz) or human p53 overexpression plasmid for 24 h. Then, the 293T cells were cotransfected with a mixture of the TET3 promoter plasmid and the pRL‐SV40 plasmid containing renilla luciferase gene.

Techniques:

Journal: MedComm

Article Title: Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5‐Hydroxymethylcytosine‐Mediated Sp1 Transcription Factor Expression

doi: 10.1002/mco2.70261

Figure Lengend Snippet: Regulation of TET3 on SP1‐ETS‐1 axis. (A) Changes in the total distribution of 5‐hmC in the genome in TET3 knockout/PQ‐treated HUVECs based on hMeDIP‐seq. (B) Distribution of DhMSs. (C) Overlapping DhMGs associated with senescence are shown using a Venn diagram. (D) Levels of KAT5 and SP1 mRNA after TET3 knockdown ( n = 3 for each group, unpaired two‐sided t ‐test). (E) Level of SP1 protein after TET3 knockdown ( n = 3 for each group, unpaired two‐sided t ‐test). (F) Level of 5‐hmC in SP1 protomer after TET3 knockdown. (G) The combination of SP1 and ETS‐1 after TET3 knockdown. (H, I) Protein and mRNA levels of p16, p21, and p53 after ETS‐1 knockdown ( n = 3 for each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test). All data are expressed as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: HEK 293T cells were transfected with p53 shRNA plasmid (sc‐29435‐SH, Santa Cruz) or human p53 overexpression plasmid for 24 h. Then, the 293T cells were cotransfected with a mixture of the TET3 promoter plasmid and the pRL‐SV40 plasmid containing renilla luciferase gene.

Techniques: Knock-Out, Knockdown

Journal: MedComm

Article Title: Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5‐Hydroxymethylcytosine‐Mediated Sp1 Transcription Factor Expression

doi: 10.1002/mco2.70261

Figure Lengend Snippet: Role of SP1 and EST1 on p53 expression. (A) SP1 binding enrichment on the p53 promoter ( n = 3 for each group, unpaired two‐sided t ‐test). EST1 binding enrichment on the p53 promoter in PQ‐induced (B) or replicative (C) senescent HUVECs detected using ChIP assay ( n = 3 for each group, unpaired two‐sided t ‐test). (D, E) Binding enrichment of SP1 and ETS‐1 on the p53 promoter after TET3 knockdown ( n = 3 for each group, unpaired two‐sided t ‐test). (F, G) Levels of SP1 and p53 in the senescent heart and aorta tissues of TET3‐heterozygous mice treated with PQ ( n = 3 for each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test, scale bars = 100 µm). All data are expressed as the mean ± SEM. * p < 0.05; ** p < 0.01.

Article Snippet: HEK 293T cells were transfected with p53 shRNA plasmid (sc‐29435‐SH, Santa Cruz) or human p53 overexpression plasmid for 24 h. Then, the 293T cells were cotransfected with a mixture of the TET3 promoter plasmid and the pRL‐SV40 plasmid containing renilla luciferase gene.

Techniques: Expressing, Binding Assay, Knockdown

Journal: MedComm

Article Title: Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5‐Hydroxymethylcytosine‐Mediated Sp1 Transcription Factor Expression

doi: 10.1002/mco2.70261

Figure Lengend Snippet: Role of p53 in regulating senescence in vivo and vitro. (A, B) Protein and mRNA levels of p16, p21, and p53 after p53 knockdown ( n = 3 for each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test). (C, D) Expression of p53, p16, and p21 mRNAs in the heart and aorta tissues of p53 + / − ‐ heterozygous mice treated with PQ ( n = 5 for C, and 3 for D each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test). (E) Protein levels of p53, p16, and p21 in the heart tissues of p53 + / − ‐heterozygous mice treated with PQ ( n = 3 for each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test). (F, G) Levels of p16, p21, and β‐galactosidase in the heart and aorta tissues of p53 + / − ‐heterozygous mice treated with PQ ( n = 3 for each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test, scale bars = 100 µm). All data are expressed as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: HEK 293T cells were transfected with p53 shRNA plasmid (sc‐29435‐SH, Santa Cruz) or human p53 overexpression plasmid for 24 h. Then, the 293T cells were cotransfected with a mixture of the TET3 promoter plasmid and the pRL‐SV40 plasmid containing renilla luciferase gene.

Techniques: In Vivo, Knockdown, Expressing

Journal: MedComm

Article Title: Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5‐Hydroxymethylcytosine‐Mediated Sp1 Transcription Factor Expression

doi: 10.1002/mco2.70261

Figure Lengend Snippet: Regulation of TET3 expression by p53. The capacity of the p53 protein (A) or RNA polymerase II (B) to bind to the TET3 promoter, determined using the ChIP assay in HUVECs ( n = 3 for each group, unpaired two‐sided t ‐test). (C, D) Luciferase reporter analysis of TET3 activity after p53 overexpression or silencing ( n = 3 for each group, unpaired two‐sided t ‐test). (E) Protein level of TET3 after p53 knockdown. (F) Protein level of TET3 in the heart tissues of p53 + / − ‐heterozygous mice treated with PQ ( n = 3 for each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test). (G, H) Levels of TET3 in the heart and aorta tissues of p53 + / − ‐heterozygous mice treated with PQ (n = 3 for each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test, scale bars = 100 µm). Levels of p‐γH2AX and 5‐hmc in the heart (I, scale bars = 100 µm) and aorta tissues (J, scale bars = 400 µm) of p53 + / − ‐heterozygous mice treated with PQ ( n = 9 for I, and 3 for J each group, one‐way analysis of variance followed by Dunnett's multiple comparisons test). All data are expressed as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: HEK 293T cells were transfected with p53 shRNA plasmid (sc‐29435‐SH, Santa Cruz) or human p53 overexpression plasmid for 24 h. Then, the 293T cells were cotransfected with a mixture of the TET3 promoter plasmid and the pRL‐SV40 plasmid containing renilla luciferase gene.

Techniques: Expressing, Luciferase, Activity Assay, Over Expression, Knockdown

Journal: Investigative and Clinical Urology

Article Title: DRG2 levels in prostate cancer cell lines predict response to PARP inhibitor during docetaxel treatment

doi: 10.4111/icu.20240263

Figure Lengend Snippet: Cell lines and status of DRG2, p53, and BRCA1/2

Article Snippet: Small interfering RNAs (siRNAs) targeting human DRG2 (siDRG2; sc-93839), human p53 (sip53; sc-29435), and control siRNA (scRNA; sc-37007) were purchased from Santa Cruz Biotechnology.

Techniques:

Journal: Investigative and Clinical Urology

Article Title: DRG2 levels in prostate cancer cell lines predict response to PARP inhibitor during docetaxel treatment

doi: 10.4111/icu.20240263

Figure Lengend Snippet: Recovery of prostate cancer cell lines after docetaxel treatment. (A) DRG2 and p53 expressions in prostate cancer cell lines as determined via western blot (W.B) analysis (full length blots; ). (B) DRG2 expression in prostate cancer cell lines as determined using real-time polymerase chain reaction (PCR). (C) IC50 values of docetaxel on prostate cancer cells were determined using a CCK assay. Cells were exposed to 0–10 µM docetaxel for 72 hours. (D) Cell viability of the prostate cancer cell lines after docetaxel treatment for 72 hours, determined using a CCK assay (during docetaxel treatment [left], after docetaxel treatment [right]). DRG2, developmentally regulated GTP-binding protein 2; OD, optical density. ** p<0.01; *** p<0.001.

Article Snippet: Small interfering RNAs (siRNAs) targeting human DRG2 (siDRG2; sc-93839), human p53 (sip53; sc-29435), and control siRNA (scRNA; sc-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Binding Assay

Journal: Investigative and Clinical Urology

Article Title: DRG2 levels in prostate cancer cell lines predict response to PARP inhibitor during docetaxel treatment

doi: 10.4111/icu.20240263

Figure Lengend Snippet: Differences in cell cycles depending on the presence or absence of DRG2 and p53 expression. (A) Cell viability assay in PC3 cells treated with APR-246 and docetaxel for 72 hours. (B) Cell viability assay in DU145 cells transfected with scRNA or sip53 after docetaxel treatment for 72 hours. (C) Changes in DRG2 expression in the cytoplasm and nuclear extracts after docetaxel treatment for 72 hours analyzed via western blot analysis (full length blots; ). (D) Immunofluorescence microscopy images of PC3 cells after docetaxel treatment for 72 hours. (E) Cell viability assay in DU145-pcDNA6-V5 and DU145-DRG2/pcDNA6-V5 cells after docetaxel treatment for 72 hours. (F) Flow cytometry analysis showing the difference in cell cycles of DU145-pcDNA6-V5 and DU145-DRG2/pcDNA6-V5 cells after treatment with docetaxel for 72 hours. (G) Cell viability assay in PC3 cells transfected with scRNA or siDRG2 after docetaxel treatment for 72 hours. (H) Flow cytometry analysis showing the difference in cell cycles of PC3 transfected with scRNA or siDRG2 after treatment with docetaxel for 72 hours. Cell viability was determined using the CCK assay. DRG2, developmentally regulated GTP-binding protein 2; OD, optical density; FITC, fluorescein isothiocyanate; DAPI, 4’,6-diamidino-2-phenylindole; ns, no significant. ** p<0.01; *** p<0.001; **** p<0.0001.

Article Snippet: Small interfering RNAs (siRNAs) targeting human DRG2 (siDRG2; sc-93839), human p53 (sip53; sc-29435), and control siRNA (scRNA; sc-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Viability Assay, Transfection, Western Blot, Immunofluorescence, Microscopy, Flow Cytometry, Binding Assay

Journal: Biomolecules

Article Title: Hepatitis B Virus X Protein Induces Reactive Oxygen Species Generation via Activation of p53 in Human Hepatoma Cells

doi: 10.3390/biom14101201

Figure Lengend Snippet: Figure 1. HBV infection induces higher levels of ROS generation in the presence of p53. HepG2-NTCP and Hep3B-NTCP cells were infected with HBV at the indicated MOI for 24 h in DMEM containing

Article Snippet: Scrambled (SC) short hairpin RNA (shRNA) (Cat No. sc-37007) and p53 shRNA (Cat No. sc-29435) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection

Journal: Biomolecules

Article Title: Hepatitis B Virus X Protein Induces Reactive Oxygen Species Generation via Activation of p53 in Human Hepatoma Cells

doi: 10.3390/biom14101201

Figure Lengend Snippet: Figure 3. HBx increases ROS levels by activating p53 during HBV replication. (A–D) HepG2 and Hep3B cells were transfected with the indicated amounts of HBx expression plasmid along with scrambled (SC) shRNA, p53 shRNA, or p53 expression plasmid for 48 h, followed by western blotting

Article Snippet: Scrambled (SC) short hairpin RNA (shRNA) (Cat No. sc-37007) and p53 shRNA (Cat No. sc-29435) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, shRNA, Western Blot

Journal: Biomolecules

Article Title: Hepatitis B Virus X Protein Induces Reactive Oxygen Species Generation via Activation of p53 in Human Hepatoma Cells

doi: 10.3390/biom14101201

Figure Lengend Snippet: Figure 4. HBx requires p53 transcriptional activity to upregulate ROS levels. (A,B) HepG2 and Hep3B cells were transfected with the indicated amounts of HBx expression plasmid along with p53 WT and

Article Snippet: Scrambled (SC) short hairpin RNA (shRNA) (Cat No. sc-37007) and p53 shRNA (Cat No. sc-29435) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation

Journal: Biomolecules

Article Title: Hepatitis B Virus X Protein Induces Reactive Oxygen Species Generation via Activation of p53 in Human Hepatoma Cells

doi: 10.3390/biom14101201

Figure Lengend Snippet: Figure 5. HBx downregulates catalase levels but upregulates Mn-SOD levels via activation of p53. (A,C) HepG2 and Hep3B cells were transfected with an HBx expression plasmid for 48 h and treated with the indicated concentration of NAC for 24 h before harvesting, followed by western blotting. (D,F) HepG2-NTCP and Hep3B-NTCP cells were infected with HBV for 24 h and incubated for an additional 3 days, and treated with the indicated concentration of NAC for 24 h before harvesting, followed by western blotting. (B,E) Levels of ROS in cells prepared in (A,D) were determined (n = 4). The protein bands of p53 and γ-tubulin in (A,D) were quantified using Image J image analysis software version 1.8.0 (NIH) to show the level of p53 relative to the loading control (γ-tubulin) (n = 1 for (B) and 3 for (E)). (G,I) HepG2 and Hep3B cells were transfected with HBx expression plasmid for 48 h and treated with PFT-α for 24 h before harvesting, followed by western blotting. (H) HepG2 cells were transfected with HBx expression plasmid and p53 expression plasmid for 48 h, followed by western blotting. Original western blots are available in the Supplementary Materials.

Article Snippet: Scrambled (SC) short hairpin RNA (shRNA) (Cat No. sc-37007) and p53 shRNA (Cat No. sc-29435) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Concentration Assay, Western Blot, Infection, Incubation, Software, Control

Journal: Biomolecules

Article Title: Hepatitis B Virus X Protein Induces Reactive Oxygen Species Generation via Activation of p53 in Human Hepatoma Cells

doi: 10.3390/biom14101201

Figure Lengend Snippet: Figure 6. The Ser-101 residue of HBx is critical for ROS amplification via activation of p53. (A–D) HepG2 and Hep3B cells were transfected with an empty vector or an expression plasmid encoding HBx variants, HBX3, hbx2, or hbx2 P101S, for 48 h. (A,C) Levels of the indicated proteins in cell lysates were determined by western blotting. (B,D) Levels of ROS were determined with the indicated conditions (n = 4). (E,G) HepG2-NTCP cells were infected with either HBV WT or HBV S101P for 24 h and incubated for an additional 3 days. (E) Levels of the intracellular proteins in cell lysates were determined by western blotting. (F) Levels of extracellular HBV DNA in the culture media were determined (n = 4). (G) Levels of ROS were determined (n = 4) at the indicated conditions. Original western blots are available in the Supplementary Materials.

Article Snippet: Scrambled (SC) short hairpin RNA (shRNA) (Cat No. sc-37007) and p53 shRNA (Cat No. sc-29435) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Residue, Amplification, Activation Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Infection, Incubation

Journal: Advanced Science

Article Title: FGF12 Positively Regulates Keratinocyte Proliferation by Stabilizing MDM2 and Inhibiting p53 Activity in Psoriasis

doi: 10.1002/advs.202400107

Figure Lengend Snippet: FGF12 is upregulated in the epidermis of patients with psoriasis and IMQ‐induced mouse model. A) The mRNA level of Fgf12 in the skin from non‐lesion skin and lesion skin of psoriasis patients was analyzed using the Gene Expression Omnibus (GEO) database. B) Immunofluorescent and quantitative analysis of FGF12 in the skin from healthy controls and psoriatic patients. Nuclei were stained with DAPI (blue) (n = 5). Scale bar = 200 µm. C) Immunofluorescent and quantitative analysis of FGF12 in the skin from Vaseline and IMQ‐mice. Nuclei were stained with DAPI (blue) (n = 5). Scale bar = 100 µm. D) Immunoblotting and quantitative analysis of FGF12 expression in the skin from Vaseline and IMQ‐mice. β‐Actin was used as a loading control (n = 5). E) Immunoblotting and quantitative analysis of FGF12 protein level in NHEK cells that were treated with PBS or M5 for 12 h. β‐Actin was used as a loading control (n = 5). Error bars show the mean ± SEM. *p < 0.05; ***p < 0.001. The p value was determined using two‐tailed unpaired Student's t test (A – E). All numbers (n) are biologically independent experiments.

Article Snippet: Additionally, Fgf12 siRNA (Santa Cruz Biotechnology, sc‐39466), p53 siRNA (Santa Cruz Biotechnology, sc‐29435), MDM 2 siRNA (Santa Cruz Biotechnology, sc‐29394), and β‐Trcp siRNA (Santa Cruz Biotechnology, sc‐37178) were utilized.

Techniques: Gene Expression, Staining, Western Blot, Expressing, Control, Two Tailed Test

Journal: Advanced Science

Article Title: FGF12 Positively Regulates Keratinocyte Proliferation by Stabilizing MDM2 and Inhibiting p53 Activity in Psoriasis

doi: 10.1002/advs.202400107

Figure Lengend Snippet: FGF12 ablation in keratinocytes ameliorates the psoriasiform phenotype. A) Representative images of the dorsal back from mice, and mice PASI scores were depicted (n = 5). B) Representative histological sections of the dorsal back from Krt14 +/+ ‐Fgf12 f/f and Krt14 Cre/+ ‐Fgf12 f/f mice treated by Vaseline or IMQ for 9 days stained with H&E, and quantification of the epidermal thickness and the infiltrating cells (n = 5). Scale bars = 100 µm. C) Immunoblotting analysis of Cyclin A1, Cyclin D1, and Cyclin E1 protein levels in Krt14 +/+ ‐Fgf12 f/f and Krt14 Cre/+ ‐Fgf12 f/f mice were treated with Vaseline or IMQ for 9 days. β‐Actin was used as a loading control (n = 6). D) Immunofluorescent and quantitative analysis of Ki‐67 positive cells in the skin from Krt14 +/+ ‐Fgf12 f/f and Krt14 Cre/+ ‐Fgf12 f/f mice were treated with Vaseline or IMQ for 9 days. Nuclei were stained with DAPI (blue) (n = 5). Scale bar = 100 µm. E) Immunofluorescent and quantitative analysis of K6 in the skin from Krt14 +/+ ‐Fgf12 f/f and Krt14 Cre/+ ‐Fgf12 f/f mice were treated with Vaseline or IMQ for 9 days. Nuclei were stained with DAPI (blue) (n = 5). Scale bar = 100 µm. F) qRT‐PCR analysis for IL‐17 , CCL2, CXCL2, S100A8 and IL‐1β mRNA levels in the skin from Krt14 +/+ ‐Fgf12 f/f and Krt14 Cre/+ ‐Fgf12 f/f mice were treated with IMQ for 9 days (n = 5). Error bars show the mean ± SEM. **p < 0.01; ***p < 0.001. &&& p < 0.001. The p value was determined using two‐tailed unpaired Student's t test (A and F) or one‐way ANOVA (B‐E). All numbers (n) are biologically independent experiments.

Article Snippet: Additionally, Fgf12 siRNA (Santa Cruz Biotechnology, sc‐39466), p53 siRNA (Santa Cruz Biotechnology, sc‐29435), MDM 2 siRNA (Santa Cruz Biotechnology, sc‐29394), and β‐Trcp siRNA (Santa Cruz Biotechnology, sc‐37178) were utilized.

Techniques: Staining, Western Blot, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Advanced Science

Article Title: FGF12 Positively Regulates Keratinocyte Proliferation by Stabilizing MDM2 and Inhibiting p53 Activity in Psoriasis

doi: 10.1002/advs.202400107

Figure Lengend Snippet: FGF12 is required for proliferation and cell cycle transition of keratinocytes. A) RNA‐Sequence analyzes the differentially regulated genes between si‐Scr group and si‐ Fgf12 group treated with M5 for 12 h. Chord diagram showing the top 20 enriched GO clusters. B) Immunoblotting and quantitative analysis of FGF12 protein level in NHEK cells that were transfected with si‐Scr or si‐ Fgf12 . β‐Actin was used as a loading control (n = 5). C) Immunoblotting and quantitative analysis of Cyclin A1, Cyclin D1, and Cyclin E1 protein levels in NHEK cells that were treated with si‐Scr or si‐ Fgf12 and stimulated with or without M5 for 12 h. β‐Actin was used as a loading control (n = 4). D) Immunofluorescent and quantitative analysis of Ki‐67 + in NHEK cells that were treated with si‐Scr or si‐ Fgf12 and stimulated with or without M5 for 12 h (n = 5). Scale bar = 50 µm. E) Immunofluorescent and quantitative analysis of EdU + in HaCaT cells that were treated with si‐Scr or si‐ Fgf12 and stimulated with or without M5 for 12 h. Nuclei were stained with Hoechst (blue) (n = 5). Scale bar = 50 µm. F) Flow cytometric plots of cell‐cycle analysis performed with PI staining on HaCaT cells treated with si‐Scr or si‐ Fgf12 and stimulated with or without M5 for 12 h (left). Quantification the percentage of cells that fall into the sub G0/G1, S, or G2/M gates (right) (n = 5). Error bars show the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. & p < 0.05; && p < 0.01; &&& p < 0.001. The p value was determined using two‐tailed unpaired Student's t test (B) or one‐way ANOVA (C‐F). All numbers (n) are biologically independent experiments.

Article Snippet: Additionally, Fgf12 siRNA (Santa Cruz Biotechnology, sc‐39466), p53 siRNA (Santa Cruz Biotechnology, sc‐29435), MDM 2 siRNA (Santa Cruz Biotechnology, sc‐29394), and β‐Trcp siRNA (Santa Cruz Biotechnology, sc‐37178) were utilized.

Techniques: Sequencing, Western Blot, Transfection, Control, Staining, Cell Cycle Assay, Two Tailed Test

Journal: Advanced Science

Article Title: FGF12 Positively Regulates Keratinocyte Proliferation by Stabilizing MDM2 and Inhibiting p53 Activity in Psoriasis

doi: 10.1002/advs.202400107

Figure Lengend Snippet: FGF12 promotes proliferation and cell cycle transition of keratinocytes through p53 signaling. A–C) KEGG analysis for the significantly upregulated signaling by the interference of si‐Scr or si‐ Fgf12 in HaCaT cells treated by M5 for 12 h. The Top 15 upregulated GO signal pathways were listed. D) GSEA showing the significant enrichment of p53 signaling in M5 treated HaCaT cells under FGF12 interference. E) Immunoblotting and quantitative analysis of p53 and p21 protein levels in NHEK cells that were treated with si‐Scr or si‐ Fgf12 and stimulated with M5 for 12 h. β‐Actin was used as a loading control (n = 4). F) Immunoblotting and quantitative analysis of p53 and p21 levels in Krt14 +/+ ‐Fgf12 f/f and Krt14 Cre/+ ‐Fgf12 f/f mice were treated with IMQ. β‐Actin was used as a loading control (n = 6). Error bars show the mean ± SEM. **p < 0.01; ***p < 0.001. The p value was determined using two‐tailed unpaired Student's t test (E and F). All numbers (n) are biologically independent experiments.

Article Snippet: Additionally, Fgf12 siRNA (Santa Cruz Biotechnology, sc‐39466), p53 siRNA (Santa Cruz Biotechnology, sc‐29435), MDM 2 siRNA (Santa Cruz Biotechnology, sc‐29394), and β‐Trcp siRNA (Santa Cruz Biotechnology, sc‐37178) were utilized.

Techniques: Western Blot, Control, Two Tailed Test

Journal: Advanced Science

Article Title: FGF12 Positively Regulates Keratinocyte Proliferation by Stabilizing MDM2 and Inhibiting p53 Activity in Psoriasis

doi: 10.1002/advs.202400107

Figure Lengend Snippet: FGF12 deficiency‐driven amelioration of keratinocyte proliferation is p53 dependent. A) Immunoblotting and quantitative analysis of p53 protein level in NHEK cells that were transfected in si‐Scr or si‐ p53 . β‐Actin was used as a loading control (n = 5). B) Immunoblotting and quantitative analysis of Cyclin A1, Cyclin D1, and Cyclin E1 protein levels in NHEK cells that si‐ p53 or si‐Scr was transfected in FGF12‐interference cells treated with M5 for 12 h. β‐Actin was used as a loading control (n = 4). C) Flow cytometric plots of cell‐cycle analysis performed with PI staining on HaCaT cells that si‐ p53 or si‐Scr was transfected in FGF12‐interference cells treated with M5 for 12 h (left). Quantification the percentage of cells that fall into the sub G0/G1, S, or G2/M gates (right) (n = 5). D) Immunofluorescent and quantitative analysis (bottom) of Ki‐67 + in NHEK cells that si‐ p53 or si‐Scr was transfected in FGF12‐interference cells treated with M5 for 12 h. Nuclei were stained with DAPI (blue) (n = 5). Scale bar = 50 µm. E) Immunofluorescent and quantitative analysis (bottom) of EdU + in HaCaT cells that si‐ p53 or si‐Scr was transfected in FGF12‐interference cells treated with M5 for 12 h. Nuclei were stained with Hoechst (blue) (n = 5). Scale bar = 50 µm. Error bars show the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. & p < 0.05; && p < 0.01; &&& p < 0.001. The p value was determined using two‐tailed unpaired Student's t test (A) or one‐way ANOVA (B‐E). All numbers (n) are biologically independent experiments.

Article Snippet: Additionally, Fgf12 siRNA (Santa Cruz Biotechnology, sc‐39466), p53 siRNA (Santa Cruz Biotechnology, sc‐29435), MDM 2 siRNA (Santa Cruz Biotechnology, sc‐29394), and β‐Trcp siRNA (Santa Cruz Biotechnology, sc‐37178) were utilized.

Techniques: Western Blot, Transfection, Control, Cell Cycle Assay, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: FGF12 Positively Regulates Keratinocyte Proliferation by Stabilizing MDM2 and Inhibiting p53 Activity in Psoriasis

doi: 10.1002/advs.202400107

Figure Lengend Snippet: FGF12 represses expression of p53 through MDM2. A) Immunoblotting and quantitative analysis of p53 and p21 protein levels in NHEK cells that si‐ MDM2 or si‐Scr was transfected in Flag‐ Fgf12 cells treated with M5. β‐Actin was used as a loading control (n = 4). B) Immunofluorescent and quantitative analysis (down) of p53 in HaCaT cells that si‐ MDM2 or si‐Scr was transfected in Flag‐ Fgf12 cells treated with M5. Nuclei were stained with DAPI (blue) (n = 5). Scale bar = 50 µm. C) qRT‐PCR analysis for p53 and p21 mRNA levels in HaCaT cells that si‐ MDM2 or si‐Scr was transfected in Flag‐ Fgf12 cells treated with M5 for 12 h (n = 5). D) p53‐dependent transcriptional activity of p53 determined by performing dual‐luciferase assays with HEK293 cells overexpressing FGF12 in the presence of si‐Scr, or si‐ MDM2 (n = 5). E) Immunoblotting and quantitative analysis of Cyclin A1, Cyclin D1, and Cyclin E1 protein levels in NHEK cells that Vector or His‐ MDM2 was transfected in FGF12‐interference cells treated with M5 for 12 h. β‐Actin was used as a loading control (n = 4). F) Immunofluorescent and quantitative analysis of Ki‐67 + in NHEK cells that Vector or His‐ MDM2 was transfected in FGF12‐interference cells treated with M5 for 12 h. Nuclei were stained with DAPI (blue) (n = 5). Scale bar = 50 µm. Error bars show the mean ± SEM. **p < 0.01; ***p < 0.001. & p < 0.05; && p < 0.01. The p value was determined using one‐way ANOVA (A‐F). All numbers (n) are biologically independent experiments.

Article Snippet: Additionally, Fgf12 siRNA (Santa Cruz Biotechnology, sc‐39466), p53 siRNA (Santa Cruz Biotechnology, sc‐29435), MDM 2 siRNA (Santa Cruz Biotechnology, sc‐29394), and β‐Trcp siRNA (Santa Cruz Biotechnology, sc‐37178) were utilized.

Techniques: Expressing, Western Blot, Transfection, Control, Staining, Quantitative RT-PCR, Activity Assay, Luciferase, Plasmid Preparation

Journal: Advanced Science

Article Title: FGF12 Positively Regulates Keratinocyte Proliferation by Stabilizing MDM2 and Inhibiting p53 Activity in Psoriasis

doi: 10.1002/advs.202400107

Figure Lengend Snippet: FGF12 stabilizes MDM2 by hindering its K48‐linked ubiquitination. A) Immunoblotting and quantitative analysis of MDM2 protein level that Flag‐ Fgf12 or Vector were transfected in HEK293 cells. β‐Actin was used as a loading control (n = 5). B) qRT‐PCR analysis for MDM2 mRNA level that si‐Scr or si‐ Fgf12 were transfected in HEK293 cells treated with M5 for 12 h (n = 5). C) Immunoblotting and quantitative analysis of MDM2 protein levels that Flag‐ Fgf12 or Vector was transfected in HEK293 cells treated with cyclohexamide (CHX) for 0, 2, 4, and 6 h. β‐Actin was used as a loading control (n = 5). D) Immunoblotting of MDM2 protein level that si‐Scr or si‐ Fgf12 were transfected in HaCaT cells treated with MG132 or CQ for 6 h. β‐Actin was used as a loading control. E) The NHEK cells were transfected with si‐Scr and si‐ Fgf12 and then treated with M5 for 12 h. Cells were treated with MG132 (10 µM) for 6 h before lysation. The cell lysates were immunoprecipitated by anti‐MDM2 antibody, and then western blot assay with anti‐Ub, anti‐FGF12, and anti‐MDM2 antibody. F) The HaCaT cells were transfected with si‐Scr and si‐ Fgf12 and then treated with M5 for 12 h. Cells were treated with MG132 (10 µM) for 6 h before lysation. The cell lysates were immunoprecipitated by anti‐MDM2 antibody, and then western blot assay with anti‐Ub, anti‐FGF12, and anti‐MDM2 antibody. G) The HaCaT cells were transfected with Vector and Flag‐ Fgf12 and then treated with M5 for 12 h. Cells were treated with MG132 (10 µM) for 6 h before lysation. The cell lysates were immunoprecipitated by anti‐MDM2 antibody, and then western blot assay with anti‐Ub, anti‐Flag, and anti‐MDM2 antibody. H) The HEK293 cells were co‐transfected with Flag‐ Fgf12 , His‐ MDM2 , and HA‐ Ub plasmids. Cells were treated with MG132 (10 µM) for 6 h before lysation. The cell lysates were immunoprecipitated by anti‐His antibody, and then western blot assay with anti‐HA, anti‐His, and anti‐Flag antibody. I) The HEK293 cells were co‐transfected with Flag‐ Fgf12 , His‐ MDM 2, and HA‐ Ub‐K48 plasmids. Cells were treated with MG132 (10 µM) for 6 h before lysation. The cell lysates were immunoprecipitated by anti‐His antibody, and then western blot assay with anti‐HA, anti‐His, and anti‐Flag antibody. J) The HEK293 cells were co‐transfected with Flag‐ Fgf12 , His‐ MDM2 , and HA‐ Ub‐K63 plasmids. Cells were treated with MG132 (10 µM) for 6 h before lysation. The cell lysates were immunoprecipitated by anti‐His antibody, and then western blot assay with anti‐HA, anti‐His, and anti‐Flag antibody. Error bars show the mean ± SEM. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001. The p value was determined using two‐tailed unpaired Student's t test (A and B) or one‐way ANOVA (C). All numbers (n) are biologically independent experiments.

Article Snippet: Additionally, Fgf12 siRNA (Santa Cruz Biotechnology, sc‐39466), p53 siRNA (Santa Cruz Biotechnology, sc‐29435), MDM 2 siRNA (Santa Cruz Biotechnology, sc‐29394), and β‐Trcp siRNA (Santa Cruz Biotechnology, sc‐37178) were utilized.

Techniques: Ubiquitin Proteomics, Western Blot, Plasmid Preparation, Transfection, Control, Quantitative RT-PCR, Immunoprecipitation, Two Tailed Test

Journal: Advanced Science

Article Title: FGF12 Positively Regulates Keratinocyte Proliferation by Stabilizing MDM2 and Inhibiting p53 Activity in Psoriasis

doi: 10.1002/advs.202400107

Figure Lengend Snippet: FGF12 interacts with MDM2 to block binding of β‐Trcp. A,B) The HEK293 cells were co‐transfected with Flag‐ Fgf1 2 and His‐ MDM 2 plasmids for 48 h. The lysates of cells were immunoprecipitated with His‐tag antibody and then western blot assay with Flag‐tag antibody (A); immunoprecipitated with Flag‐tag antibody and immunoblotted with His‐tag antibody (B). C) The HaCaT cell lysates were immunoprecipitated with IgG and anti‐MDM2 antibody and the expression of FGF12 and MDM2 were detected by western blot. D) Immunofluorescent staining (left) of FGF12 and MDM2 in HaCaT cells. Nuclei were stained with DAPI (blue). Scale bar = 20 µm. White lines in merged images (right) indicate the area where the distances between FGF12, MDM2, and DAPI were analyzed using ImageJ. E) The schematic diagram showed structural domains of MDM2 protein. F) HEK293 cells were transfected with Flag‐ Fgf12 and several MDM2 deletion mutants. The whole‐cell lysates were immunoprecipitated with anti‐His beads and immunoblotted with anti‐Flag and anti‐His antibodies. G) HaCaT cells and stimulated with M5 for 0, 12, 24, 48 h. Whole‐cell lysates were IP with anti‐MDM2 then subjected to immunoblot analysis with the anti‐FGF12, anti‐MDM2 antibodies. H) HaCaT cells and stimulated with M5 for 0, 12, 24, 48 h. Whole‐cell lysates were immunoprecipitated with anti‐FGF12 then subjected to immunoblot analysis with the anti‐FGF12, anti‐MDM2 antibodies. I) NHEK cells were transfected with si‐Scr and si‐ Fgf12 and then stimulated with M5 for 12 h. Cells were treated with MG132 (10 µM) for 6 h before lysation. Whole‐cell lysates were immunoprecipitated with anti‐MDM2 then subjected to immunoblot analysis with the anti‐β‐Trcp, anti‐MDM2 and anti‐FGF12 antibodies. J) Immunoblotting analysis of MDM2 protein level in NHEK cells that were transfected with si‐Scr, si‐ β‐Trcp and si‐ Fgf12 treatment by M5 for 12 h. β‐Actin was used as a loading control. K) HaCaT cells were co‐transfected with Flag‐ Fgf12 plasmid (0, 2, 4 µg) and then stimulated with M5 for 12 h. Cells were treated with MG132 (10 µM) for 6 h before lysation. Whole‐cell lysates were immunoprecipitated with anti‐MDM2 then subjected to immunoblot analysis with the anti‐β‐Trcp, anti‐MDM2 and anti‐Flag antibodies. Data are representative of three independent experiments.

Article Snippet: Additionally, Fgf12 siRNA (Santa Cruz Biotechnology, sc‐39466), p53 siRNA (Santa Cruz Biotechnology, sc‐29435), MDM 2 siRNA (Santa Cruz Biotechnology, sc‐29394), and β‐Trcp siRNA (Santa Cruz Biotechnology, sc‐37178) were utilized.

Techniques: Blocking Assay, Binding Assay, Transfection, Immunoprecipitation, Western Blot, FLAG-tag, Expressing, Staining, Control, Plasmid Preparation

Journal: Advanced Science

Article Title: FGF12 Positively Regulates Keratinocyte Proliferation by Stabilizing MDM2 and Inhibiting p53 Activity in Psoriasis

doi: 10.1002/advs.202400107

Figure Lengend Snippet: Loss of p53 abolishes the mitigatory effects of FGF12 knockdown on psoriasis in mice. A) Immunofluorescence images for the skin of mice with knock‐down respective genes were labeled with the indicated antibodies. Nuclei were stained with DAPI (blue). Scar bar = 50 µm. B) Immunoblotting and quantitative analysis of p53 protein level in AAV‐ GFP and AAV‐sh‐ p53 mice. β‐Actin was used as a loading control (n = 5). C) Representative histological sections of the dorsal back from Krt14 +/+ ‐Fgf12 f/f ; AAV‐ GFP , Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐ GFP and Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐sh‐ p53 mice treated by IMQ stained with H&E, and quantification of the epidermal thickness and the infiltrating cells (n = 5). Scale bars = 100 µm. D) Immunofluorescent and quantitative analysis of K6 in the skin from Krt14 +/+ ‐Fgf12 f/f ; AAV‐ GFP , Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐ GFP and Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐sh‐ p53 mice induced by IMQ. Nuclei were stained with DAPI (blue) (n = 5). Scale bars = 50 µm. E) Immunofluorescent and quantitative analysis of Ki‐67 positive cells in the skin from Krt14 +/+ ‐Fgf12 f/f ; AAV‐ GFP , Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐ GFP and Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐sh‐ p53 mice stimulated by IMQ. Nuclei were stained with DAPI (blue) (n = 5). Scale bars = 100 µm. F) Immunoblotting of Cyclin A1, Cyclin D1, and Cyclin E1 protein levels in the skin from Krt14 +/+ ‐Fgf12 f/f ; AAV‐ GFP , Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐ GFP and Krt14 Cre/+ ‐Fgf12 f/f ; AAV‐sh‐ p53 mice treated by IMQ. β‐Actin was used as a loading control (n = 6). Error bars show the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. & p < 0.05; && p < 0.01; &&& p < 0.001. The p value was determined using two‐tailed unpaired Student's t test (B) or one‐way ANOVA (C‐F). All numbers (n) are biologically independent experiments.

Article Snippet: Additionally, Fgf12 siRNA (Santa Cruz Biotechnology, sc‐39466), p53 siRNA (Santa Cruz Biotechnology, sc‐29435), MDM 2 siRNA (Santa Cruz Biotechnology, sc‐29394), and β‐Trcp siRNA (Santa Cruz Biotechnology, sc‐37178) were utilized.

Techniques: Knockdown, Immunofluorescence, Labeling, Staining, Western Blot, Control, Two Tailed Test

Journal: Cancer Genomics & Proteomics

Article Title: P53 Status Influences the Anti-proliferative Effect Induced by IFITM1 Inhibition in Estrogen Receptor-positive Breast Cancer Cells

doi: 10.21873/cgp.20468

Figure Lengend Snippet: TamR cells showed decreased IFITM1 and p53 expression. (A) For colony formation assay, MCF-7 and TamR cells were kept for 10 days after treatment with tamoxifen and then stained using crystal violet and imaged. (B) The viability of MCF-7 cells decreased after treatment with tamoxifen (3 and 9 μM) for 72 h, whereas that of TamR cells did not change. (C) The protein expression levels of p53, IFITM1, ERα, STAT1, and phosphorylated STAT1 were determined using western blotting in MCF-7 and TamR cells. (D) The mRNA levels of p53 and IFITM1 were measured using RT-PCR in MCF-7 and TamR cells. Western blotting (E) and RT-PCR (F) were used to measure p53 and IFITM1 expression in MCF-7 cells after treatment with tamoxifen (3 and 9 μM) for 48 h. Data are expressed as mean±standard deviation (SD). p-Values were calculated using unpaired t-test (B and D) and one-way ANOVA with Turkey’s post hoc test (F). *p<0.01, ** p<0.001 and ***p<0.0001.

Article Snippet: MCF-7 and TamR were transiently transfected with p53 siRNA (#sc-29435, Santa Cruz), IFITM1 siRNA (#sc-44549, Santa Cruz), and negative control siRNA (#sc-37007, Santa Cruz) using the Lipofectamine ® RNAiMAX reagent (#13778-075, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Colony Assay, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal: Cancer Genomics & Proteomics

Article Title: P53 Status Influences the Anti-proliferative Effect Induced by IFITM1 Inhibition in Estrogen Receptor-positive Breast Cancer Cells

doi: 10.21873/cgp.20468

Figure Lengend Snippet: IFITM1 inhibition did not affect cell survival in TamR cells. (A) After transfection of siControl (CTL) and siIFITM1 in TamR cells, cells were treated with 9 μM tamoxifen for 48 h, and then cell viability was measured using the WST-8 assay. (B) Cell viability of TamR cells was measured after treatment with tamoxifen (9 μM) and ruxolitinib (20 μM) for 48 h. (C) Cellular apoptosis in TamR cells was evaluated using flow cytometry after treatment with tamoxifen (9 μM) and ruxolitinib (20 μM) for 48 h. Data are expressed as mean±standard deviation (SD). p-Values were calculated using unpaired t-test (A) and one-way ANOVA with Turkey’s post hoc test (B and C). *p<0.01, **p<0.001 and ***p<0.0001.

Article Snippet: MCF-7 and TamR were transiently transfected with p53 siRNA (#sc-29435, Santa Cruz), IFITM1 siRNA (#sc-44549, Santa Cruz), and negative control siRNA (#sc-37007, Santa Cruz) using the Lipofectamine ® RNAiMAX reagent (#13778-075, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Inhibition, Transfection, Flow Cytometry, Standard Deviation

Journal: Cancer Genomics & Proteomics

Article Title: P53 Status Influences the Anti-proliferative Effect Induced by IFITM1 Inhibition in Estrogen Receptor-positive Breast Cancer Cells

doi: 10.21873/cgp.20468

Figure Lengend Snippet: P53 status can affect IFITM1 expression and cell death after tamoxifen treatment or IFITM1 inhibition in MCF-7 cells. (A) After transfection of siControl (CTL), sip53, and siIFITM1 in MCF-7 cells, the mRNA levels of p53, IFITM1, and IRF9 were measured using RT-PCR. (B) The protein expression levels of p53, IFITM1, SOCS1, and SOCS3 were measured using western blotting 48 h after the transfection of siControl (CTL), sip53, and siIFITM1 in MCF-7 cells. The mRNA levels of SOCS1 and SOCS3 were measured after transfection of siControl (CTL), sip53, and siIFITM1 in MCF-7 (C) and TamR cells (D). (E) Decreased protein and mRNA levels of IFITM1 were observed in T47D cells with mutant p53 compared with those in MCF-7 cells. (F) IFITM1 and IRF9 mRNA levels were decreased and SOCS3 mRNA levels were increased in breast cancer with mutant p53 compared with those in breast cancer with wild-type p53. Data are expressed as mean±standard deviation (SD). p-Values were calculated using unpaired t-test (A, C, D, and E). *p<0.05, **p<0.005 and ***p<0.0005.

Article Snippet: MCF-7 and TamR were transiently transfected with p53 siRNA (#sc-29435, Santa Cruz), IFITM1 siRNA (#sc-44549, Santa Cruz), and negative control siRNA (#sc-37007, Santa Cruz) using the Lipofectamine ® RNAiMAX reagent (#13778-075, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Inhibition, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Mutagenesis, Standard Deviation