Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Molecular mechanisms of thalidomide effectiveness on COVID-19 patients explained: ACE2 is a new ΔNp63α target gene

doi: 10.1007/s00109-024-02485-x

Figure Lengend Snippet: Thal and Len at clinically relevant concentrations reduce ACE2 expression. A HaCaT, A431, and A549 cells were treated for 24 h with Len (1 or 5 µM) or Thal (10 or 100 µM). Cell extracts were prepared and analyzed by WB with anti-ACE2 or anti-p63 Abs. Actin was used as the loading control, and one representative experiment is shown. B p63-null U-2 OS cells were treated for 24 h with increasing concentrations of Thal (10, 50, 100 µM) or Len (1, 5, 15, 50, 80, 100 µM). Cell extracts were analyzed by WB with anti-ACE2 Ab. Actin was used as the loading control, and one representative experiment is shown. C HaCaT, A431, A549, and H1299 cells were treated with Thal 100 µM or Len 5 µM for 8 h; DMSO was used as the control (NT). Lower panel: cell extracts were prepared and analyzed by WB with anti-p63 Ab; GAPDH was used as the loading control, and one representative experiment is shown. Upper panel: compared to the relative Ctrl, ACE2 mRNA levels decreased in p63-proficient HaCaT, and A431 and A459 cells were treated with Thal or Len but not in p63-deficient H1299 cells. ACE2 mRNA was evaluated by qRT-PCR, mRNA levels were normalized using GAPDH, and the means ± standard errors (SE) of three replicates are shown

Article Snippet: For transient transfection, 5 × 10 4 A431 cells were plated in 24-well multi-plates and on the next day transfected with Lipofectamine 2000 (Invitrogen #11,668,019) with increasing amount of CEREBLON (CRBN) encoding plasmid or with four different p63-small hairpin RNA (sh-p63) vectors with sequence homology to four different regions of p63 mRNA (OriGene Technologies # TF308688); shRNA-SCRAMBLED (sh-SCRB) was used as a control.

Techniques: Expressing, Control, Quantitative RT-PCR

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Molecular mechanisms of thalidomide effectiveness on COVID-19 patients explained: ACE2 is a new ΔNp63α target gene

doi: 10.1007/s00109-024-02485-x

Figure Lengend Snippet: ΔNp63α and ACE2 parallel modulation at both protein and mRNA levels. A HaCaT and A431 cells were transiently transfected with 100 and 250 ng of CRBN encoding plasmid. After 24 h from transfection, cell extracts were prepared and analyzed by WB with anti-ACE2, anti-p63, and anti-CRBN Abs. Actin was used as the loading control; one representative experiment is shown. Two bands of the CRBN protein are evident: °stays for endogenous CRBN, *for transfected CRBN that has higher molecular weight due to an HA tag. B p63-null U-2 OS cells were transiently transfected with 10 and 25 ng of ΔNp63α expression vector. After 24 h from transfection, cell extracts were prepared and analyzed by WB with anti-ACE2 or anti-p63 Abs. Actin was used as the loading control, and one representative experiment is shown. C ACE2 mRNA levels were evaluated by qRT-PCR, and mRNA levels were normalized using GAPDH as housekeeping gene. Means ± SE of three replicates are shown. D HaCaT and A431 cells were transiently transfected with 100 ng of small hairpin RNA (shRNA) plasmids targeting p63 mRNA; four different p63 shRNA vectors with sequence homology to four different regions of p63 mRNA were used (#1, #2, #3, #4); the cells were also transfected with a control (100 ng), shRNA-SCR, that encodes for an RNA that is not complementary to any mRNA sequence. After 24 h from transfection, cell extracts were prepared and analyzed by WB with anti-ACE2 and anti-p63 Abs. Actin was used as the loading control; one representative experiment is shown. E A431 cells were stably transfected with both p63shRNA#3 and p63shRNA#4, and polyclonal populations were isolated by puromycin selection. As the control, cells were also transfected with a shRNA-SCRB vector. Cell extracts were prepared and analyzed by WB with anti-ACE2 and anti-p63 Abs, and parallel samples were used for ACE2 mRNA level evaluation by qRT-PCR; mRNA levels were normalized using GAPDH as housekeeping gene. Means ± SE of three replicates of three cultures are shown

Article Snippet: For transient transfection, 5 × 10 4 A431 cells were plated in 24-well multi-plates and on the next day transfected with Lipofectamine 2000 (Invitrogen #11,668,019) with increasing amount of CEREBLON (CRBN) encoding plasmid or with four different p63-small hairpin RNA (sh-p63) vectors with sequence homology to four different regions of p63 mRNA (OriGene Technologies # TF308688); shRNA-SCRAMBLED (sh-SCRB) was used as a control.

Techniques: Transfection, Plasmid Preparation, Control, Molecular Weight, Expressing, Quantitative RT-PCR, shRNA, Sequencing, Stable Transfection, Isolation, Selection

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Molecular mechanisms of thalidomide effectiveness on COVID-19 patients explained: ACE2 is a new ΔNp63α target gene

doi: 10.1007/s00109-024-02485-x

Figure Lengend Snippet: ACE2 is a new ΔNp63α target gene. A HaCaT cells were treated with 5 ng/mL of TNF-α or with 0.5 µg/mL of LPS for the indicated times. Cell extracts were prepared and analyzed by WB with an anti-ACE2 and anti-p63 Abs. Actin was used as the loading control, and one representative experiment is shown. The cell supernatants were collected, and IL-8 levels were quantified by the ELISA assay. B The schematic representation and sequence of the p53/p63RE were identified in the first intron of ACE2. C A431 cells were treated or not with LPS 0.5 μg/mL for 2 h. Cell extracts were prepared and analyzed by WB using anti-ACE2 and anti-p63 Abs. Actin was used as the loading control, and one representative experiment is shown. D , E A431 cells were treated with LPS for 2 h and then collected for chromatin immunoprecipitation (ChIP) analysis

Article Snippet: For transient transfection, 5 × 10 4 A431 cells were plated in 24-well multi-plates and on the next day transfected with Lipofectamine 2000 (Invitrogen #11,668,019) with increasing amount of CEREBLON (CRBN) encoding plasmid or with four different p63-small hairpin RNA (sh-p63) vectors with sequence homology to four different regions of p63 mRNA (OriGene Technologies # TF308688); shRNA-SCRAMBLED (sh-SCRB) was used as a control.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Sequencing, Chromatin Immunoprecipitation

Journal: Molecular Medicine Reports

Article Title: Prognostic and predictive roles of microRNA-411 and its target STK17A in evaluating radiotherapy efficacy and their effects on cell migration and invasion via the p53 signaling pathway in cervical cancer

doi: 10.3892/mmr.2019.10826

Figure Lengend Snippet: Reverse transcription-quantitative polymerase chain reaction primer sequences.

Article Snippet: The membranes were incubated with phosphorylated (p)-STK17A antibody (cat. no. 14433-1-AP; Proteintech, Wuhan, China; 1:1,000), p-p21 WAF1 antibody (cat. no. AP01654PU-N; Origene; 1:10,000), p-p53 antibody (cat. no. MABE518; Merck KGaA; 1:1,000), TAp63 antibody (cat. no. TA311397; Origene; 1:1,000), and GAPDH antibody (cat. no. 10494-1-AP; Proteintech; 1:2,000) at 4°C overnight.

Techniques: Polymerase Chain Reaction, Sequencing

Journal: Molecular Medicine Reports

Article Title: Prognostic and predictive roles of microRNA-411 and its target STK17A in evaluating radiotherapy efficacy and their effects on cell migration and invasion via the p53 signaling pathway in cervical cancer

doi: 10.3892/mmr.2019.10826

Figure Lengend Snippet: miR-411 activates the p53 signaling pathway and negatively regulates STK17A in cervical cancer cells. (A) Determination by reverse transcription-quantitative polymerase chain reaction analysis demonstrated that ectopic expression of miR-411 and siRNA-mediated knockdown of STK17A decreased the mRNA expression of STK17A, but increased the mRNA expression of p53, p21 WAF1 and TAp63; miR-411 inhibitor increased the mRNA expression of STK17A, but decreased the mRNA expression of p53, p21 WAF1 and TAp63. (B) Determination by western blot analysis and (C) quantification demonstrated that ectopic expression of miR-411 and siRNA-mediated knockdown of STK17A decreased the protein expression of STK17A, but increased the protein expression of p53, p21 WAF1 and TAp63; miR-411 inhibitor increased the protein expression of STK17A, but decreased the protein expression of p53, p21 WAF1 and TAp63. *P<0.05, vs. NC group; # P<0.05, vs. miR-411 inhibitor + siRNA-STK17A group. The experiment was repeated three times and data were compared by one-way analysis of variance and analyzed by Tukey's post hoc test. NC, negative control; miR-411, microRNA-411; STK17A, serine/threonine kinase 17a; siRNA, small interfering RNA.

Article Snippet: The membranes were incubated with phosphorylated (p)-STK17A antibody (cat. no. 14433-1-AP; Proteintech, Wuhan, China; 1:1,000), p-p21 WAF1 antibody (cat. no. AP01654PU-N; Origene; 1:10,000), p-p53 antibody (cat. no. MABE518; Merck KGaA; 1:1,000), TAp63 antibody (cat. no. TA311397; Origene; 1:1,000), and GAPDH antibody (cat. no. 10494-1-AP; Proteintech; 1:2,000) at 4°C overnight.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Western Blot, Negative Control, Small Interfering RNA

Journal: Oncology Reports

Article Title: HDAC inhibitors suppress the proliferation, migration and invasiveness of human head and neck squamous cell carcinoma cells via p63-mediated tight junction molecules and p21-mediated growth arrest

doi: 10.3892/or.2021.7997

Figure Lengend Snippet: Antibodies.

Article Snippet: p63 , mAb , , 1:200 , , OriGene; Technologies, Inc. (cat. no. TA802078).

Techniques:

Journal: Oncology Reports

Article Title: HDAC inhibitors suppress the proliferation, migration and invasiveness of human head and neck squamous cell carcinoma cells via p63-mediated tight junction molecules and p21-mediated growth arrest

doi: 10.3892/or.2021.7997

Figure Lengend Snippet: H&E and immunohistochemical staining of p63, HDAC1, JAM-A and claudin-1 in tissues of patients with human head and neck squamous cell carcinoma, including the adjacent dysplastic regions. Scale bar, 200 µm. H&E, hematoxylin and eosin. HDAC, histone deacetylase; JAM-A, junctional adhesion molecule-A; CLDN-1, claudin-1; SCC, squamous cell carcinoma.

Article Snippet: p63 , mAb , , 1:200 , , OriGene; Technologies, Inc. (cat. no. TA802078).

Techniques: Immunohistochemical staining, Staining, Histone Deacetylase Assay

Journal: Oncology Reports

Article Title: HDAC inhibitors suppress the proliferation, migration and invasiveness of human head and neck squamous cell carcinoma cells via p63-mediated tight junction molecules and p21-mediated growth arrest

doi: 10.3892/or.2021.7997

Figure Lengend Snippet: Western blot analysis of p63, JAM-A and claudin-1 expression in Detroit 562 cells (A) transfected with p63 siRNA and (B) treated with HDAC inhibitor TSA, or HDAC1 and 6 inhibitors; 1 and 10 µM. (C) Immunocytochemical staining for p63, HDAC1, JAM-A and claudin-1 in Detroit 562 cells treated with HDAC inhibitors at 10 µM. Scale bar, 10 µm. HDAC, histone deacetylase; iHDAC, HDAC inhibitor; JAM-A, junctional adhesion molecule-A; CLDN-1, claudin-1; Ac-tub, acetylated tubulin; TSA, trichostatin A; siRNA, small interfering RNA; cont, control.

Article Snippet: p63 , mAb , , 1:200 , , OriGene; Technologies, Inc. (cat. no. TA802078).

Techniques: Western Blot, Expressing, Transfection, Staining, Histone Deacetylase Assay, Small Interfering RNA, Control

Journal: Oncology Reports

Article Title: HDAC inhibitors suppress the proliferation, migration and invasiveness of human head and neck squamous cell carcinoma cells via p63-mediated tight junction molecules and p21-mediated growth arrest

doi: 10.3892/or.2021.7997

Figure Lengend Snippet: (A) Cell cycle and (B) cell counting assays using Detroit 562 cells treated with TSA at 10 µM. (C) Western blot analysis for p63, p21 and cyclin D1 in Detroit 562 cells treated with 1 or 10 µM HDAC inhibitors. (D) Western blot analysis of p63, EGFR, phosphorylated-EGFR, ERK1/2 and phosphorylated-ERK1/2 in Detroit 562 cells treated with 10 µM TSA. **P<0.01 vs. the control. TSA, trichostatin A; iHDAC, HDAC inhibitor; EGFR, epidermal growth factor receptor; Ac-tub, acetylated tubulin; cont, control.

Article Snippet: p63 , mAb , , 1:200 , , OriGene; Technologies, Inc. (cat. no. TA802078).

Techniques: Cell Counting, Western Blot, Control

Journal: Oncology Reports

Article Title: HDAC inhibitors suppress the proliferation, migration and invasiveness of human head and neck squamous cell carcinoma cells via p63-mediated tight junction molecules and p21-mediated growth arrest

doi: 10.3892/or.2021.7997

Figure Lengend Snippet: (A) Cell cycle and (B) cell counting assays, (C) and western blot analysis for p63, EGFR, phosphorylated-EGFR, ERK1/2, phosphorylated-ERK1/2, p21 and cyclin D1 in Detroit 562 cells transfected with p63 siRNA. (D) Cell cycle assay, (E) cell counting assay and (F) western blotting for EGFR, phosphorylated-EGFR, ERK1/2, phosphorylated-ERK1/2, p21 and cyclin D1 in Detroit 562 cells treated with the EGFR inhibitor AG1478. *P<0.05 and **P<0.01 vs. the control. EGFR, epidermal growth factor receptor; siRNA, small interfering RNA; AG, AG1478; cont, control.

Article Snippet: p63 , mAb , , 1:200 , , OriGene; Technologies, Inc. (cat. no. TA802078).

Techniques: Cell Counting, Western Blot, Transfection, Cell Cycle Assay, Control, Small Interfering RNA

Journal: Oncology Reports

Article Title: HDAC inhibitors suppress the proliferation, migration and invasiveness of human head and neck squamous cell carcinoma cells via p63-mediated tight junction molecules and p21-mediated growth arrest

doi: 10.3892/or.2021.7997

Figure Lengend Snippet: (A) Immunocytochemical staining for CK7, p63 and ΔNp63 in primary cultured cancer cells derived from human head and neck squamous cell carcinoma tissues. Scale bar, 10 µm. (B) Phase-contrast images. Scale bar, 50 µm. (C) Western blotting for p63, JAM-A and claudin-1, and (D) immunocytochemical staining for JAM-A and claudin-1 in primary cultured cancer cells treated with 10 µM HDAC inhibitors. Scale bar, 10 µm. (E) Wound-healing assays and (G) western blotting for p63, p21 and cyclin D1 in primary cultured cancer cells treated with HDAC inhibitors at 10 µM. Scale bar, 200 µm. (F) Quantification of the results in (E). (H) Western blotting for p63, EGFR, phosphorylated-EGFR, ERK1/2 and phosphorylated-ERK1/2 in primary cultured cancer cells treated with 10 µM TSA. (I) Western blotting for p63, p21 and cyclin D1 in primary cultured cancer cells transfected with p63siRNA of. **P<0.01 vs. the control. CK7, anti-cytokeratin 7; HDAC, histone deacetylase; iHDAC, HDAC inhibitor; JAM-A, junctional adhesion molecule-A; CLDN-1, claudin-1; Ac-tub, acetylated tubulin; TSA, trichostatin A; siRNA, small interfering RNA; cont, control.

Article Snippet: p63 , mAb , , 1:200 , , OriGene; Technologies, Inc. (cat. no. TA802078).

Techniques: Staining, Cell Culture, Derivative Assay, Western Blot, Transfection, Control, Histone Deacetylase Assay, Small Interfering RNA

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Molecular mechanisms of thalidomide effectiveness on COVID-19 patients explained: ACE2 is a new ΔNp63α target gene

doi: 10.1007/s00109-024-02485-x

Figure Lengend Snippet: Thal and Len at clinically relevant concentrations reduce ACE2 expression. A HaCaT, A431, and A549 cells were treated for 24 h with Len (1 or 5 µM) or Thal (10 or 100 µM). Cell extracts were prepared and analyzed by WB with anti-ACE2 or anti-p63 Abs. Actin was used as the loading control, and one representative experiment is shown. B p63-null U-2 OS cells were treated for 24 h with increasing concentrations of Thal (10, 50, 100 µM) or Len (1, 5, 15, 50, 80, 100 µM). Cell extracts were analyzed by WB with anti-ACE2 Ab. Actin was used as the loading control, and one representative experiment is shown. C HaCaT, A431, A549, and H1299 cells were treated with Thal 100 µM or Len 5 µM for 8 h; DMSO was used as the control (NT). Lower panel: cell extracts were prepared and analyzed by WB with anti-p63 Ab; GAPDH was used as the loading control, and one representative experiment is shown. Upper panel: compared to the relative Ctrl, ACE2 mRNA levels decreased in p63-proficient HaCaT, and A431 and A459 cells were treated with Thal or Len but not in p63-deficient H1299 cells. ACE2 mRNA was evaluated by qRT-PCR, mRNA levels were normalized using GAPDH, and the means ± standard errors (SE) of three replicates are shown

Article Snippet: For this type of experiment, we used four different p63 shRNA vectors (OriGene) with sequence homology to four different regions of the p63 mRNA, with the shp63#3 and the shp63#4 vectors resulting in the strongest effect on ΔNp63α silencing and a concomitant decrease of ACE2 protein levels, in both cell lines (Fig. D).

Techniques: Expressing, Control, Quantitative RT-PCR

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Molecular mechanisms of thalidomide effectiveness on COVID-19 patients explained: ACE2 is a new ΔNp63α target gene

doi: 10.1007/s00109-024-02485-x

Figure Lengend Snippet: ΔNp63α and ACE2 parallel modulation at both protein and mRNA levels. A HaCaT and A431 cells were transiently transfected with 100 and 250 ng of CRBN encoding plasmid. After 24 h from transfection, cell extracts were prepared and analyzed by WB with anti-ACE2, anti-p63, and anti-CRBN Abs. Actin was used as the loading control; one representative experiment is shown. Two bands of the CRBN protein are evident: °stays for endogenous CRBN, *for transfected CRBN that has higher molecular weight due to an HA tag. B p63-null U-2 OS cells were transiently transfected with 10 and 25 ng of ΔNp63α expression vector. After 24 h from transfection, cell extracts were prepared and analyzed by WB with anti-ACE2 or anti-p63 Abs. Actin was used as the loading control, and one representative experiment is shown. C ACE2 mRNA levels were evaluated by qRT-PCR, and mRNA levels were normalized using GAPDH as housekeeping gene. Means ± SE of three replicates are shown. D HaCaT and A431 cells were transiently transfected with 100 ng of small hairpin RNA (shRNA) plasmids targeting p63 mRNA; four different p63 shRNA vectors with sequence homology to four different regions of p63 mRNA were used (#1, #2, #3, #4); the cells were also transfected with a control (100 ng), shRNA-SCR, that encodes for an RNA that is not complementary to any mRNA sequence. After 24 h from transfection, cell extracts were prepared and analyzed by WB with anti-ACE2 and anti-p63 Abs. Actin was used as the loading control; one representative experiment is shown. E A431 cells were stably transfected with both p63shRNA#3 and p63shRNA#4, and polyclonal populations were isolated by puromycin selection. As the control, cells were also transfected with a shRNA-SCRB vector. Cell extracts were prepared and analyzed by WB with anti-ACE2 and anti-p63 Abs, and parallel samples were used for ACE2 mRNA level evaluation by qRT-PCR; mRNA levels were normalized using GAPDH as housekeeping gene. Means ± SE of three replicates of three cultures are shown

Article Snippet: For this type of experiment, we used four different p63 shRNA vectors (OriGene) with sequence homology to four different regions of the p63 mRNA, with the shp63#3 and the shp63#4 vectors resulting in the strongest effect on ΔNp63α silencing and a concomitant decrease of ACE2 protein levels, in both cell lines (Fig. D).

Techniques: Transfection, Plasmid Preparation, Control, Molecular Weight, Expressing, Quantitative RT-PCR, shRNA, Sequencing, Stable Transfection, Isolation, Selection

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Molecular mechanisms of thalidomide effectiveness on COVID-19 patients explained: ACE2 is a new ΔNp63α target gene

doi: 10.1007/s00109-024-02485-x

Figure Lengend Snippet: ACE2 is a new ΔNp63α target gene. A HaCaT cells were treated with 5 ng/mL of TNF-α or with 0.5 µg/mL of LPS for the indicated times. Cell extracts were prepared and analyzed by WB with an anti-ACE2 and anti-p63 Abs. Actin was used as the loading control, and one representative experiment is shown. The cell supernatants were collected, and IL-8 levels were quantified by the ELISA assay. B The schematic representation and sequence of the p53/p63RE were identified in the first intron of ACE2. C A431 cells were treated or not with LPS 0.5 μg/mL for 2 h. Cell extracts were prepared and analyzed by WB using anti-ACE2 and anti-p63 Abs. Actin was used as the loading control, and one representative experiment is shown. D , E A431 cells were treated with LPS for 2 h and then collected for chromatin immunoprecipitation (ChIP) analysis

Article Snippet: For this type of experiment, we used four different p63 shRNA vectors (OriGene) with sequence homology to four different regions of the p63 mRNA, with the shp63#3 and the shp63#4 vectors resulting in the strongest effect on ΔNp63α silencing and a concomitant decrease of ACE2 protein levels, in both cell lines (Fig. D).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Sequencing, Chromatin Immunoprecipitation

Journal: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: Col10a1-TAp63γ expression plasmid and establishment of stable TAp63γ expressing ATDC5 cell lines. ( A ) pCMV- TAp63γ and its derivative pCol10a1-TAp63γ expression plasmids are shown. Enzyme restriction sites for cloning are also shown. ( B ) Enzyme digestion confirmed the integration of TAp63γ in designated stable cell lines. ( C ) PCR using p63 and Taq sequence-specific primers confirmed the integration of TAp63γ into the stable cell lines: pCMV- TAp63γ and Col10a1-TAp63γ . ( D ) Western blot results further confirmed expression of TAp63γ in designated stable lines.

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: Expressing, Plasmid Preparation, Cloning, Stable Transfection, Sequencing, Western Blot

Journal: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: TAp63γ upregulates Col10a1 expression in ATDC5 cells. ( A ) ATD5C cells were harvested for RNA isolation on day zero or after 4 days in culture. Col10a1 showed significant elevation in Col10a1-TAp63γ stable cell lines compared with the controls after 4 days in culture. ( B ) ATD5C cells were harvested for RNA isolation on day zero or after 4 days in culture. Col10a1 showed significant elevation in pCMV- TAp63γ stable cell lines compared with the controls after 4 days in culture. ( C ) The protein levels of Col10a1 in TAp63γ stable cell lines also showed upregulation of Col10a1 compared to blank and pCMV controls by western blot analysis.

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: Expressing, Isolation, Stable Transfection, Western Blot

Journal: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: In vitro effect of TAp63γ on chondrocyte proliferation. ( A ) ATD5C cells were cultured for 0, 4, 7, 14, or 21 days and stained with Alcian blue. After 7 days in culture, the staining intensity of TAp63γ stable cell lines was much stronger than the blank and vector controls. Scale bar, 25 μm. ( B ) Sum object area of the staining by densitometry analysis (n=3, * p<0.05, ** p<0.01).

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: In Vitro, Cell Culture, Staining, Stable Transfection, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: TAp63γ promotes hypertrophic differentiation of ATDC5 cells. ( A ) ATD5C cells were cultured for 0, 4, 7, 14, or 21 days and stained for ALP (alkaline phosphatase). After 7 days in culture, the staining intensity of TAp63γ stable cell lines was much stronger than the blank and vector controls. Scale bar, 50 μm. ( B ) Sum object area of the staining by densitometry analysis (n=3, * p<0.05, ** p<0.01).

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: Cell Culture, Staining, Stable Transfection, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: In vitro effect of TAp63γ on matrix mineralization. ( A ) ATD5C cells were cultured for 0, 4, 7, 14, or 21 days and stained with Alizarin red. After 4 and 7 days in culture, enhanced Alizarin red staining was observed in both Col10a1-TAp63γ and pCMV- TAp63γ stable cell lines compared with the blank and vector controls. Scale bar, 25 μm. ( B ) Sum object area of the staining by densitometry analysis (n=3, * p<0.05, ** p<0.01).

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: In Vitro, Cell Culture, Staining, Stable Transfection, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: Accelerated ossification in Col10a1-TAp63γ transgenic mice. ( A ) Col10a1 distal promoter and a shorter Col10a1 basal promoter (ShXBP) (from −220 to +45 bp) were used to generate a p63-expressing transgenic construct. ( B ) PCR genotyping was performed for the Col10a1-TAp63γ transgenic mice using DNA prepared from skin. ( C ) For mice at postnatal day 1 (P1), ossification signals of the fore- and hind-limb digits were evaluated. Tail ossification signals were observed up to the 11 th caudal vertebra in transgenic mice and up to the 8 th caudal vertebra in WT mice. ( D ) The statistical analyses of the ossified caudal vertebrae from four Col10a1-TAp63γ transgenic mouse lines at P1 are presented. Line 2: n = 9; Line 5: n = 9; Line 7: n = 10.

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: Transgenic Assay, Expressing, Construct

Journal: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: Histological and immunofluorescent analysis of Col10a1-TAp63γ transgenic mice. ( A ) The proliferative and hypertrophic zones of the limb cartilage in Col10a1-TAp63γ transgenic and WT mice were evaluated by hematoxylin and eosin (H&E) staining. Scale bar, 100 μm. ( B ) Sagittal sections of the distal humerus from both WT and transgenic mouse limbs at postnatal day 1 were subjected to immunofluorescent analysis using an anti-Sox9 antibody. Scale bar, 50 μm. ( C ) Sagittal sections of the distal humerus from both WT and transgenic mouse limbs at postnatal day 1 were subjected to immunofluorescent analysis using an anti-Runx2 antibody (label as Sox9 and some description of the findings). Scale bar, 50 μm.

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: Transgenic Assay, Staining

Journal: Journal of Biological Chemistry

Article Title: Phosphorylated TP63 Induces Transcription of RPN13, Leading to NOS2 Protein Degradation

doi: 10.1074/jbc.m110.158642

Figure Lengend Snippet: FIGURE 1. Cisplatin induces RPN13 expression at the RNA level. Wild- type Np63 and Np63-S385G cells were treated with control medium () or 10 g/ml cisplatin (CIS; ) for 12 h. GAPDH was used as a loading control. A, RT-PCR analysis for RPN13 transcription. B, qPCR. Values for RPN13 (in relative units (RU)) were normalized to values for GAPDH, and values obtained from the control untreated samples were designated as 1. Experiments were performed in triplicate. FIGURE 2. Schematic representation of the human RPN13 gene pro- moter. The sequence of the human 1500-bp RPN13 promoter was found on the UCSC Genome Bioinformatics human genome web site, and certain potential TF-responsive elements (RE) were defined using TFSEARCH soft- ware. TF sequences are shown in boldface. The TSS is shown as an uppercase letter. The following responsive elements were located in the RPN13 pro- moter: TP63 (1376/1354, 1231/1216, 1189/1167, and 500/ 481), NF-Y/DDIT3 (1267/1246, 93/71, and 65/37), NF-B (995/ 985 and 724/716), STAT (857/843), and GAS (gamma-activated site; 971/862).

Article Snippet: Antibodies—We used a rabbit anti- Np63 polyclonal antibody (Ab-1, EMD Chemicals); a rabbit anti-TP63 (TP73L) monoclonal antibody (clone Y289, NB110-57309) and a rabbit anti-DDIT3 (DNA damage-inducible transcript 3) polyclonal antibody (NB100-78344) (Novus Biologicals); mouse monoclonal antibodies against -actin (Sigma) and NF-YA (Rockland Immunochemicals); and rabbit polyclonal antibodies against DNA topoisomerase II (TOP2A, ab74715), RPN13 (ab91567), NF- B p65 subunit (ab32536), NF- B p50 subunit (ab7549), STAT3 (signal transduction activator of transcription 3; ab32500), and UCH37 (ab38528) (Abcam).

Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Sequencing