Journal: Scientific reports

Article Title: Assessment of the toxic effect of benzalkonium chloride on human limbal stem cells.

doi: 10.1038/s41598-025-96919-2

Figure Lengend Snippet: Fig. 3. (A) Agarose gel electropherogram of amplification products (passage 1). Lane 1: DNA ladder (M), lanes 2–3: KRT3 (101 bp), lanes 4–5: KRT12 (190 bp), lane 6: DNA ladder (M), lanes 7–8: TP63 (96 bp), lanes 9–10: ABCG2 (144 bp), lanes 11–12: ACTβ (131 bp), lane 13: DNA ladder (M). NC, negative control. (B) Visualization of lack of expression of CK3, and CK12 and expression of positive markers p63 and ABCG2, in LSCs cultured in a medium supplemented with H/I/E. FITC-conjugated specific antibodies (green); DAPI- stained cell nuclei (blue). IC, isotype control. Scale bars = 15 μm.

Article Snippet: After rinsing with PBS, the cells were incubated with blocking buffer for 1 h, rinsed again, and labelled with rabbit anti-ABCG2 polyclonal (1:50; 27286-AP; Proteintech, USA), rabbit anti-p63 polyclonal (1:50; 12143-1-AP; Proteintech, USA), mouse anti-CK3 monoclonal (1:50; ab68260; Abcam, UK), and rabbit anti-CK12 monoclonal (1:50; ab185627; Abcam, UK) primary antibodies at 4 °C overnight.

Techniques: Agarose Gel Electrophoresis, Amplification, Negative Control, Expressing, Cell Culture, Staining, Control

Journal: The Journal of Cell Biology

Article Title: Transcriptional repression induces a slowly progressive atypical neuronal death associated with changes of YAP isoforms and p73

doi: 10.1083/jcb.200509132

Figure Lengend Snippet: YAPΔC and phosphorylated p73 are expressed in striatal neurons of Htt transgenic mice. Immunohistochemical analyses of mutant Htt transgenic (R6/2) and sibling control mice were performed at 4 wk with anti-p73 (full-length form), antiphosphorylated p73, or anti-YAPΔC antibodies. Arrows indicate immunoreactive striatal neurons. Expression of phosphorylated p73 was increased in R6/2 transgenic mice (middle), whereas the total amount of p73 was similar in transgenic and control mice (left). Immunoreactivities of YAPΔC were slightly increased in transgenic mice (right).

Article Snippet: The filter was blotted with the anti-p73 goat polyclonal antibody (S-20; 1:1,000; Santa Cruz Biotechnology, Inc.) or antiphosphorylated p73 rabbit polyclonal antibodies (1:1,000; Cell Signaling) followed by HRP-coupled detection.

Techniques: Transgenic Assay, Immunohistochemical staining, Mutagenesis, Control, Expressing

Journal: The Journal of Cell Biology

Article Title: Transcriptional repression induces a slowly progressive atypical neuronal death associated with changes of YAP isoforms and p73

doi: 10.1083/jcb.200509132

Figure Lengend Snippet: YAPΔC and phosphorylated p73 are coexpressed in striatal neurons of human HD brain. (A) Immunoreactivities of phosphorylated p73 and YAPΔC isoforms were increased in striatal neurons of HD patients (arrows). Postmortem brain tissues, including the caudate nucleus, were prepared from three HD patients and three controls. (B) Double staining with anti-p73 rabbit polyclonal antibody specific for full-length p73 but not reactive to ΔNp73 (H-79; 1:500; Santa Cruz Biotechnology, Inc.) and with antiphospho-p73 rabbit polyclonal antibody (1:500; Cell Signaling) showed colocalization of the two signals in most striatal neurons of HD patients (top, white arrows). It suggests that the full-length p73 is phosphorylated in striatal neurons. Bottom panels show that YAPΔC isoforms were colocalized with phosphorylated p73 in striatal neurons (bottom, white arrows). However, a minor portion of neurons expresses only p73 (red arrows).

Article Snippet: The filter was blotted with the anti-p73 goat polyclonal antibody (S-20; 1:1,000; Santa Cruz Biotechnology, Inc.) or antiphosphorylated p73 rabbit polyclonal antibodies (1:1,000; Cell Signaling) followed by HRP-coupled detection.

Techniques: Double Staining

Journal: The Journal of Cell Biology

Article Title: Transcriptional repression induces a slowly progressive atypical neuronal death associated with changes of YAP isoforms and p73

doi: 10.1083/jcb.200509132

Figure Lengend Snippet: YAPΔC isoforms repress apoptosis and the TRIAD. (A) p73-mediated transcriptional activation by cisplatin (CDDP) was repressed by YAPΔCs. Luciferase assays were performed with MCF-7 cells 24 h after transfection of a p21/WAF1 reporter plasmid containing the p73 consensus cis-element and a YAPΔC expression vector (left). 25 μM CDDP was added 2 h after transfection. CDDP increased the transcription level to about threefold. Expression of YAPΔC isoforms (ins13, ins25, and ins61) remarkably repressed transcriptional activation by CDDP. FL-YAP (YAP) did not enhance the transcriptional activation, suggesting that endogenous YAP function was saturated. The expression of YAPs was checked simultaneously (right). n = 6. (B) YAPΔCs suppressed CDDP-induced apoptosis of MCF-7 cells. 25 μM CDDP was added to the medium 24 h after infection of adenovirus vectors, and cell death assay was performed with annexin-V in six wells after another 16 h. Adenovirus expression vectors are abbreviated as follows: AxCA, empty adenovirus vector AxCAwt; YAP, AxCAYAP-FL; ins13, AxCAins13; ins25, AxCAins25; and ins61, AxCAins61. Right panel shows the expression of YAP insert forms in cortical neurons. (C) YAPΔCs suppressed TRIAD of cortical neurons. 24 h after infection of adenovirus vectors of YAP isoforms, 25 μg/ml AMA was added. Cell death was assayed with annexin-V (six wells) at 4 d. Right panel shows expression of YAPΔCs in cortical neurons. (D) YAPΔC suppression by siRNA specific to a YAPΔC common sequence (siYAPΔ) enhanced AMA-induced TRIAD of cortical neurons, supporting the idea that YAPΔCs repress TRIAD of cortical neurons. Right panel shows specific repression of YAPΔCs by siYAPΔ. sc, siRNA of a scrambled sequence. 0.5 μg/well siRNA was transfected into cortical neurons (2 × 10 4 cells/well of 24-well dish), and 25 μM AMA was added to the medium 12 h later. Cell death was quantified by trypan blue staining in six wells at 4 d. (right) Bottom numbers represent relative intensities of the endogenous YAPΔC bands. (A–D) Asterisks indicate significant differences from controls (P < 0.01, t test). Error bars represent SD. (E) p73 was activated in TRIAD of cortical neurons. Right panel shows fold increase of phosphorylated p73 by AMA treatment (25 μg/ml).

Article Snippet: The filter was blotted with the anti-p73 goat polyclonal antibody (S-20; 1:1,000; Santa Cruz Biotechnology, Inc.) or antiphosphorylated p73 rabbit polyclonal antibodies (1:1,000; Cell Signaling) followed by HRP-coupled detection.

Techniques: Activation Assay, Luciferase, Transfection, Plasmid Preparation, Expressing, Infection, Sequencing, Staining

Journal: The Journal of Cell Biology

Article Title: Transcriptional repression induces a slowly progressive atypical neuronal death associated with changes of YAP isoforms and p73

doi: 10.1083/jcb.200509132

Figure Lengend Snippet: Relevance of YAPΔC isoforms and p73 to Htt-induced pathology. (A) YAPΔCs repressed Htt-induced cell death of cortical neurons. Primary cortical neurons were coinfected by adenovirus vectors for mutant Htt (AxCAHtt111) and a YAPΔC (AxCAins13, AxCAins25, or AxCAins61). Cell death was assayed with trypan blue at 4 d. As a control, empty vector (AxCA) was used. Expression of mutant Htt was equivalent among infections (not depicted). (B) Suppression of YAPΔCs by YAPΔC sequence-specific siRNA (siYAPΔ) enhanced mutant Htt-induced cell death of cortical neurons. 0.5 μg/well siRNA was transfected into primary cortical neurons (2 × 10 4 cells/well of 24-well dish) and infected with adenovirus vectors for mutant htt (AxCAHtt111) 12 h later. Cell death was quantified by trypan blue in six wells at 4 d. (C) Phosphorylation of p73 was induced in cortical neurons expressing mutant Htt. Cortical neurons were harvested 48 h after infection of empty adenovirus vector (AxCA) or mutant Htt adenovirus vector (htt111). Immunoblotting was performed with anti-p73, antiphosphorylated p73, or anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody (left). Relative values of phosphorylated p73 to total p73 were compared between AxCA-infected and AxCAHtt111-infected neurons (right). (D) Suppression of p73 by siRNA repressed Htt-induced cell death of cortical neurons (left). siRNA transfection and AxCAHtt111 infection were performed similarly to that in B. sip73, siRNA of p73; sc, siRNA of a scrambled sequence; Mock, mock treatment without siRNA. Cell death was quantified by trypan blue staining in four independent wells at 4 d after infection. Right panel shows expression of p73 and GAPDH at the time point of infection of AxCAHtt111 and indicates suppression of p73 by siRNA. (A, B, and D) Asterisks indicate significant reduction of cell death in four independent assays (P < 0.01, t test). Error bars represent SD. (E) p73 phosphorylation was enhanced in the brain of human HD patients. Cerebral cortex tissues of three HD patients (lanes 4–6) and three controls (lanes 1–3) were analyzed similarly (left). Relative values of phosphorylated p73 to total p73 were calculated (right).

Article Snippet: The filter was blotted with the anti-p73 goat polyclonal antibody (S-20; 1:1,000; Santa Cruz Biotechnology, Inc.) or antiphosphorylated p73 rabbit polyclonal antibodies (1:1,000; Cell Signaling) followed by HRP-coupled detection.

Techniques: Mutagenesis, Control, Plasmid Preparation, Expressing, Sequencing, Transfection, Infection, Phospho-proteomics, Western Blot, Staining

Journal: Human molecular genetics

Article Title: Androgen receptor-induced tumor suppressor, KLLN, inhibits breast cancer growth and transcriptionally activates p53/p73-mediated apoptosis in breast carcinomas.

doi: 10.1093/hmg/ddt077

Figure Lengend Snippet: Figure 3. KLLN inhibits breast cancer cell growth and induces apoptosis by transcriptional activation of p53 and p73. (A) MCF-7 and MDA-MB-453 cells were transfected with control vector or KLLN plasmid. After 48 h, cells were treated with 40 ng/ml nocodazole for 12 h. Cells were then fixed and stained by PI for flow cytometry to assess the cell cycle. (B) After KLLN was overexpressed (+) in MCF-7 or MDA-MB-453 cells, cell lysates were harvested for western blots with the shown antibodies. (C) Total RNA was extracted from MCF-7 cells after overexpression of KLLN. After reverse transcription, the cDNAs were used as templates for quantitative PCR (the Mann–Whitney test, ∗P ¼ 0.01, ∗∗P ¼ 0.0026). (D) KLLN was expressed in MCF-7 cells, and cell lysates were subjected to ChIP assay using anti-KLLN antibodies. Pull-down from ChIP assay was quantified and normalized to 3% input in qRT-PCR by specific primers covering different regions of the TP53 (left panel) and TP73 (right panel) promoters.

Article Snippet: Rabbit polyclonal anti- p73, CCNA and Lamin A antibodies are from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Transfection, Control, Plasmid Preparation, Staining, Cytometry, Western Blot, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Quantitative RT-PCR