Journal: Frontiers in Cell and Developmental Biology

Article Title: Irradiation Induces Epithelial Cell Unjamming

doi: 10.3389/fcell.2020.00021

Figure Lengend Snippet: Ionizing radiation does not disrupt normal epithelial cell functions. (A) On ALI days 7, 10, and 14, TEER measured 1 h before (pre-exposure, open symbols) and 1 h after (post-exposure, closed symbols) indicates that IR exposure did not reduce TEER. Black lines represent TEER measured from two independent wells (Control A and Control B) of control and red lines represent TEER measured from two independent wells (Irradiated A and Irradiated B) of irradiated cells from one donor. (B) On ALI day 14, the percent change in TEER comparing 1 h pre-, and 1 h post-IR exposure showed no difference between control (white bar) and irradiated (black bar) cells, indicating that IR did not reduce TEER. Error bars represent the standard error of the mean from four independent donors ( n = 4). (C) Methanol treatment used as a positive control for cell death resulted in prominent cell death as indicated by EthD-1 staining (red), whereas neither control nor IR exposure induced cell death (scale bars = 50 μm). Representative images (D) of MUC5AC, FOXJ1, p63α (20X), and β4-tubulin (63X). RT-qPCR (E) showing mRNA expression of MUC5AC, FOXJ1, TEKT1 , and TP63 indicate no differences in cellular differentiation between control and irradiated cells. Scale bar = 50 μm (top 8 panels), 20 μm (bottom 2 panels) in (D) and error bars represent the standard deviation from two representative donors ( n = 2) in (E) .

Article Snippet: To observe the differentiation of the ALI cultures, we stained fixed cells with primary antibodies for MUC5AC (45M1, ThermoFisher Scientific), FOXJ1 (2A5, ThermoFisher Scientific), β4-tubulin (ONS, Millipore Sigma), and p63α (D2K8X, Cell Signaling Technology).

Techniques: Control, Irradiation, Positive Control, Staining, Quantitative RT-PCR, Expressing, Cell Differentiation, Standard Deviation

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: PLOS Pathogens

Article Title: Identification and characterization of a ubiquitin E3 RING ligase of the Chlamydia -like bacterium Simkania negevensis

doi: 10.1371/journal.ppat.1013626

Figure Lengend Snippet: (A, B) A FLAG-tagged version of the SneRING was overexpressed in U2OS cells using PEI MAX transfection. 24 h later, cells were infected with Sne at an MOI of 1. Non-transfected cells served as a control. 48 h p.i., samples were collected and immunoprecipitation was performed using FLAG-magnetic beads, followed by mass spectrometry analysis. The graphs show identified proteins, with significance (-log 10 p-value calculated by two-tailed T-test, n = 3) plotted against the log 2 fold change (log 2 FC) of transfected/infected U2OS cells (U2OS+Sne + SneRING) relative to non-transfected/infected controls (U2OS+Sne) (A) or of transfected/infected U2OS cells (U2OS+Sne + SneRING) relative to transfected/non-infected controls (U2OS+SneRING) (B) . Enriched proteins are labeled in red, reduced proteins are shown in blue, and grey represents unchanged proteins. Only host cell proteins are shown. (C) Samples were prepared, and immunoprecipitation was performed as in A. Input and elution fractions were analyzed by SDS-PAGE and western blot using antibodies against Sne heat shock protein SnGroEL (star indicates that the signal represents a cross-reactive band observed at 130 kDa), Mic60, CKAP4, Erlin2, PHB, Cox5a, Tom70, and FLAG. Mic60, mitochondrial contact site and cristae organizing system 60; CKAP4, cytoskeleton‐associated protein 4; Erlin2, ER Lipid Raft Associated 2; PHB, Prohibitin; Cox5a, cytochrome c oxidase subunit 5A; Tom70, translocase of the outer mitochondrial membrane 70.

Article Snippet: The CKAP4 antibody (Cat. No. 16686-1-AP) was purchased from Proteintech (Rosemont, USA).

Techniques: Transfection, Infection, Control, Immunoprecipitation, Magnetic Beads, Mass Spectrometry, Two Tailed Test, Labeling, SDS Page, Western Blot, Membrane

Journal: PLOS Pathogens

Article Title: Identification and characterization of a ubiquitin E3 RING ligase of the Chlamydia -like bacterium Simkania negevensis

doi: 10.1371/journal.ppat.1013626

Figure Lengend Snippet: (A, B) A reaction containing purified recombinant SneRING, recombinant E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme (UbcH5b), ubiquitin, and ATP was mixed with the respective substrate (purified recombinant GST (A) or His-CKAP4 (B) ) for the indicated periods at 37 °C. Reactions without the ligase and/or substrate served as controls. Samples were analyzed by SDS-PAGE and western blot, using antibodies against SneRING, GST, ubiquitin, and CKAP4. Asterisks indicate ubiquitinated CKAP4. Ponceau staining of the western blot membranes is shown for the loading control.

Article Snippet: The CKAP4 antibody (Cat. No. 16686-1-AP) was purchased from Proteintech (Rosemont, USA).

Techniques: Purification, Recombinant, Ubiquitin Proteomics, SDS Page, Western Blot, Staining, Control

Journal: Oncogene

Article Title: Role for c-Abl and p73 in the radiation response of male germ cells.

doi: 10.1038/sj.onc.1204568

Figure Lengend Snippet: Figure 1 Expression of c-Abl, p73 and p63 in the testis. Arrowheads show spermatogonia, Ley: Leydig cells, Ser: Sertoli cells, PL: preleptotene spermatocytes, L: leptotene spermatocytes, Z: zygotene spermatocytes, Ps: pachytene spermatocytes, Rs: round spermatids, Es: elongating spermatids, Rb: residual bodies. Insets show magni®cation of a spermatogonium (a, b) and a spermatocyte (b) or a tubule containing residual bodies (g, h, i). Stages of the seminiferous epithelium are indicated with Roman numerals. DAB staining shows expression of c-Abl in the cytoplasm of spermatogonia, spermatocytes, Leydig cells and residual bodies (a), p73 in the cytoplasm of spermatogonia, early spermatocytes and Leydig cells and in the nucleus of in round spermatids (b), and nuclear p63 in spermatocytes, round spermatids and Sertoli cells (c). Confocal images also show expression of both p63 (d) and p73 (g), and of c-Abl (e, h). Merged pictures show co-localization of c-Abl with p73 (i) and not p63 (f). Note nuclear expression of p73 in late spermatocytes (stage IX) (g, i). Magni®cation: bar represents 20 mm

Article Snippet: The slides were then incubated with a rabbit polyclonal c-Abl antibody (K-12, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), a mouse monoclonal c-Abl antibody (Ab-3, Oncogene Science, Cambridge, MA, USA), a rabbit polyclonal p73 antibody (H79, Santa Cruz Biotechnology Inc.), a goat polyclonal p73a antibody (C-17, Santa Cruz Biotechnology Inc.) or a mouse monoclonal p63 antibody (4A4, Santa Cruz Biotechnology Inc.) diluted 1 : 25 in PBS including 1% BSA in a humidi®ed chamber overnight at 48C.

Techniques: Expressing, Staining

Journal: Oncogene

Article Title: Role for c-Abl and p73 in the radiation response of male germ cells.

doi: 10.1038/sj.onc.1204568

Figure Lengend Snippet: Figure 2 Western blot analysis of p63, p73 and c-Abl in total testis lysates before and after treatment with irradiation showing an increase of c-Abl in response to ionizing irradiation. Arrow indicates bands that remain detectable when c-Abl is immuno- precipitated using K-12 and detected using Ab-3 (Figure 3). PVDF membranes stained for c-Abl were stripped and reprobed using an antibody against a-tubulin

Article Snippet: The slides were then incubated with a rabbit polyclonal c-Abl antibody (K-12, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), a mouse monoclonal c-Abl antibody (Ab-3, Oncogene Science, Cambridge, MA, USA), a rabbit polyclonal p73 antibody (H79, Santa Cruz Biotechnology Inc.), a goat polyclonal p73a antibody (C-17, Santa Cruz Biotechnology Inc.) or a mouse monoclonal p63 antibody (4A4, Santa Cruz Biotechnology Inc.) diluted 1 : 25 in PBS including 1% BSA in a humidi®ed chamber overnight at 48C.

Techniques: Western Blot, Irradiation, Staining

Journal: Journal of Neuroinflammation

Article Title: Cardiac arrest triggers IL-17-mediated neuroinflammation and astrocyte polarization: insights into pathogenesis and intervention

doi: 10.1186/s12974-025-03600-6

Figure Lengend Snippet: Cardiac arrest promotes IL-17 A/F expression and secretion, upregulates IL-17RA/C in hippocampal astrocytes, and induces inflammation. a Quantification of hippocampal IL-17 A-F mRNA levels at 24 h post ROSC ( n = 5). b , c The levels of IL-17 A in hippocampus and serum were detected by ELISA assays ( n = 6). d Quantitative analysis of hippocampal IL-17RA-RF mRNA levels at 24 h after ROSC ( n = 5). e Western blot analyzed the expression of IL-17RA/RC in the hippocampal tissues ( n = 3). f Immunofluorescence staining of GFAP/IBA1/NeuN (green), IL-17RA (red), and DAPI (blue) in hippocampus of CA rats. g The mRNA levels of IL-1β , IL-6 , IL-10 , TNF-α , and GM-CSF in the hippocampus. h , i The levels of IL-1β , IL-6 , IL-10 , TNF-α , and GM-CSF in the hippocampus and serum were analyzed using Elisa assays respectively ( n = 5). Biologically independent samples were used. Statistical analysis for panels a ( IL-17B , C , D ), b, d ( IL-17RA , RB , RD , RE ), g ( IL-1β , TNF-α ), h and i (IL-6, IL-10) was performed with unpaired Student’s t test (two-tailed). Data in a ( IL-17 A , E , F ), c, d ( IL-17RC ), g (IL-6 , IL-10 , GM-CSF ) and i (IL-1β, TNF-α, GM-CSF) were analyzed using the non-parametric Mann-Whitney U test. P < 0.05 considered statistically significant. * P < 0.05, ** P < 0.01, NS - not significant, error bars are mean ± SEM

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against: GFAP (1:250, Abcam, Cat# ab7206), IBA1 (1:500, Abcam, Cat# ab178846), NeuN (1:300, Abcam, Cat# ab177487), IL-17RA (1:200, Biorbyt, Cat# orb1536865), p-P65 (1:200, Zenbio, Cat# 310013), p-ERK (1:200, Zenbio, Cat# 310065), CD31 (1:250, Abcam, Cat# ab81289), ZO-1 (1:100, Abcam, Cat# ab307799).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Neuroinflammation

Article Title: Cardiac arrest triggers IL-17-mediated neuroinflammation and astrocyte polarization: insights into pathogenesis and intervention

doi: 10.1186/s12974-025-03600-6

Figure Lengend Snippet: IL-17RA neutralization mitigates IL-17 A-mediated astrocyte polarization, activation of MAPK/NF-κB pathway, and inflammation. a , b The protein and mRNA levels of activated astrocyte markers GFAP and S100B in human astrocyte SVG P12 cells treated with IL-17 A recombinant protein alone or in combination with an anti-IL-17RA. c , d The A1 astroyte marker ( C3 , FbIn5 , Ligp1 , and Serping1 ) and A2 astrocyte marker ( Clcf1 , Tgm1 , Cd109 , Ptx3 , and S100A10 ) in human astrocyte SVG P12 cells were detected by real-time PCR ( n = 3). e Western blot analysis showed the expression of NF-κB and MAPK pathways changes in SVG P12 cells treated with IL-17 A recombinant protein or in combination with an anti-IL-17RA. f The levels of IL-1β , IL-6 , IL-10 , TNF-α , and GM-CSF in SVG P12 cells were analyzed using Real-time PCR ( n = 3). Biologically independent samples were used. Statistical significance was determined by unpaired Student’s t test (two-tailed) in (b, c, d, g) with P < 0.05 considered statistically significant. * P < 0.05, ** P < 0.01, NS - not significant, error bars are mean ± SEM

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against: GFAP (1:250, Abcam, Cat# ab7206), IBA1 (1:500, Abcam, Cat# ab178846), NeuN (1:300, Abcam, Cat# ab177487), IL-17RA (1:200, Biorbyt, Cat# orb1536865), p-P65 (1:200, Zenbio, Cat# 310013), p-ERK (1:200, Zenbio, Cat# 310065), CD31 (1:250, Abcam, Cat# ab81289), ZO-1 (1:100, Abcam, Cat# ab307799).

Techniques: Neutralization, Activation Assay, Recombinant, Marker, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: Cardiac arrest triggers IL-17-mediated neuroinflammation and astrocyte polarization: insights into pathogenesis and intervention

doi: 10.1186/s12974-025-03600-6

Figure Lengend Snippet: The proposed model describing the mechanisms underlying CA-induced neuroinflammation and brain injury. CA promoted Th17 cell differentiation, disrupted the blood-brain barrier (BBB), and enhanced IL-17 A/F secretion. Subsequently, Th17 cells and their secreted IL-17 A/F infiltrated the brain parenchyma through the compromised BBB. In addition, IL-17 A/F specifically binds to the IL-17RA/RC receptors on astrocytes, activating the NF-κB and MAPK signaling pathways. This activation drives astrocyte polarization toward the neurotoxic A1 phenotype, promotes the expression and secretion of proinflammatory cytokines (including IL-1β, IL-6, IL-10, TNF-α, and GM-CSF), and ultimately perpetuates CA-induced neuroinflammation. Importantly, IL-17 A-neutralizing antibody treatment reversed A1 astrocyte polarization, alleviated neuroinflammatory responses, reduced neuronal apoptosis, and improved survival in CA model rats. These findings highlight the IL-17 A pathway as a promising therapeutic target for mitigating CA-induced neurological dysfunction

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against: GFAP (1:250, Abcam, Cat# ab7206), IBA1 (1:500, Abcam, Cat# ab178846), NeuN (1:300, Abcam, Cat# ab177487), IL-17RA (1:200, Biorbyt, Cat# orb1536865), p-P65 (1:200, Zenbio, Cat# 310013), p-ERK (1:200, Zenbio, Cat# 310065), CD31 (1:250, Abcam, Cat# ab81289), ZO-1 (1:100, Abcam, Cat# ab307799).

Techniques: Cell Differentiation, Protein-Protein interactions, Activation Assay, Expressing

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

Journal: Journal of Virology

Article Title: Molecular mechanism of resistance to lonafarnib conferred by mutations in the cysteine-rich region of respiratory syncytial virus fusion glycoprotein and discovery of a lonafarnib-derived antiviral PROTAC

doi: 10.1128/jvi.01487-25

Figure Lengend Snippet: Evaluation of the antiviral activity of compound 0179841 in a human airway organoid model. ( A ) Establishment of HBEC-derived apical-out airway organoids. Representative white-light images of organoids cultured over the long term are presented, along with confocal images demonstrating the expression of respiratory epithelial markers. The confocal images illustrate the presence and spatial distribution of distinct cell types within the organoids, as identified by their specific markers: basal cells labeled with P63 (purple), goblet cells labeled with MUC5AC (green), and ciliated cells labeled with acetylated tubulin (red). ( B–E ) Organoids were infected with RSV ON1-GFP at an MOI of 1 and cultured in media supplemented with 0.1% DMSO or compound 0179841 at the indicated concentrations (5, 1, and 0.5 µM) for 96 h. ( B ) The cell culture supernatant of infected organoids was collected, and a TCID 50 assay was performed. ( C ) The RSV ON1-GFP-infected organoids were visualized via microscopy. ( D ) The GFP intensity in five randomly selected fields per treatment was measured using ImageJ analysis software and normalized to the intensity in the DMSO condition. ( E ) The RSV ON1-GFP-infected organoids were visualized using a confocal microscope. The graphs shown are representative of three independent experiments. The differences between the experimental and DMSO groups were determined by Student’s t -test for comparing two groups of data (*** P < 0.001).

Article Snippet: The organoids were fixed in parallel in 4% PFA, permeabilized with 0.5% Triton X-100 (1 h), blocked with 5% BSA (Macklin, cat#B824162; 2 h), and incubated overnight at 4°C with primary antibodies: Goat anti-Human p63/TP73L polyclonal antibody (R&D Systems, cat#AF1916), Rabbit anti-Human Mucin 5AC polyclonal antibody (Abways, cat#CY6826), and Mouse anti-acetylated tubulin monoclonal antibody (Sigma-Aldrich, cat#T7451).

Techniques: Activity Assay, Derivative Assay, Cell Culture, Expressing, Labeling, Infection, Microscopy, Software