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: Oral surgery, oral medicine, oral pathology and oral radiology

Article Title: Transforming growth factor-β1 activates ΔNp63/c-Myc to promote oral squamous cell carcinoma.

doi: 10.1016/j.oooo.2016.05.018

Figure Lengend Snippet: Fig. 1. Different grades of oral squamous cell carcinoma (OSCC) express proliferating cell nuclear antigen (PCNA), Ki67, cyclin A, DNp63, and transforming growth factor-b1 (TGF-b1) in comparison to E-cadherin. (A, E, I, M): Proteins expression in normal oral gingival mucosa (red): PCNA, Ki67, and DNp63 revealed nuclear positivity staining in epithelia cells, and cyclin A expressed in both the epithelial cells and extracellular matrix (ECM). (B, C, F, G, J, K, N, O): Proteins expression in well- and moderately differentiated OSCC (red): All of them showed a very similar pattern as they were in normal oral gingival mucosa, but their expression decreased in the cells toward the center of the tumor nest. (D, H, L, P): Proteins expression in poorly differentiated OSCC (red): In the tumor nest, these proteins expressed only in a small portion of cancer cells (red), showing a significantly

Article Snippet: To activate, we transfected cells with DNp63 and c-Myc, full-length DNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, Cambridge, MA) for 24 hours by themselves followed by an additional 24 hours with TGF-b1.

Techniques: Comparison, Expressing, Staining

Journal: Oral surgery, oral medicine, oral pathology and oral radiology

Article Title: Transforming growth factor-β1 activates ΔNp63/c-Myc to promote oral squamous cell carcinoma.

doi: 10.1016/j.oooo.2016.05.018

Figure Lengend Snippet: Fig. 2. Transforming growth factor-b1 (TGF-b1) regulates proliferating cell nuclear antigen (PCNA), Ki67, cyclin E2, DNp63, and E-cadherin expression in UMSCC38 cells (A). Column (a): PCNA, Ki67, cyclin E2, and DNp63 revealed negative staining in untreated UMSCC38 cells. Column (b): All the cells grown in 10% FBS/DMEM (positive control) were nuclear positive stained by PCNA, Ki67, cyclin E2, or DNp63. Columns (c-h): PCNA, Ki67, cyclin E2, and DNp63 expression in TGF-b1-treated cells. The number of nuclei positively stained cells increased in a time and dose-dependent manner, compared with untreated groups. Furthermore, as indicated by E-cadherin staining along the cell membrane (red), the cobblestone morphology was preserved in all treated UMSCC38 cells. TGF-b1 regulates PCNA, Ki67, cyclin E2, DNp63, and E-cadherin expression in UMSCC11B cells. Columns (a-b): PCNA, Ki67, cyclin E2, and DNp63 expressed very similarly as that in UMSCC38 untreated and positive control cells. Columns (c-e): 24 h TGFb1 treatment increased the number and intensity of PCNA, Ki67, cyclin E2, and DNp63 (green) expression in UMSCC11B cells (B) compared to untreated control groups. Also, the expression pattern of the epithelial marker, E- cadherin (red), was concentrated on the cell membrane, forming a continuous membranous, similar to that in the untreated cells. Columns (f-h): When treated with TGF-b1 for 48 hours, the number of positive cells decreased significantly, and the membranous expression of E-cadherin (red) was reduced and interrupted thereafter. (C): Percentage of immunofluorescence positively stained UMSCC38 and UMSCC11B cells. For both UMSCC38 and UMSCC11B cells, only very limited untreated cells express PCNA, Ki67, cyclin E2, or DNp63. (a-d): Percentage of positively stained UMSCC38 cells for PCNA (a), DNp63 (b), Ki67 (c), and cyclin E2 (d). Over 90% of UMSCC38 cells grown in 10% fetal bovine serum (FBS) were positively stained for these proteins. When cells were treated with TGF-b1 for 24 hours, the percentages increased as the dilutions of TGF-b1 were raised. At 48 hours, the percentages raised to very similar level with that of positive control (10% FBS) groups. (e-h): Percentage of positively stained UMSCC11B cells for PCNA (e), DNp63 (f), Ki67 (g), and cyclin E2 (h). Almost 95% of cells grown in 10% FBS were positive stained for PCNA, DNp63, Ki67, and cyclin E2. When cells were treated with TGF-b1 for 24 hours, the percentages increased obviously as the dilutions of TGF-b1 were raised. But at 48 hours, the percentages of positively stained cells reduced obviously, very similar to that of untreated cells.

Article Snippet: To activate, we transfected cells with DNp63 and c-Myc, full-length DNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, Cambridge, MA) for 24 hours by themselves followed by an additional 24 hours with TGF-b1.

Techniques: Expressing, Negative Staining, Positive Control, Staining, Membrane, Control, Marker

Journal: Oral surgery, oral medicine, oral pathology and oral radiology

Article Title: Transforming growth factor-β1 activates ΔNp63/c-Myc to promote oral squamous cell carcinoma.

doi: 10.1016/j.oooo.2016.05.018

Figure Lengend Snippet: Fig. 4. Pathways used by transforming growth factor-b1 (TGF-b1) during UMSCC cell proliferation. A, TGF-b1 uses both Smad- dependent and Smad-independent pathways during UMSCC cell proliferation. pSmad2 was expressed in UMSCC38 cells, but the expression was indifferent with TGF-b1 treatment of different dosages; however, its expression in the UMSCC11B was negligible in 2 and 5 ng/mL treatments and very low in the 10 ng/mL treatment. Subsequently, Western blot analysis of phosphorylated proteins was performed to determine the effects of TGF-b1 on Smad-independent pathways. pAKT showed no expression in UMSCC38 cells with any doses of TGF-b1 treatments. UMSCC11B showed similarly increased expression of pAKT with no difference in the TGF-b1 treatment conditions. DNp63 expression in both UMSCC38 and UMSCC11B were comparable and showed increased expression in a dose-dependent manner in comparison to control (actin). B, All three doses of TGF-b1 treatment groups differed significantly among themselves and between two cell lines (P .05, as indicated by *) from the untreated control cells (0.2% fetal bovine serum [FBS]) (P .005, as indicated by **). Results from the blots (see Figure 4A) and the intensity of the bands were measured using the Carestream Molecular Imaging Software version 5.3.1 (Rochester, NY). To perform a t test analysis of mean intensity measurements, a region of interest analysis was done from the data to Microsoft Excel software. Data points for all samples are paired by spatial arrangement on gel and compared pairwise to minimize the impact of subtle background artifacts on image analysis. C, Regulation of cyclin mRNA by TGF-b1. Cyclin D, E, A, and B mRNA expressions were determined by RT- PCR in TGF-b1 (5 ng/mL)etreated UMSCC38 and UMSCC11B cells every 12 hours for 48 hours. TGF-b1etreated cells had higher cyclins (D, E, A, and B) mRNA expression levels compared with the untreated control (0.2% FBS). Compared with UMSCC11B, TGF-b1 had chronologically higher cyclins (D, E, A, and B) mRNA levels in UMSCC38 cells in a time-dependent fashion till 48 hours. However, the levels sharply dropped after 24 hours in the UMSCC11B cell lines and continued to decline until 48 hours. D, Regulation of cyclin D mRNA by TGF-b1 in UMSCC cells: The levels of cyclin D mRNAs in response to TGF- b1, when DNp63 and c-Myc are either activated (by full-length cDNA) or repressed (by pRetrosuper-shRNA), was measured. To induce cyclins, TGF-b1 functions on both DNp63 and c-Myc, as activation and repression of DNp63 and c-Myc significantly affect cyclin D mRNA levels. The change in mRNA levels was determined by comparison to untreated control (UnTr, 0.2% FBS) and plotted as fold change/s (mean SD; n ¼ 3; *P < .05 compared with controls; **P < .005 compared with TGF-b1 treatments.

Article Snippet: To activate, we transfected cells with DNp63 and c-Myc, full-length DNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, Cambridge, MA) for 24 hours by themselves followed by an additional 24 hours with TGF-b1.

Techniques: Expressing, Western Blot, Comparison, Control, Imaging, Software, Reverse Transcription Polymerase Chain Reaction, shRNA, Activation Assay

Journal: Oral surgery, oral medicine, oral pathology and oral radiology

Article Title: Transforming growth factor-β1 activates ΔNp63/c-Myc to promote oral squamous cell carcinoma.

doi: 10.1016/j.oooo.2016.05.018

Figure Lengend Snippet: Fig. 4. (continued) E, TGF-b1 stimulates DNp63 gene activity in UMSCC cells: TGF-b1 increased DNp63 promoter activity chronologically, starting at 6 hours (five- and sixfold in UMSCC38 and UMSCC11B, respectively) and reaching the peak at 24 hours (103-fold and 98-fold in UMSCC38 and UMSCC11B, respectively). However, when both Smad-dependent and Smad- independent pathways were blocked, DNp63 promoter activity was repressed in both UMSCC38 and UMSCC11B cell lines. The results are shown as a mean SD obtained from three independent chromatin preparations (P .05 as indicated by * and P .005 as indicated by **).

Article Snippet: To activate, we transfected cells with DNp63 and c-Myc, full-length DNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, Cambridge, MA) for 24 hours by themselves followed by an additional 24 hours with TGF-b1.

Techniques: Activity Assay

Journal: Oral surgery, oral medicine, oral pathology and oral radiology

Article Title: Transforming growth factor-β1 activates ΔNp63/c-Myc to promote oral squamous cell carcinoma.

doi: 10.1016/j.oooo.2016.05.018

Figure Lengend Snippet: Fig. 6. Schematic diagram of the proposed mechanism of oral squamous cell carcinoma (OSCC) proliferation and invasion. On the basis of our data, we hypothesized that in the initial stage, UMSCC cell proliferation (both in the primary tumor, UMSCC38, and the secondary tumors, UMSCC11B cell lines) is achieved by TGF-b1/Smads/DNp63/c-Myc pathway with higher proliferative attributes in UMSCC38. However, subsequently, TGF-b1 switches its signaling via the PI3K/ AKT/DNp63 pathway at the inception stage for EMT/inva- sive, primarily in the secondary tumor, as seen in the UMSCC11B cell lines (that resulted from relapse and recur- rence at the primary site). We further conclude that although TGF-b1 has distinct functions in cancer progression, its downstream signaling partners and transcription factors regulate ultimate cancer cell fate and achieve switch from tumor growth to invasion.

Article Snippet: To activate, we transfected cells with DNp63 and c-Myc, full-length DNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, Cambridge, MA) for 24 hours by themselves followed by an additional 24 hours with TGF-b1.

Techniques: