Proliferating cell nuclear antigen (pcna)

SCC25 and SCC4 cells were seeded in 6-wells at a density of 1.5 × 10⁶ per well. After overnight incubation, the cells were treated with CDDP, 5-MTP, or both at indicated concentrations for 24 h. The cells were washed with phosphate-buffered saline and lysed by adding ice-cold RIPA buffer supplemented with protease inhibitor and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). The cell lysates were centrifuged, and the supernatant was separated in 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto PVDF membranes. The membranes were blocked with blocking reagent (Leadgene Biomedical, Taiwan), incubated with primary antibodies, and diluted with TBS containing 0.1% Tween 20 supplemented with 5% nonfat dried milk. The target proteins were probed with specific antibodies against p-STAT3 (ABclonal; 1:1000, AP0705), p-Akt (ABclonal; 1:1000, AP0637), p-p65 (Cell Signaling; 1:1000, 3033T), p-p38 (ABclonal; 1:1000, AP0526), PCNA (GeneTex; 1:3000, GTX100539), GAPDH (GeneTex; 1:10000, GTX100118). Enhanced chemiluminescence was used to detect the signals. The protein abundance of the samples was quantified using Image-J software following densitometric scanning.

Tissues were fixed with 4% paraformaldehyde at 4 °C overnight, embedded with paraffin, cut into 5 μm-thick sections, and mounted onto slides. The slides were dehydrated by graded ethanol. Antigens were retrieved by microwave heating for 20 min in sodium citrate-hydrochloric acid buffer. Tissue sections were incubated with an antibody against SNTB1 (1:100; Thermo fisher Scientific) or PCNA (1:800; Genetex). Background staining was assessed by omitting the primary antibody. The intensity of staining was evaluated using a scoring system described in detail in “Tissue microarray” section. The overall staining score was calculated by multiplying the intensity score and percentage score.

DAB quantification of slides stained for proliferation cell nuclear antigen (PCNA) (GeneTex Inc., Irvine, USA) and counterstained with hematoxylin was performed by semiquantitative analyses. Ten random, non-overlapping fields (magnification, ×200) of tumor tissue from each specimen were photographed, and pictures were automated single cell counted for DAB using the “Fiji” version of ImageJ from http://fiji.sc (68 (link)). The following adjustments for automatic counting were used: Color Deconvolution DAB, threshold 150, particle size 150–6,000, Circularity 0.14–1.00. From all fields, the mean counts were averaged to yield the final score for each specimen.

Western blotting was performed by standard procedures after tissue lysis in Laemmli buffer. Nitrocellulose blotted membranes were probed with the following antibodies: H3K27me (Abcam, ab6002), H3K9me3 (Abcam, ab8898), H4K20me3 (Abcam, ab9053), H3K4me3 (Abcam, ab8580), H3 (Cell Signalling), PCNA (GeneTex, GTX124496), γH2AX (Genetex, GTX127343), and α-Tubulin (Cell Signalling, 3873S). Development was performed by Odyssey CLX reader (LI-COR). Densitometry analysis was performed using LI-COR software. Signal intensity from three independent Western blots loaded with lysates derived from different individuals was used for densitometry calculations normalized to young samples.

Sodium cholate hydrate, cholesterol, D-(+)-glucose, D-(−)-fructose, 2,4-dinitrophenylhydrazine (DNPH), and 4-hydroxynonenal (4HNE) were purchased from Sigma (St. Louis, MO, USA). Antibodies to nephrin (sc-376522), NGAL (sc-515876), IL-1β (sc-52012), IL-6 (sc-57315), TNF-α (sc-52746), and actin (sc-8432) were acquired from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). KIM-1 (GTX85067), Nrf2 (GTX103322), Keap1 (GTX60660), NFκB p65 (GTX102090), IκB (GTX82797), and PCNA (GTX100539) were from GeneTex (Irvine, CA, USA). Goat anti-rabbit and goat anti-mouse IgG-HRP were purchased from Cell Signaling Technology (Danvers, MA, USA). All other chemicals used were of the highest purity available.

Antibodies against alpha smooth muscle actin (ACTA2), Cav1 were from Santa Cruz (Santa Cruz, CA), against total AKT, phospho-Cav1, Ccdn1 and Src were from Cell Signaling Technology (Danvers, MA, Germany), against Tagln from Proteintech (Chicago, IL), against FAP from Abcam (Cambridge, MA), against Pcna from GeneTex (Irvine, CA) and against beta actin (clone AC-74, A2228) from Sigma-Aldrich (St. Louis, MO).

Embryos were collected at 48 hpf, fixed in 4% paraformaldehyde at 4°C overnight. For the proliferation assay, the embryos were digested using Collagenase, Type II (Life technologies, USA) at room temperature, and blocking was performed for one hour at room temperature, and followed by incubating with the primary antibody against p-H3(S10) (Abacam, USA) or PCNA (Genetex, USA) at 4°C overnight. Second antibody were from the series of Alexa Fluor (Life technologies, USA). For the apoptosis assay, In Situ Cell Death Detection kit, TMR red (Roche, USA) was used and all procedures were performed as the instruction manual described. Finally, embryo hearts were collected under a Leica M205C stereomicroscope and were imaged using a Leica SP8 microscope.