Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: Silencing of LINC01013 inhibited hypoxia-induced hPASMC proliferation and inflammation. A , B hPASMC proliferation was determined by CCK8 and EdU incorporation assays ( n = 6). EdU (red), DAPI (blue), scale bar, 50 μm. C Western blotting analysis of PCNA, cyclin A and cyclin D in hPASMCs ( n = 6). D RT‒qPCR analysis showed the mRNA levels of TNF-α and IL-6 after LINC01013 knockdown, β-actin was used as a internal reference gene ( n = 6). E Western blotting analysis of TNF-α and IL-6 in hPASMCs. ( n = 6). All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; Hyp, hypoxia; NC, negative control; si, siRNA

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Western Blot, Knockdown, Negative Control

Journal: World Journal of Gastroenterology

Article Title: Danggui-Baishao herb pair protects against dextran sulfate sodium-induced colitis by modulating the Wnt/β-catenin pathway

doi: 10.3748/wjg.v32.i5.113024

Figure Lengend Snippet: Herb pair restored abnormal epithelial proliferation in colitis mice. A and B: Western blotting analysis of protein expression of leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) in colon tissues; C: Quantitative polymerase chain reaction analysis of mRNA expression of Lgr5, SRY-box transcription factor 9, homeodomain-only protein homeobox, achaete scute-like 2, telomerase reverse transcriptase, Mucin 2, lysozyme 1, and Chromogranin A in colon tissues; D: Representative images of immunohistochemical (IHC) of Lgr5, proliferating cell nuclear antigen, Ki67 in colon tissues; E-G: Quantitative analysis of IHC results of Lgr5, proliferating cell nuclear antigen, Ki67. For western blotting analysis: n = 3, for quantitative polymerase chain reaction analysis: n = 5-6, for IHC measurement: n = 3. a P < 0.05 vs dextran sulfate sodium group, b P < 0.01 vs dextran sulfate sodium group, c P < 0.001 vs dextran sulfate sodium group. Lgr5 : Leucine-rich repeat-containing G-protein coupled receptor 5; Sox9 : SRY-box transcription factor 9; Hopx : Homeodomain-only protein homeobox; Ascl2 : Achaete scute-like 2; Tert : Telomerase reverse transcriptase; Muc2 : Mucin 2; Lyz1 : Lysozyme 1; Chga : Chromogranin A; DSS: Dextran sulfate sodium; PCNA: Proliferating cell nuclear antigen.

Article Snippet: They were incubated with anti-CD11b (1:500, ab133357, ABCAM, United Kingdom), anti-F4/80 (1:500, ab111101, ABCAM, United Kingdom), anti-Lgr5 (1:500, ab75850, ABCAM, United Kingdom), anti-proliferating cell nuclear antigen (PCNA) (1:500, 60097-1-Ig, Proteintech, IL, United States), anti-Ki67 (1:100, MA514520 , Thermo Fisher Scientific, MA, United States), and anti-β-catenin (1:500, ab32572, ABCAM, United Kingdom) overnight at 4 °C.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription, Immunohistochemical staining

Journal: ACS Omega

Article Title: Pterostilbene-Isothiocyanate Inhibits Proliferation of Human MG-63 Osteosarcoma Cells via Abrogating β-Catenin/TCF-4 Interaction—A Mechanistic Insight

doi: 10.1021/acsomega.3c02732

Figure Lengend Snippet: Effect of PTER-ITC on transcription and translation of different factors. (A) Effect of PTER-ITC on transcription level of proliferation ( PCNA ), migration ( MMP-2/9 ), metastatic ( E/N-cadherin ), and apoptotic ( Caspase-3 and PARP-1 ) markers. (B, C) Representative immunoblot images showing the effect of PTER-ITC and ZA treatments on PCNA, MMP-2/9, E/N-cadherin, active Caspase-3, and cleaved PARP-1 markers (B) along with their corresponding quantitative densitometric analysis (C). Data represented as mean ± SE of three independent experiments. Statistical significance was determined through two-way ANOVA and represented as *, **, and *** for p < 0.05, 0.01, and 0.001, respectively, when compared to respective control.

Article Snippet: Antibodies against proliferative cell nuclear antigen (PCNA) (E-AB-10010), cleaved poly [ADP-ribose] polymerase-1 (PARP-1) (E-AB-30059), matrix metalloproteinase-2 (MMP-2) (E-AB-32054), matrix metalloproteinase-9 (MMP-9) (E-AB-70247), active Caspase-3 (E-AB-22115), TCF-4 (E-AB-60206), and β-actin (E-AB-20058) were purchased from Elabscience (Texas, USA).

Techniques: Migration, Western Blot, Control

Journal: ACS Omega

Article Title: Pterostilbene-Isothiocyanate Inhibits Proliferation of Human MG-63 Osteosarcoma Cells via Abrogating β-Catenin/TCF-4 Interaction—A Mechanistic Insight

doi: 10.1021/acsomega.3c02732

Figure Lengend Snippet: List of Primers Used in This Study

Article Snippet: Antibodies against proliferative cell nuclear antigen (PCNA) (E-AB-10010), cleaved poly [ADP-ribose] polymerase-1 (PARP-1) (E-AB-30059), matrix metalloproteinase-2 (MMP-2) (E-AB-32054), matrix metalloproteinase-9 (MMP-9) (E-AB-70247), active Caspase-3 (E-AB-22115), TCF-4 (E-AB-60206), and β-actin (E-AB-20058) were purchased from Elabscience (Texas, USA).

Techniques: Sequencing

Journal: eLife

Article Title: The scaffold protein Nde1 safeguards the brain genome during S phase of early neural progenitor differentiation

doi: 10.7554/elife.03297

Figure Lengend Snippet: Figure 7. Identification of a nuclear pool of Nde1 that interacts with the cohesin complex. (A) Double immunohistological staining with antibodies to Nde1 (red) and Pax6 (green) reveals the presence of Nde1 in the nucleus of cells in the neocortical VZ. Notice the detection of Ndel1 in the cortical neurons of the Nde1−/− brain due to the cross reactivity of anti-NDE1/Nde1 to Ndel1. (B) Immunofluorescence confocal image of GFP-Nde1 (green) transfected HeLa cells showing nuclear targeting. Cell nuclei are highlighted by co-staining with Nucleoporin p62 at the nuclear envelope (red) as well as with Hoechst (blue). (C) Immunohistological analysis reveals enhanced nuclear Nde1 (red) in S phase neural progenitors (identified by Pcna foci in green). (D) Co-immunoprecipitation of the cohesin complex with Nde1. Myc-Nde1 was transfected in 293T cells and immunoprecipitated by the anti-myc 9E10 antibody or mouse IgG. The presence of SMC3, SMC1, and RAD21 in the Myc-Nde1 immunocomplex is shown by immunoblotting. (E) Binding of SMC3 NBD with Nde1. Flag-NBD of SMC3 was co-transfected with GFP-Nde1 in 293T cells and immunoprecipitated by the Flag antibody and mouse IgG. The presence of GFP-Nde1 and the absence SMC3 in the Flag-NBD immunocomplex are shown by immunoblotting. A diagram of the cohesin complex and the Nde1 binding domain (NBD) of SMC3 is included. (F) Flow cytometry analysis of cell cycle DNA content of 293T cells transfected with the vector Figure 7. Continued on next page

Article Snippet: The following antibodies were used: γH2AX, PH3, p53, NeuN (Millipore, Billerica, MA); phospho-p53 (Ser15), Rad50, γH2AX (Cell Signaling Tech, Beverly, MA); SMC3, Tbr2, BU1/75, Foxp2, NPM1 (Abcam, Cambridge, MA); PCNA, SMC1, 53PB1 (Novus Biologicals Littleton, CO); Cux1, DCX, BRCA1,Cdc25A (Santa Cruz, Dallas, TX); Pax6 (Thermo, Waltham, MA); B44 (BD Biosciences, San Jose, CA); phospho-vimentin 4A4 (MBL International, Woburn, MA); SNF2h (Active Motif, Carlsbad, CA); RAD21 (Bethyl Lab, Montgomery, TX); parvalbumin (Sigma, St. Louis, MO); Tuj1 (Covance, Princeton, NJ).

Techniques: Staining, Immunofluorescence, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Flow Cytometry, Plasmid Preparation

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Transdermal lovastatin enhances fracture repair in rats.

doi: 10.1359/jbmr.080603

Figure Lengend Snippet: FIG. 4. Representative histological sections comparing vehicle-treated vs. TD LV (2.5 mg/kg/d for 5 days) after 8 days of fracture. Several sections 500 m proximal and distal to the fracture site were stained for the analy- sis. Ten regions as described in the Materials and Methods section were analyzed in each section. The table under the images shows the results of the histomorphometric analysis. Os- teoclast and osteoblast surface as well as the number of PCNA+ cells were counted, and the ratio of PCNA+ cells to total cells was calculated and expressed as a percentage.

Article Snippet: Immunohistochemistry: PCNA antibody was obtained from Novus Biologicals.

Techniques: Staining

Journal: Scientific Reports

Article Title: Nucleocapsid Interacts with NPM1 and Protects it from Proteolytic Cleavage, Enhancing Cell Survival, and is Involved in PEDV Growth

doi: 10.1038/srep39700

Figure Lengend Snippet: Western blot analysis of nuclear and cytoplasmic fractions of PEDV-infected Vero E6 cells with an anti-GAPDH mAb ( A ), anti-PCNA mAb ( B ), and anti-N mAb ( C ). ( A,B and C ) Nuc, nuclear fraction of PEDV-infected cells; Cyt, cytoplasmic fraction of PEDV-infected cells. ( C ) Moc, mock-infected cells; Inf, PEDV-infected cells. The arrowheads indicate purified bands that are the same sizes as GAPDH ( A ), PCNA ( B ), and N ( C ) proteins. ( D ) Western blot analysis of N protein in nuclear and cytoplasmic fractions of PEDV-infected Vero E6 cells at different times with an anti-GAPDH mAb, anti-PCNA mAb, and anti-N mAb. Nuc, nuclear fraction of PEDV-infected cells; Cyt, cytoplasmic fraction of PEDV-infected cells.

Article Snippet: The membrane was soaked in blocking buffer (PBS containing 5% nonfat milk) for 2 h and then reacted with the indicated antibodies: mouse anti-GAPDH mAb (1:10,000) (G8795; Sigma), mouse anti-PCNA mAb (1:200) (BM0104; BOSTER), mouse anti-N mAb (1:500), mouse anti-GST mAb (1:1000) (AG768; Beyotime), mouse anti-actin mAb (1:5000) (A5441; Sigma), mouse anti-GFP mAb (1:10,000) (66002–1–1 g; Proteintech), rabbit anti-Flag mAb (1:2000) (20543–1-AP; Proteintech), or mouse anti Myc mAb (1:2000) (66004–1–1 g; Proteintech), rabbit anti-caspase-3 mAb (1:1000) (AC030; Beyotime).

Techniques: Western Blot, Infection, Purification

Journal: Scientific Reports

Article Title: Nucleocapsid Interacts with NPM1 and Protects it from Proteolytic Cleavage, Enhancing Cell Survival, and is Involved in PEDV Growth

doi: 10.1038/srep39700

Figure Lengend Snippet: ( A ) The NPM1 fusion protein as a marker of the nucleolus is colored red. The nucleolar localization of N in transfected cells was clearly observed at 42–42.5 hpt. As shown, a small amount of N was observed in the nucleolus at 42 hpt (t = 30 min). N protein accumulated continuously in the nucleolus of transfected cells until t = 60 min and was exported from the nucleolus at t = 61–65 min. This figure shows snapshots of the cells from the time-lapse movie ( in the ). Data are representative of one of three independent experiments. Real-time visualization of the kinetics of the nucleolar localization of N protein indicated that the process was rapid, taking only 30 min in total. ( B ) Knockdown of NPM1 protein levels following siRNA treatment. Vero E6 cells transfected with no siRNA (Mock), scrambled siRNA (siScr), left untreated (No treat) or with different concentrations (mM) of siRNAs targeting NPM1 (siNPM1) (as indicated at the top of each lane) were harvested 48 hpt. Endogenous NPM1 protein levels were detected by immunoblotting using antibodies directed against the indicated proteins. ( C and D ) Western blot analysis of Myc-N protein in nuclear and cytoplasmic fractions of NPM1-knockdown cells ( C ) or Ectopic NPM1-overexpression cells ( D ) at 48 hpt with anti-GAPDH mAb, anti-PCNA mAb, anti-NPM1 mAb, anti-Myc mAb and anti-Flag mAb. Nuc, nuclear fraction; Cyt, cytoplasmic fraction; cell, whole cells. Densitometric data for Nuc/Cell (Myc-N) from three independent experiments are expressed as mean ± SD.

Article Snippet: The membrane was soaked in blocking buffer (PBS containing 5% nonfat milk) for 2 h and then reacted with the indicated antibodies: mouse anti-GAPDH mAb (1:10,000) (G8795; Sigma), mouse anti-PCNA mAb (1:200) (BM0104; BOSTER), mouse anti-N mAb (1:500), mouse anti-GST mAb (1:1000) (AG768; Beyotime), mouse anti-actin mAb (1:5000) (A5441; Sigma), mouse anti-GFP mAb (1:10,000) (66002–1–1 g; Proteintech), rabbit anti-Flag mAb (1:2000) (20543–1-AP; Proteintech), or mouse anti Myc mAb (1:2000) (66004–1–1 g; Proteintech), rabbit anti-caspase-3 mAb (1:1000) (AC030; Beyotime).

Techniques: Marker, Transfection, Knockdown, Western Blot, Over Expression