Journal: Nature Communications

Article Title: Impaired Plakophilin-2 in obesity breaks cell cycle dynamics to breed adipocyte senescence

doi: 10.1038/s41467-023-40596-0

Figure Lengend Snippet: a A comprehensive pipeline was settled to sequentially validate altered cell cycle and enhanced senescence in MA under either defective PKP2 (si- PKP2 ) or enduring sustained exposure to MCM, also characterized by impaired PKP2 gene expression. In the latter, we used plasmids to restore PKP2 levels (MCM + OE_PKP2). Bar plots show the change for a set of genes previously identified as related to impaired PKP2 , including those (i) directly or (ii) inversely related to senescence, and (iii) biomarkers downstream E2F signaling, also bound to cell cycle, in b si-PKP2 and c MCM-treated adipocytes. SA-β-gal staining in MA under different conditions. Pictures show three representative images sorted out from four biological replicates in which measures of absorbance (546 nm) allowed the quantitative assessment plotted at the bottom right corner. e By using a BD Accuri C6 flow cytometer, the amounts of SA-β-gal positive adipocytes were assessed. Representative flow cytometric curves are provided for each treatment. The gray line shows cell distribution in unmarked control cells. The gating strategy is exemplified in supplementary Fig. , . The bean plot on the right indicates the amounts of SA-β-gal positive adipocytes for each condition ( n = 4 biological replicates/condition). f Western blot analysis for PKP2, Cyclin D1 and PAPPA in non-targeting controls (NTC) and si-PKP2-treated MA. The bar plots below indicate β-actin-normalized relative amounts of each protein. g The box plot (median values plus 25 th and 75 th percentiles, and whiskers bound to maximum and minimum values in each group) shows concentrations of hydrogen peroxide (H 2 O 2 ) measured in conditioned media supernatants collected from cell cultures of either NTC or si-PKP2 adipocytes (left boxes), or MCM-inflamed adipose cells (right boxes) containing a plasmid with an empty cassette (reference) or coding for PKP2 ( n = 8 biological replicates/condition). h Representative 20x immunofluorescent images (the scale bars denote 100 μm length) showing cell cycle driver Cyclin D1 signal in adipocyte cultures. The bean plot at the right-hand shows the Cyclin D1 signal after correcting for blue fluorescent stain DAPI in four biological replicates/conditions. Below, i the percentage of nuclei showing Protein Kinase C alpha (PKCα) immunofluorescence staining in adipocyte cultures following the treatments depicted above. Representative 20x immunofluorescent images are provided in Fig. (scale bars of 100 μm), and an example of image handling and data collection is shown in Fig. . j Gene expression patterns and k Western blot analysis of PKP2, PAPPA, and Cyclin D1 in cultures of adipocytes maintained undisturbed (Ctrl) and inflamed (MCM) adipocytes in which we used plasmids to restore PKP2 levels (MCM + OE_PKP2). Ctrl and MCM groups were treated with the negative empty control vector for pReceiver-M90 and transfection reagent, as per proper control conditions. Bar plots show results assessed in four biological replicates/conditions, and the mean ± SEM Statistical significance was assessed by two-tailed Fisher’s exact t -test. * p < 0.05; ** p < 0.01. Source data are provided as a Source data file.

Article Snippet: Fixed cells were incubated with primary antibodies against Cyclin D1 (Cell Signaling, #55506, clone E3P5S) or PKCα (#2056), followed by incubation with goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor™ 488 (Invitrogen, #A-11034).

Techniques: Expressing, Staining, Flow Cytometry, Western Blot, Plasmid Preparation, Immunofluorescence, Transfection, Two Tailed Test

Journal: OncoTargets and therapy

Article Title: A novel synthetic Asiatic acid derivative induces apoptosis and inhibits proliferation and mobility of gastric cancer cells by suppressing STAT3 signaling pathway

doi: 10.2147/OTT.S121619

Figure Lengend Snippet: Transfection with STAT3 shRNA and STAT3 expression plasmid in SGC7901 modulated the expression of proteins, which were induced by AA-PMe. Notes: SGC7901 cells (2×10 6 /mL) were transfected with either shSTAT3 or STAT3 plasmid. After 24 h, cells were treated with 5, 10, and 50 μM AA-PMe for another 24 h and the whole-cell extracts were subjected to Western blot analysis for STAT3, pSTAT3, Bax, Bcl-2, c-Myc, cyclin D1, and MMP-2, and MMP-9 ( A ). The density of A was analyzed according to GAPDH ( B ). Representative of three independent experiments. Significant differences are denoted by * P <0.05, ** P <0.01, and *** P <0.001. Abbreviations: AA-PMe, asiatic acid- N -(2α,3β,23-acetoxyurs-12-en-28-oyl)-L-proline methyl ester; STAT3, signal transducers and activators of transcription 3; MMP, matrix metalloproteinase.

Article Snippet: Rabbit polyclonal primary antibodies to phospho-STAT3(Tyr 705 ), JAK2, phospho-JAK2, c-Myc, MMP-2, and MMP-9, and mouse monoclonal primary antibody against cyclin D1 were all obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, shRNA, Expressing, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: MicroRNA-34a Mediates the Autocrine Signaling of PAR 2 -Activating Proteinase and Its Role in Colonic Cancer Cell Proliferation

doi: 10.1371/journal.pone.0072383

Figure Lengend Snippet: A549 cells were treated with PAR 2 activating peptide (PAR 2 -AP, 50 µM) or reverse peptide (PAR 2 -RP) for 24 h. A. DNA synthesis was measured by BrdU labeling assay.B. Levels of Cyclin D1 and Cyclin E mRNA were detected with PCR after treatment with or without PAR 2 -AP. C. Protein level of Cyclin D1 was determined by Western blot. D. A549 cells were treated with PAR 2 -AP (50 µM) 24 h after transient transfection with E2F-luciferase (1 µg/well). The data were normalized with tk-RL and shown as the percentage of the control group. E. HT29 cells were treated with PIC (proteinase inhibitor cocktail) or T9128 (trypsin inhibitor). DNA synthesis was measured by BrdU labeling assay. F. Protein (left) and mRNA (right) levels of Cyclin D1 in PAR 2 knockout cells were measured. * p <0.05, *** p <0.001 All data are shown as mean±SEM. N = 3–6. All experiments have been repeated at least 3 times independently.

Article Snippet: The membranes were probed overnight at 4°C with primary antibodies against human Cyclin D1 (1∶1,000; Cell Signaling Technology), PAR 2 (N-19, 1∶1000; Santa Cruz Biotechnology) or β-catenin (Cell Signaling Technology), followed by incubation with peroxidase-conjugated secondary antibodies (Cell Signaling Technology) at 1∶10,000 dilution for 1 h. The signal was visualized with ECL (Millipore).

Techniques: DNA Synthesis, Labeling, Western Blot, Transfection, Luciferase, Knock-Out

Journal: PLoS ONE

Article Title: MicroRNA-34a Mediates the Autocrine Signaling of PAR 2 -Activating Proteinase and Its Role in Colonic Cancer Cell Proliferation

doi: 10.1371/journal.pone.0072383

Figure Lengend Snippet: A549 cells were pretreated with actinomycin D (2 µg/ml) 30 min before challenge with PAR 2 -AP (50 µM) for 24 h. (A) mRNA and (B) protein levels of Cyclin D1 were detected by real time PCR and Western Blot. siRNA targeting beta-catenin was transfected into A549 cells 24 h before treatment with PAR 2 -AP. (C) mRNA and (D) protein levels of Cyclin D1 were detected by real time PCR and Western Blot, respectively.

Article Snippet: The membranes were probed overnight at 4°C with primary antibodies against human Cyclin D1 (1∶1,000; Cell Signaling Technology), PAR 2 (N-19, 1∶1000; Santa Cruz Biotechnology) or β-catenin (Cell Signaling Technology), followed by incubation with peroxidase-conjugated secondary antibodies (Cell Signaling Technology) at 1∶10,000 dilution for 1 h. The signal was visualized with ECL (Millipore).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Transfection

Journal: PLoS ONE

Article Title: MicroRNA-34a Mediates the Autocrine Signaling of PAR 2 -Activating Proteinase and Its Role in Colonic Cancer Cell Proliferation

doi: 10.1371/journal.pone.0072383

Figure Lengend Snippet: (A) The expression of trypsinogen and KLK14 in human CRC tumor samples (T) and paired normal tissue (N) were measured with PCR. Actin was used as an internal control. The mRNA expression of PAR 2 , CCND1 and the level of miR-34a in human CRC samples were tested with real time PCR. They were compared according to (B) the grade or (C) the status of lymph node metastasis. The data are shown as the fold change of tumor sample (T) to paired normal tissue (N).

Article Snippet: The membranes were probed overnight at 4°C with primary antibodies against human Cyclin D1 (1∶1,000; Cell Signaling Technology), PAR 2 (N-19, 1∶1000; Santa Cruz Biotechnology) or β-catenin (Cell Signaling Technology), followed by incubation with peroxidase-conjugated secondary antibodies (Cell Signaling Technology) at 1∶10,000 dilution for 1 h. The signal was visualized with ECL (Millipore).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: DCB-3503, a Tylophorine Analog, Inhibits Protein Synthesis through a Novel Mechanism

doi: 10.1371/journal.pone.0011607

Figure Lengend Snippet: ( A ) Structure of DCB-3503. ( B ) and ( C ) HepG2 cells and HeLa cells were treated with DCB-3503 for the indicated time and dose. Whole cell lysates were probed with antibodies as indicated. β-Actin blot was used as internal loading control. D. Down-regulation of cyclin D1 by DCB-3503 in Huh7 and MCF-7 cells. B , C , and D are representatives from at least three separate experiments. (* Indicating non-specific band).

Article Snippet: Western blot analysis was done using primary antibodies against cyclin D1 (Cell Signaling Technology), cyclin E (Santa Cruz Biotechnology, Santa Cruz, CA), β-catenin (Cell Signaling Technology), survivin (Cell Signaling Technology), p53 (Santa Cruz Biotechnology), p21 (Santa Cruz Biotechnology), phospho-p70 S6 kinase (Thr 389 ) (Cell Signaling Technology), phospho-S6 ribosomal protein (Ser 235/236 ) (Cell Signaling Technology), phospho-4E-BP1 (Ser 65 ) (Cell Signaling Technology), phospho-4E-BP1 (Thr 37/46 ) (Cell Signaling Technology), 4E-BP1 (Cell Signaling Technology), and β-actin (Sigma-Aldrich) at optimal dilution.

Techniques:

Journal: PLoS ONE

Article Title: DCB-3503, a Tylophorine Analog, Inhibits Protein Synthesis through a Novel Mechanism

doi: 10.1371/journal.pone.0011607

Figure Lengend Snippet: A. HepG2 cells were treated with either DCB-3503 or CHX for the indicated time. mRNA levels of cyclin D1, survivin, β-catenin, p53, and p21 were analyzed by real-time RT-PCR using specific primers and probes and were normalized to that of β-actin. Values are depicted as mean ± S.D. from at least three separate experiments and are expressed relative to the basal mRNA level in the corresponding untreated control cells. B. mRNA level of cyclin D1 was analyzed by Northern blot under DCB-3503 treatment. Ribosomal RNA gel image on the bottom panel shows equal loading of total RNA. C. Expressions of cyclin D1, survivin, β-catenin, p53, and p21 under treatment of CHX, DCB-3503, and CHX and DCB-3503 in combination were examined by Western blot analysis in HepG2 cells. β-Actin blot was used as internal loading control. D. ( a ) 2 µM MG-132 reverses suppression of cyclin D1, survivin, β-catenin, p53, and p21 by DCB-3503 within 4-hour culture, ( b ) 2 µM MG-132 does not block cytotoxicity of DCB-3503 after 24-hour treatment. B , C , and D are representatives from at least three separate experiments. Numbers marked in C are normalized band intensity. (* Indicating non-specific band).

Article Snippet: Western blot analysis was done using primary antibodies against cyclin D1 (Cell Signaling Technology), cyclin E (Santa Cruz Biotechnology, Santa Cruz, CA), β-catenin (Cell Signaling Technology), survivin (Cell Signaling Technology), p53 (Santa Cruz Biotechnology), p21 (Santa Cruz Biotechnology), phospho-p70 S6 kinase (Thr 389 ) (Cell Signaling Technology), phospho-S6 ribosomal protein (Ser 235/236 ) (Cell Signaling Technology), phospho-4E-BP1 (Ser 65 ) (Cell Signaling Technology), phospho-4E-BP1 (Thr 37/46 ) (Cell Signaling Technology), 4E-BP1 (Cell Signaling Technology), and β-actin (Sigma-Aldrich) at optimal dilution.

Techniques: Quantitative RT-PCR, Northern Blot, Western Blot, Blocking Assay

Journal: PLoS ONE

Article Title: DCB-3503, a Tylophorine Analog, Inhibits Protein Synthesis through a Novel Mechanism

doi: 10.1371/journal.pone.0011607

Figure Lengend Snippet: A. DCB-3503 inhibited [ 3 H]-amino acid incorporation in both time- ( a ) and dose-dependent ( b ) manner in HepG2 cells. B. DCB-3503 inhibited [ 3 H]-amino acid incorporation in HeLa cells ( a ) and PANC-1 cells ( b ) in a dose-dependent fashion. A and B results from three separate experiments are presented as mean ± S.D. C. Inhibitory effect of DCB-3503 on global protein synthesis and cyclin D1 protein synthesis assessed by [ 35 S]-methionine/cysteine incorporation. This is a representative from three separate experiments.

Article Snippet: Western blot analysis was done using primary antibodies against cyclin D1 (Cell Signaling Technology), cyclin E (Santa Cruz Biotechnology, Santa Cruz, CA), β-catenin (Cell Signaling Technology), survivin (Cell Signaling Technology), p53 (Santa Cruz Biotechnology), p21 (Santa Cruz Biotechnology), phospho-p70 S6 kinase (Thr 389 ) (Cell Signaling Technology), phospho-S6 ribosomal protein (Ser 235/236 ) (Cell Signaling Technology), phospho-4E-BP1 (Ser 65 ) (Cell Signaling Technology), phospho-4E-BP1 (Thr 37/46 ) (Cell Signaling Technology), 4E-BP1 (Cell Signaling Technology), and β-actin (Sigma-Aldrich) at optimal dilution.

Techniques:

Journal: PLoS ONE

Article Title: DCB-3503, a Tylophorine Analog, Inhibits Protein Synthesis through a Novel Mechanism

doi: 10.1371/journal.pone.0011607

Figure Lengend Snippet: A. Effects of DCB-3503 and CHX on translation of uncapped and capped poly(A) tail cyclin D1 mRNAs in HeLa cell free lysate system. B. Effects of DCB-3503, CHX, and rapamycin on translation of capped luciferase mRNAs with or without 3'poly(A) tail in HeLa cell free lysate system. Values are depicted as mean ± S.D. from at least three separate experiments. C. Western blot analysis of phosphorylated p70 S6Kinase T 389 , S6 S 235/236 , phosphorylated 4EBP1 S 65 and T 37/46 under DCB-3503 and rapamycin treatment for 2 hours in HepG2 cells. 4EBP1 and β-Actin blots were used as internal loading controls. A and C are representatives from three separate experiments.

Article Snippet: Western blot analysis was done using primary antibodies against cyclin D1 (Cell Signaling Technology), cyclin E (Santa Cruz Biotechnology, Santa Cruz, CA), β-catenin (Cell Signaling Technology), survivin (Cell Signaling Technology), p53 (Santa Cruz Biotechnology), p21 (Santa Cruz Biotechnology), phospho-p70 S6 kinase (Thr 389 ) (Cell Signaling Technology), phospho-S6 ribosomal protein (Ser 235/236 ) (Cell Signaling Technology), phospho-4E-BP1 (Ser 65 ) (Cell Signaling Technology), phospho-4E-BP1 (Thr 37/46 ) (Cell Signaling Technology), 4E-BP1 (Cell Signaling Technology), and β-actin (Sigma-Aldrich) at optimal dilution.

Techniques: Luciferase, Western Blot

Journal: PLoS ONE

Article Title: DCB-3503, a Tylophorine Analog, Inhibits Protein Synthesis through a Novel Mechanism

doi: 10.1371/journal.pone.0011607

Figure Lengend Snippet: A. DCB-3503 treatment induced accumulation of polysomes in HepG2 and HeLa cells after 1-hour treatment. B. DCB-3503 induced accumulation of mRNAs of cyclin D1 and β-Actin in polysomal fractions in the sucrose gradient in HepG2 cells. A and B were representatives from three separate experiments with similar results.

Article Snippet: Western blot analysis was done using primary antibodies against cyclin D1 (Cell Signaling Technology), cyclin E (Santa Cruz Biotechnology, Santa Cruz, CA), β-catenin (Cell Signaling Technology), survivin (Cell Signaling Technology), p53 (Santa Cruz Biotechnology), p21 (Santa Cruz Biotechnology), phospho-p70 S6 kinase (Thr 389 ) (Cell Signaling Technology), phospho-S6 ribosomal protein (Ser 235/236 ) (Cell Signaling Technology), phospho-4E-BP1 (Ser 65 ) (Cell Signaling Technology), phospho-4E-BP1 (Thr 37/46 ) (Cell Signaling Technology), 4E-BP1 (Cell Signaling Technology), and β-actin (Sigma-Aldrich) at optimal dilution.

Techniques: