Journal: Cancers

Article Title: Short-Term TERT Inhibition Impairs Cellular Proliferation via a Telomere Length-Independent Mechanism and Can Be Exploited as a Potential Anticancer Approach

doi: 10.3390/cancers15102673

Figure Lengend Snippet: Telomerase reverse transcriptase (TERT) inhibition downregulated MYC proto-oncogene bHLH transcription factor (MYC) levels. ( A , B ) Cells were treated with BIBR at indicated concentrations or with DMSO as a control for 24 h. Levels of relative mRNA expression for MYC and TERT in 4134/Late ( A ) and BL41 ( B ) cells are shown. Data represent the mean and SD (bar) from three separate experiments. ( C , D ) Cells treated with BIBR at indicated concentrations or DMSO as a control for 24 h were processed to obtain cytoplasmic and nuclear extracts. Representative Western blots showing cytoplasmic and nuclear protein levels of TERT, MYC, TRF2, and α-tubulin in 4134/Late ( C ) and BL41 ( D ) cells are shown. α-tubulin and TRF2 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. The original Western blots are shown in . Graphs next to the blots show the values in arbitrary units of densitometric analysis performed with ImageJ software. Data represent the mean and SD (bar) from three separate experiments. A significant difference between values in BIBR-treated vs. DMSO-treated cells is shown: * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Article Snippet: The expression of TERT, MYC, p65, phosphorylated p65 (p-p65), cyclin-dependent kinase inhibitor 1A (CDKN1A, P21), telomeric repeat binding factor 2 (TRF2), and α-tubulin were evaluated by the following antibodies: TERT (ab32020, Abcam, Cambridge, UK; abx120550, Abexxa, Arlington, TX, USA), NF-κB p65 (ab32536, Abcam), phospho-NF-κB p65 (Ser536) (93H1, Cell Signaling), MYC (ab32072, Abcam), P21 (ab109520, Abcam), TRF2 (NB110-57130, Novus Biological, Littleton, CO, USA), GAPDH (GTX100118, GeneTex, Alton Pkwy, Irvine, CA, USA), and α-tubulin (Sigma-Aldrich).

Techniques: Reverse Transcription, Inhibition, Control, Expressing, Western Blot, Software

Journal: Cancers

Article Title: Short-Term TERT Inhibition Impairs Cellular Proliferation via a Telomere Length-Independent Mechanism and Can Be Exploited as a Potential Anticancer Approach

doi: 10.3390/cancers15102673

Figure Lengend Snippet: p65 inhibition recapitulated the effects of TERT inhibition on MYC and the cell cycle. ( A , B ) Representative Western blots showing total protein levels of p-p65 and MYC in 4134/Late ( A ) and BL41 ( B ) cells upon treatment with Ammonium pyrrolidine dithiocarbamate (PDTC) at the indicated concentrations for 24 h. α-tubulin was used as a loading control. The original Western blots are shown in . Graphs on the right show the values in arbitrary units of densitometric analysis performed with ImageJ software. Data represent the mean and SD (bar) from two separate experiments. ( C , D ) 4134L/Late ( C ) and BL41 ( D ) cells were treated with 30 μM BIBR, PDTC at indicated concentrations, or DMSO as a control for 24 h, labelled with propidium iodide (PI) and analysed by flow cytometry for cell cycle profiles. Panels from a representative experiment are shown. Graphs on the right show percentages of cells in G1, S, and G2/M-phases, respectively. Values show the means and SD (bar) of three separate experiments. A significant difference between values in BIBR-treated or PDTC-treated cells vs. DMSO-treated cells is shown: * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The expression of TERT, MYC, p65, phosphorylated p65 (p-p65), cyclin-dependent kinase inhibitor 1A (CDKN1A, P21), telomeric repeat binding factor 2 (TRF2), and α-tubulin were evaluated by the following antibodies: TERT (ab32020, Abcam, Cambridge, UK; abx120550, Abexxa, Arlington, TX, USA), NF-κB p65 (ab32536, Abcam), phospho-NF-κB p65 (Ser536) (93H1, Cell Signaling), MYC (ab32072, Abcam), P21 (ab109520, Abcam), TRF2 (NB110-57130, Novus Biological, Littleton, CO, USA), GAPDH (GTX100118, GeneTex, Alton Pkwy, Irvine, CA, USA), and α-tubulin (Sigma-Aldrich).

Techniques: Inhibition, Western Blot, Control, Software, Flow Cytometry

Journal: Cancers

Article Title: Short-Term TERT Inhibition Impairs Cellular Proliferation via a Telomere Length-Independent Mechanism and Can Be Exploited as a Potential Anticancer Approach

doi: 10.3390/cancers15102673

Figure Lengend Snippet: BIBR treatment downregulated the expression of zebrafish myca and mycb and upregulated p21 . Twelve hours post fertilization (hpf), zebrafish wild-type (WT) and tert mutant ( tert hu3430/hu3430 ; tert −/−) embryos were treated with 2 μM BIBR or DMSO as a control for 12 h, from 12 to 24 hpf. Levels of relative mRNA expression for the indicated genes in WT ( A – C ) and tert mutant ( D – F ) embryos are shown. Data represent the mean and SD (bar) from three separate experiments. ( G ) Representative Western blots showing total protein levels of Myc in WT and tert mutant zebrafish embryos upon treatment with 2 μM BIBR for 12 h. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as a loading control. The original Western blots are shown in . The graph shows the values in arbitrary units of densitometric analysis performed with ImageJ software. Data represent the mean and SD (bar) from two separate experiments. A significant difference between values in BIBR-treated embryos vs. DMSO-treated embryos is shown: * p < 0.05; *** p < 0.001; ns: not significant.

Article Snippet: The expression of TERT, MYC, p65, phosphorylated p65 (p-p65), cyclin-dependent kinase inhibitor 1A (CDKN1A, P21), telomeric repeat binding factor 2 (TRF2), and α-tubulin were evaluated by the following antibodies: TERT (ab32020, Abcam, Cambridge, UK; abx120550, Abexxa, Arlington, TX, USA), NF-κB p65 (ab32536, Abcam), phospho-NF-κB p65 (Ser536) (93H1, Cell Signaling), MYC (ab32072, Abcam), P21 (ab109520, Abcam), TRF2 (NB110-57130, Novus Biological, Littleton, CO, USA), GAPDH (GTX100118, GeneTex, Alton Pkwy, Irvine, CA, USA), and α-tubulin (Sigma-Aldrich).

Techniques: Expressing, Mutagenesis, Control, Western Blot, Software

Journal: International Journal of Molecular Sciences

Article Title: In Vitro and In Vivo Validation of Endothelium-Derived Potential Therapeutics for Myocardial Ischemia/Reperfusion Injury Identified by an AI-Enhanced Single-Cell and Virtual-Cell Paradigm

doi: 10.3390/ijms27062743

Figure Lengend Snippet: Andrographolide inhibits the S100A8 downstream IL-17 signaling pathway and reduces the inflammatory response of H/R-induced CMECs. ( A , B ) The expressions of IL-17A, IL-17RA, Act1, and NF-κB proteins were determined by Western blot ( n = 3). ( C – E ) The levels of IL-1β, IL-6, and TNF-α were determined through ELISA. ( F – H ) The expressions of IL-17A, IL-17RA and Act1 proteins were determined by Western blot in co-transfection with the overexpression S100A8 plasmid ( n = 3). Data are presented as mean ± SD ( n = 6). “Con or C” stands for the control group, “H/R” or “H” indicates the H/R group, and “H + AG or H + A” means the H/R group treated with AG. “H + A + S” means the H/R group treated with AG and overexpression of S100A8. ** p < 0.01 compared to the Con group; # p < 0.05, ## p < 0.01 compared to the H/R or H group, @ p < 0.05, @@ p < 0.01 compared to the H + A + S group.

Article Snippet: The membranes were blocked for 1 h at room temperature with 5% skimmed milk before being incubated overnight at 4 °C with the following primary antibodies: S100A8 (A15315, Abclonal, Wuhan, China), IL-17A (A12454, Abclonal, Wuhan, China), IL-17RA (A10052, Abclonal, Wuhan, China), Act1 (A6776, Abclonal, Wuhan, China), p-NF-κB p65 (1:500, AP0124, Abclonal, Wuhan, China), NF-κB p65 (1:500, A00284-1, Boster, Wuhan, China), β-actin (1:2000, TA-09, Zhongshan Golden Bridge, Beijing, China), and GAPDH (1:2000, TA-08, Zhongshan Golden Bridge, Beijing, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Cotransfection, Over Expression, Plasmid Preparation, Control

Journal: Cell reports

Article Title: Modeling Progressive Fibrosis with Pluripotent Stem Cells Identifies an Anti-fibrotic Small Molecule

doi: 10.1016/j.celrep.2019.11.019

Figure Lengend Snippet: (A) Active TGF-β secreted during the progression of the iFA phenotype (days 2–14) in a TGF-β bioassay (n = 3). (B) Representative immunofluorescent image of iFA model stained for total SMAD3 and p-SMAD2/3 (S465/S467). (C) Quantification of cell stiffness of the cells in the iFA model at different time points (days 1–13) (n = 22). (D) Representative image of the iFA model at day 13 of culture stained with p16 Ink4 staining and counterstained with VIM. (E and F) Cytokine profiles (E) and HMGB1 (F) levels determined from conditioned media of the iFA model at day 13 relative to day 4 (n = 5 in duplicate). (G) Representative image of the iFA model at day 13 stained for NF-κB p65. HMGB1 was translocated from the nucleus to the cytoplasm. (H) Enrichment analysis of canonical pathways (1,006 MSigDB canonical pathways) in transcriptome data of RNA sequencing in day 13 versus day 4 cultures of the iFA model compared to the fibrotic organ versus healthy tissue data. Fibrotic organs include IPF versus healthy lung (n = 8), advanced (n = 6) versus early (n = 6) liver steatosis, and kidneys from chronic kidney disease (n = 46) versus healthy controls (n = 8). Related to and . Scale bars, 50 mm. Data in figures represent the mean ± SEM; ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 using two-way ANOVA and Sidak’s multiple comparisons test.

Article Snippet: Rabbit NF-kB p65 , Bioss , Cat# bs-0465R; RRID:AB_10855447.

Techniques: Staining, RNA Sequencing Assay

Journal: Cell reports

Article Title: Modeling Progressive Fibrosis with Pluripotent Stem Cells Identifies an Anti-fibrotic Small Molecule

doi: 10.1016/j.celrep.2019.11.019

Figure Lengend Snippet:

Article Snippet: Rabbit NF-kB p65 , Bioss , Cat# bs-0465R; RRID:AB_10855447.

Techniques: Recombinant, Luciferase, Imaging, Enzyme-linked Immunosorbent Assay, Hydroxyproline Assay, Software