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: Nature Communications

Article Title: Neuropeptide SP protects against colitis and linked anxiety-like behavior through the putative roles of gut microbiota and metabolite inositol

doi: 10.1038/s41467-025-67904-0

Figure Lengend Snippet: a , b Heat maps revealing differential gene expression in FMT-DSS vs FMT-CON, FMT-DSS + SP vs FMT-DSS groups. n = 4 mice/group. The pathway-related genes were selected (log2 fold change at least > 1, p < 0.05). c , d KEGG analysis of total-, up- and down-regulated genes in the FMT-DSS group compared with the FMT-CON group (Top 20). n = 4 mice/group. e , f KEGG analysis of total-, up- and down-regulated genes in the FMT-DSS + SP group vs the FMT-DSS group (Top 20). n = 4 mice/group. g Quantitative real-time PCR analysis of mRNA expressions of NF-κB signaling pathway genes ( p65 and IKBα ) in the hippocampus. n = 4 mice/group. The whiskers indicate the minimum and maximum values observed within the range of Q1 − 1.5 × IQR to Q3 + 1.5 × IQR. The box reveals the interquartile range (IQR) between the 25th (Q1) and 75th (Q3) percentiles, and the line inside the box represents the median (50th percentile). h The protein expression of p-p65 and p-IKBα in hippocampus tissue, as determined by western blotting. n = 3 independent experiments. i Quantitative real-t i me PCR analysis of mRNA expressions of GABA receptor and Ca 2+ signaling genes ( Gabra1 , Gabra3 , Gabrg2 , Gabrb2 and Camk2d ). n = 4 mice/group. The whiskers indicate the minimum and maximum values observed within the range of Q1 − 1.5 × IQR to Q3 + 1.5 × IQR. The box reveals the interquartile range (IQR) between the 25th (Q1) and 75th (Q3) percentiles, and the line inside the box represents the median (50th percentile). j Protein expressions of GABA A R, GABA B R, GAD65 and CaMKII were measured in hippocampus tissue by western blot. n = 3 independent experiments. k Representative immunofluorescence images of double-labeling for p-IKBα (red)/Iba1 (green) in hippocampus tissues. Scale bar = 50 μm. n = 3 independent experiments. I Representative immunofluorescence images of double-labeling for GABA A R (green)/GFAP (red) in hippocampus tissues. Scale bar = 50 μm. n = 3 independent experiments. m Double immunofluorescence staining for CaMKII (green)/GFAP (red) in hippocampus tissues and statistical analysis. Scale bar = 50 μm. n = 3 independent experiments. n Differential gene expressions were presented by the heatmap in microglia of three groups, and GSEA analysis of microglia from FMT-DSS + SP group vs the FMT-DSS group. n = 4 mice/group. o Heat maps of differential gene expression in astrocytes from three groups and GSEA analysis of astrocytes from FMT-DSS + SP group vs the FMT-DSS group. n = 4 mice/group. Data were presented as means ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a file.

Article Snippet: Next, primary antibody against GFAP (1:1000, A8335, 1:200, NB100-53809, USA), GABA A Rα1 (1:1000, 12708-1-AP, Proteintech, China), GABA B R2 (1:1000, 27567-1-AP, Proteintech, China), CaMKII (1:1000, 12716, Cell Signaling Technology, USA), GAD65 (1:1000, ab239372, Abcam, USA), CD206 (1:1000, ab64693, Abcam, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), CD80 (1:1000, 66406-1, Proteintech, China), p-p65 (1:1000, A00284-1, Boster, China), p-IKBα (1:1000, WLH3930, Wanleibio, China), and β-actin (50201, 1:1000, Kemei Borui Science and Technology, China) were incubated with the membranes overnight at 4 °C.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Labeling, Double Immunofluorescence Staining

Journal: Journal of neurochemistry

Article Title: A novel function of TBK1 as a target of Cdon in oligodendrocyte differentiation and myelination.

doi: 10.1111/jnc.13882

Figure Lengend Snippet: Fig. 2 Identification of TBK1 as a protein interacting with cell adhesion molecule- related, down-regulated by oncogenes (Cdon) in oligodendrocytes (OLGs) during early differentiation (a) Cdon or Goat IgG antibodies were used for immunoprecipitation (IP) in day 1 differentiated primary OLG cultures. A silver-stained sodium dodecyl sulfate– polyacrylamide gel with protein markers (M), IP input, Cdon antibody IP, and Goat IgG control IP is shown. The indicated band TBK1 was excised and analyzed by mass spectrometry. Molecular weights in kDa’s are indicated on the left. (b) Protein sequences of TBK1. The peptides identified by mass spectrometry are shown highlighted in yellow, cover ~ 14% of the protein sequence. (c) Cdon was immunoprecipitated from primary OLG cell lysates (day 1 differentiated) and the precipitated complex was analyzed by western blotting using antibodies specific for Cdon and TBK1. The experiment was reproduced with three different batches of primary OLG cultures. JLP and cAbl were shown as positive and negative control for IP.

Article Snippet: Primary antibodies were from the following suppliers: mouse monoclonal antibody (mmAb) anti-MBP (SMI-99) from Chemicon (Temecula, CA, USA); Goat IgG, Alexa Fluor 488-, 594-, Cy5-, or horseradish peroxidase-conjugated secondary antibodies from Southern Biotechnology (Birmingham, AL, USA), Jackson Immunoresearch Laboratories (Cedarlane, Hornby, ON, Canada), Bio-Rad Canada, or Invitrogen (Burlington, ON, Canada); Akt, phospho-Akt-T308, phospho-Akt-S473, phospho-ERK, ERK, JLP, GAPDH, phospho-TBK1, and TBK1 rabbit polyclonal antibodies for western blot from Cell Signaling Technology (Whitby, ON, Canada); TBK1 antibody for immunostaining from Abcam (Toronto, ON, Canada); TrueBlot conformation specific secondary antibodies from Rockland Inc. (Limerick, PA, USA); Cdon goat polyclonal antibody from R&D (Minneapolis, MN, USA); Olig2 rabbit polyclonal antibody from EMD Millipore (Darmstadt, Germany); cAbl and a-tubulin mmAb from Sigma-Aldrich; actin antibody from Santacruz (Dallas, TX, USA); Invitrogen green fluorescent protein (GFP) antibody from Thermo Fisher Scientific Inc. MAG antibody was a gift from Dr. Peter E. Braun (Emeritus Professor in Department of Biochemistry of McGill University).

Techniques: Immunoprecipitation, Staining, Control, Mass Spectrometry, Sequencing, Western Blot, Negative Control

Journal: Journal of neurochemistry

Article Title: A novel function of TBK1 as a target of Cdon in oligodendrocyte differentiation and myelination.

doi: 10.1111/jnc.13882

Figure Lengend Snippet: Fig. 4 TBK1 siRNA decreases expression of myelin-specific markers during oligodendrocyte (OLG) differentiation (a) siRNA knockdown efficiency. Forty-eight hours after siRNA treatment cells were changed into differentiation medium for 24 h followed by protein extraction. Targeted siRNA reduced levels of TBK1 protein. Densitometric quantification of the signals is shown below. (b) Two myelin- specific proteins, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG), were reduced after nucleofection with TBK1 siRNA treatment during OLG differentiation. Densitometric quantification of western blot bands is shown in (d) as a relative ratio to GAPDH expression, which was used as a loading control in both (a) and (b). (c) Representative images of non- targeted Control and TBK1 siRNA- transfected cells. GalC is shown in red. MAG in green, and Olig2 in purple. (e) Quantification of the numbers of Olig2- positive cells expressing GalC or MAG. The values represent mean SEM of triplicate samples. Statistical differences were computed using independent t-tests (*p < 0.05, **p < 0.01, ***p < 0.005).

Article Snippet: Primary antibodies were from the following suppliers: mouse monoclonal antibody (mmAb) anti-MBP (SMI-99) from Chemicon (Temecula, CA, USA); Goat IgG, Alexa Fluor 488-, 594-, Cy5-, or horseradish peroxidase-conjugated secondary antibodies from Southern Biotechnology (Birmingham, AL, USA), Jackson Immunoresearch Laboratories (Cedarlane, Hornby, ON, Canada), Bio-Rad Canada, or Invitrogen (Burlington, ON, Canada); Akt, phospho-Akt-T308, phospho-Akt-S473, phospho-ERK, ERK, JLP, GAPDH, phospho-TBK1, and TBK1 rabbit polyclonal antibodies for western blot from Cell Signaling Technology (Whitby, ON, Canada); TBK1 antibody for immunostaining from Abcam (Toronto, ON, Canada); TrueBlot conformation specific secondary antibodies from Rockland Inc. (Limerick, PA, USA); Cdon goat polyclonal antibody from R&D (Minneapolis, MN, USA); Olig2 rabbit polyclonal antibody from EMD Millipore (Darmstadt, Germany); cAbl and a-tubulin mmAb from Sigma-Aldrich; actin antibody from Santacruz (Dallas, TX, USA); Invitrogen green fluorescent protein (GFP) antibody from Thermo Fisher Scientific Inc. MAG antibody was a gift from Dr. Peter E. Braun (Emeritus Professor in Department of Biochemistry of McGill University).

Techniques: Expressing, Knockdown, Protein Extraction, Western Blot, Control, Transfection