Journal: Frontiers in Microbiology

Article Title: In vitro Type II Restriction of Bacteriophage DNA With Modified Pyrimidines

doi: 10.3389/fmicb.2020.604618

Figure Lengend Snippet: Percentage of modified cytosine, adenine and 5hmdU bases in phage genomes.

Article Snippet: Bacteriophages Xp12 and SP8 were obtained from ATCC (#35934-B1 and #15563-B1, respectively).

Techniques: Modification

Journal: Frontiers in Microbiology

Article Title: In vitro Type II Restriction of Bacteriophage DNA With Modified Pyrimidines

doi: 10.3389/fmicb.2020.604618

Figure Lengend Snippet: Modified bases found in phage T4 gt (5hmC), T4 (5gmC), Xp12 (5mC), and SP8 (5hmdU).

Article Snippet: Bacteriophages Xp12 and SP8 were obtained from ATCC (#35934-B1 and #15563-B1, respectively).

Techniques: Modification

Journal: Frontiers in Microbiology

Article Title: In vitro Type II Restriction of Bacteriophage DNA With Modified Pyrimidines

doi: 10.3389/fmicb.2020.604618

Figure Lengend Snippet: Restriction of phage Xp12 DNA. Example of 10 restriction digestions analyzed in 1% agarose gel (A) . NEBcutter predicted cleavage patterns in the absence of cytosine modification (B) . Enzyme recognition sequences and cleavage result (C) .

Article Snippet: Bacteriophages Xp12 and SP8 were obtained from ATCC (#35934-B1 and #15563-B1, respectively).

Techniques: Agarose Gel Electrophoresis, Modification

Journal: Frontiers in Microbiology

Article Title: In vitro Type II Restriction of Bacteriophage DNA With Modified Pyrimidines

doi: 10.3389/fmicb.2020.604618

Figure Lengend Snippet: Summary of Type II restrictions on phage T4 gt , T4, Xp12, and SP8 gDNA.

Article Snippet: Bacteriophages Xp12 and SP8 were obtained from ATCC (#35934-B1 and #15563-B1, respectively).

Techniques:

Journal: Frontiers in Microbiology

Article Title: In vitro Type II Restriction of Bacteriophage DNA With Modified Pyrimidines

doi: 10.3389/fmicb.2020.604618

Figure Lengend Snippet: Digestion of modified phage gDNA with MDREs.

Article Snippet: Bacteriophages Xp12 and SP8 were obtained from ATCC (#35934-B1 and #15563-B1, respectively).

Techniques: Modification

Journal: Cell death & disease

Article Title: CCN3/NOV promotes metastasis and tumor progression via GPNMB-induced EGFR activation in triple-negative breast cancer.

doi: 10.1038/s41419-023-05608-3

Figure Lengend Snippet: Fig. 5 CCN3 correlates with EGFR signaling in TNBC. A GSEA was performed with mRNA expression data from TNBC and all patients in TCGA (Cell, 2015). Plots indicate EGFR-related gene signatures were enriched in CCN3 altered patients. ES; enrichment score, NES; normalized enrichment score, FDR; false discovery rate. B Western blot analysis of whole cell lysate of CCN3 knockdown cell lines with indicated antibody. GAPDH was used as a loading control. C Western blot analysis of whole cell lysate with indicated antibody. CCN3 knockdown cell line restored their expression by transfection with shRNA-resistant CCN3 construct. β-tubulin was used as a loading control. D–E Transwell migration (D) and invasion (E) assays were assessed with Hs578Ta and MDA-MB-231 CCN3 knockdown cell lines with or without shRNA resistant CCN3. Each value was normalized based on the value of the control cell line. Mean ± SD (n = 3). P-values were calculated with a one-way ANOVA with a post-hoc Tukey multiple comparison test (**p < 0.005, ***p < 0.0005). F 2D colony formation assays were performed with Hs578T and MDA-MB- 231 CCN3 knockdown cell lines with or without shRNA resistant CCN3. Each value was normalized with the value of the control cell line. Mean ± SD (n = 3). P-values were calculated with one-way ANOVA with a post-hoc Tukey multiple comparison test (**p < 0.005, ***p < 0.0005). G Recurrence-free survival (RFS) plot of patients with EGFR high versus low expression in each dataset. Upper panel indicated patients filtered above median expression of CCN3 and bottom panel indicated patients filtered below median expression of CCN3. P-values were calculated with log-rank test.

Article Snippet: Control siRNA (sc-37007) β-catenin siRNA (sc-29209), MITF siRNA (sc-35934), TFEB siRNA (sc-38509), TFE3 siRNA (sc38507) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Knockdown, Control, Transfection, shRNA, Construct, Migration, Comparison

Journal: Cell death & disease

Article Title: CCN3/NOV promotes metastasis and tumor progression via GPNMB-induced EGFR activation in triple-negative breast cancer.

doi: 10.1038/s41419-023-05608-3

Figure Lengend Snippet: Fig. 8 CCN3 regulates GPNMB expression by Wnt signaling induced MITF activation. A Western blot analysis of lysate isolated into cytoplasm and nuclear fraction of CCN3 knockdown cell line using the indicated antibodies. β-actin and histone H1 were used as a loading control for cytosol and nuclear fraction, respectively. B Immunofluorescence assay performed with indicated antibodies (scale bar = 20 μm). C Dual luciferase assay was performed using pSUPER-TOP FLASH construct. pCMV-RL was used as an internal control. Mean ± SD (n = 3). P-values were calculated with one-way ANOVA with a post-hoc Dunnett’s multiple comparison test (*p < 0.05, ***p < 0.0005). D, E Western blot (D) and immunofluorescence (E) analysis with indicated antibody showed decreased Wnt/β-catenin signaling proteins in Hs578T and MDA- MB-231 CCN3 knockdown cell lines (scale bar = 20 μm). F, G Cyclohexamide (CHX) chase assays were performed for measuring β-catenin degradation in Hs578T and MDA-MB-231 CCN3 knockdown cell lines. Each cell line treated with CHX (100 μg/ml) at the indicated time points. Quantification of western blot band was performed using ImageJ software. Mean ± SD (n = 3). P-values were calculated with two-way ANOVA with a Bonferroni posttest (*p < 0.05, **p < 0.005, ***p < 0.0005). H Western blot analysis of whole cell lysate with indicated antibody. Hs578T and MDA-MB-231 were treated with β-catenin siRNA for 48 h. β-actin was used as a loading control. I Dual luciferase assay was performed using a pGL3-GPNMB-luc construct. pCMV-RL was used as an internal control. Mean ± SD (n = 3). P-value was calculated using the two-tailed student t-test (**p < 0.005). J Western blot analysis of lysate isolated into cytoplasm and nuclear of β-catenin siRNA treated Hs578T cell line using the indicated antibodies. β-actin and histone H1 were used as a loading control for cytosol and nuclear fraction, respectively. K Immunofluorescence assay performed with indicated antibodies (scale bar = 20 μm). L Schematic description of CCN3 induced signaling cascade in TNBC.

Article Snippet: Control siRNA (sc-37007) β-catenin siRNA (sc-29209), MITF siRNA (sc-35934), TFEB siRNA (sc-38509), TFE3 siRNA (sc38507) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Activation Assay, Western Blot, Isolation, Knockdown, Control, Luciferase, Construct, Comparison, Software, Two Tailed Test

Journal: Cell Death & Disease

Article Title: CCN3/NOV promotes metastasis and tumor progression via GPNMB-induced EGFR activation in triple-negative breast cancer

doi: 10.1038/s41419-023-05608-3

Figure Lengend Snippet: A GSEA was performed with mRNA expression data from TNBC and all patients in TCGA (Cell, 2015). Plots indicate EGFR-related gene signatures were enriched in CCN3 altered patients. ES; enrichment score, NES; normalized enrichment score, FDR; false discovery rate. B Western blot analysis of whole cell lysate of CCN3 knockdown cell lines with indicated antibody. GAPDH was used as a loading control. C Western blot analysis of whole cell lysate with indicated antibody. CCN3 knockdown cell line restored their expression by transfection with shRNA-resistant CCN3 construct. β-tubulin was used as a loading control. D – E Transwell migration ( D ) and invasion ( E ) assays were assessed with Hs578Ta and MDA-MB-231 CCN3 knockdown cell lines with or without shRNA resistant CCN3. Each value was normalized based on the value of the control cell line. Mean ± SD ( n = 3). P -values were calculated with a one-way ANOVA with a post-hoc Tukey multiple comparison test (** p < 0.005, *** p < 0.0005). F 2D colony formation assays were performed with Hs578T and MDA-MB-231 CCN3 knockdown cell lines with or without shRNA resistant CCN3. Each value was normalized with the value of the control cell line. Mean ± SD ( n = 3). P -values were calculated with one-way ANOVA with a post-hoc Tukey multiple comparison test (** p < 0.005, *** p < 0.0005). G Recurrence-free survival (RFS) plot of patients with EGFR high versus low expression in each dataset. Upper panel indicated patients filtered above median expression of CCN3 and bottom panel indicated patients filtered below median expression of CCN3. P -values were calculated with log-rank test.

Article Snippet: Control siRNA (sc-37007) β-catenin siRNA (sc-29209), MITF siRNA (sc-35934), TFEB siRNA (sc-38509), TFE3 siRNA (sc-38507) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Knockdown, Control, Transfection, shRNA, Construct, Migration, Comparison

Journal: Cell Death & Disease

Article Title: CCN3/NOV promotes metastasis and tumor progression via GPNMB-induced EGFR activation in triple-negative breast cancer

doi: 10.1038/s41419-023-05608-3

Figure Lengend Snippet: A Western blot analysis of lysate isolated into cytoplasm and nuclear fraction of CCN3 knockdown cell line using the indicated antibodies. β-actin and histone H1 were used as a loading control for cytosol and nuclear fraction, respectively. B Immunofluorescence assay performed with indicated antibodies (scale bar = 20 μm). C Dual luciferase assay was performed using pSUPER-TOP FLASH construct. pCMV-RL was used as an internal control. Mean ± SD ( n = 3). P -values were calculated with one-way ANOVA with a post-hoc Dunnett’s multiple comparison test (* p < 0.05, *** p < 0.0005). D , E Western blot ( D ) and immunofluorescence ( E ) analysis with indicated antibody showed decreased Wnt/β-catenin signaling proteins in Hs578T and MDA-MB-231 CCN3 knockdown cell lines (scale bar = 20 μm). F , G Cyclohexamide (CHX) chase assays were performed for measuring β-catenin degradation in Hs578T and MDA-MB-231 CCN3 knockdown cell lines. Each cell line treated with CHX (100 μg/ml) at the indicated time points. Quantification of western blot band was performed using ImageJ software. Mean ± SD ( n = 3). P -values were calculated with two-way ANOVA with a Bonferroni posttest (* p < 0.05, ** p < 0.005, *** p < 0.0005). H Western blot analysis of whole cell lysate with indicated antibody. Hs578T and MDA-MB-231 were treated with β-catenin siRNA for 48 h. β-actin was used as a loading control. I Dual luciferase assay was performed using a pGL3-GPNMB-luc construct. pCMV-RL was used as an internal control. Mean ± SD ( n = 3). P -value was calculated using the two-tailed student t -test (** p < 0.005). J Western blot analysis of lysate isolated into cytoplasm and nuclear of β-catenin siRNA treated Hs578T cell line using the indicated antibodies. β-actin and histone H1 were used as a loading control for cytosol and nuclear fraction, respectively. K Immunofluorescence assay performed with indicated antibodies (scale bar = 20 μm). L Schematic description of CCN3 induced signaling cascade in TNBC.

Article Snippet: Control siRNA (sc-37007) β-catenin siRNA (sc-29209), MITF siRNA (sc-35934), TFEB siRNA (sc-38509), TFE3 siRNA (sc-38507) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Isolation, Knockdown, Control, Immunofluorescence, Luciferase, Construct, Comparison, Software, Two Tailed Test

Journal: Neoplasia (New York, N.Y.)

Article Title: S-adenosylmethionine blocks tumorigenesis and with immune checkpoint inhibitor enhances anti-cancer efficacy against BRAF mutant and wildtype melanomas

doi: 10.1016/j.neo.2022.100874

Figure Lengend Snippet: Mitf is responsible for phenotype switching and controls the expression of MDAs and stemness genes in melanoma cells. (A-B) Expression (±SEM) of (A) Mitf and MDAs; and (B) master CSC marker genes ( Wnt, Sox2 and Nanog ); in B16 and YUMMER1.7 cells upon siMitf (35nM) treatment analysed using RT-qPCR. The fold change is relative to scramble control (siScr) where value of control is 1. Statistical significance was calculated using (A-B) two-way ANOVA test and are **** unless indicated. (C) Model figure summarizing the effect of SAM on phenotype switching of melanoma cells from invasive and proliferative to differentiated state which could be due to elevation in Mitf levels.

Article Snippet: siRNAs for Mitf (cat# sc-35935, Santa Cruz Biotechnology, US) containing 3 different siMitf targeting different exons of Mitf were utilized to KD the Mitf mRNA expression.

Techniques: Expressing, Marker, Quantitative RT-PCR, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: S-adenosylmethionine blocks tumorigenesis and with immune checkpoint inhibitor enhances anti-cancer efficacy against BRAF mutant and wildtype melanomas

doi: 10.1016/j.neo.2022.100874

Figure Lengend Snippet: SAM reduces tumor growth and progression, and metastasis of melanoma mouse models. (A) Tumor volume (mm 3 ) of control (PBS) and SAM treated YUMMER1.7 (n≥7/group) and B16 (n≥10/group) tumor bearing mice plotted against days post tumor injection. Data pooled from two independent repeats. Essentially, YUMMER1.7 (5 × 10 5 ) and B16 (5 × 10 5 ) cells were subcutaneously injected in C57BL/6 mice, and once the tumors were palpable treatment was initiated with either control (PBS) or SAM via oral gavage. Tumor volumes were measured at timed intervals. (B, left) Immunohistochemical images (top; lens, 1x, magnification, 10x; and bottom; lens, 40x, magnification, 400x) of the primary YUMMER1.7 (n=4 samples/group) and B16 (n≥3 samples/group) tumors stained with murine antibody against Ki67 proliferation marker (brown) from control and SAM treated group showing proliferative ability of tumor cells. The nucleus is stained blue. (B, right) Ki67 positive staining area percentage (n= 5 images/sample ±SEM). (C) Expression (±SEM) of Mitf and MDAs in YUMMER1.7 and B16 tumors from control and SAM treated group (n=4 samples/group) determined with RT-qPCR. (D) Percentage proportion of metastatic nodules (relative to control, ±SEM) and number of metastatic nodules (±SEM) on lungs of control and SAM treated mice. Representative lung images showing front and back. Essentially, B16 (2.5 × 10 5 ) cells were intravenously injected in C57BL/6 mice (n≥ 7/group) and treated with PBS (control) or SAM. Statistical significance was calculated using (A) two-way ANOVA test; (B-D) two-tailed t-test.

Article Snippet: siRNAs for Mitf (cat# sc-35935, Santa Cruz Biotechnology, US) containing 3 different siMitf targeting different exons of Mitf were utilized to KD the Mitf mRNA expression.

Techniques: Control, Injection, Immunohistochemical staining, Staining, Marker, Expressing, Quantitative RT-PCR, Two Tailed Test