Journal: Frontiers in Pharmacology

Article Title: Chick Embryo: A Preclinical Model for Understanding Ischemia-Reperfusion Mechanism

doi: 10.3389/fphar.2018.01034

Figure Lengend Snippet: Representative pictures of effect of I/R on DNA damage, cell survival, and inflammation. (A) DNA gel electrophoresis diagram. (B) The phosphorylation of H2AX in response to I/R-induced DNA damage ( ∗∗∗ P < 0.001 vs. control). Western blot analysis expression level of LC3 I/II ( ∗ P < 0.05) (C) , Beclin1 ( ∗∗∗ P < 0.001), SQSTM1 ( ∗ P < 0.05), and ATG7 ( ∗∗ P < 0.01) (D) and lysosomal associated proteins Lamp1 ( ∗∗ P < 0.01), Lamp2 ( ∗ P < 0.05), and Cathepsin B ( ∗ P < 0.05) (E) in total protein extract from RVA of I/R treated vs. control group. (F) The expression of NLRP3 ( ∗∗∗ P < 0.001), Caspase-1 ( ∗∗ P < 0.01), ASC ( ∗∗ P < 0.01), and IL-1β ( ∗∗ P < 0.01). Results represent mean ± SE ( n = 3). The graph shows the densitometry quantification of western blot bands. Here control group represented Sham group. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The primary antibodies used were rabbit polyclonal anti-HIF1α (NB100-449, Novus Biologicals), mouse polyclonal anti-LC3 (SC16756, Santa Cruz), rabbit polyclonal anti-Beclin1 (24352, SAB), rabbit polyclonal anti-SOD 1 (3458-100, Biovision), rabbit polyclonal anti-SOD 2 (NB100-1992SS, Novus Biologicals), rabbit monoclonal Caspase-1 (ab179515, Abcam), mouse monoclonal Caspae-3 (NB100-56708SS, Novus Biologicals), mouse monoclonal Cathepsin B (ab58802, Abcam), rat monoclonal LAMP1 (ab25245, Abcam), mouse monoclonal LAMP-2 (NBP2-22217SS, Novus Biologicals), rabbit monoclonal IFNγ (ab133566), rat monoclonal anti-NLRP3 (MAB7578-SP, Novus Biologicals), rabbit polyclonal anti-ASC (PA5-50915, Invitrogen), mouse monoclonal anti-IL-1β (701304, Invitrogen), rabbit polyclonal anti-Ambra 1 (GTX17003, Genetex), rabbit polyclonal anti-ATG7 (PA535203, Themofisher), rabbit polyclonal anti-SQSTM1 (PA520839, Invitrogen), rabbit polyclonal anti-ORP150 (NBP2-14113, Novus Biologicals), rabbit polyclonal anti-NF-Kβ (51-0500, Invitrogen), rabbit polyclonal anti-TNFα (NB600-587SS, Novus Biologicals), and rabbit polyclonal anti-GAPDH (ITI5052, GBIO).

Techniques: DNA Gel Electrophoresis, Western Blot, Expressing

Journal: iScience

Article Title: Genome-wide CRISPR-Cas9 screens identify mechanisms of BET bromodomain inhibitor sensitivity

doi: 10.1016/j.isci.2021.103323

Figure Lengend Snippet: Validation of whole-genome JQ1 sensitivity screen (A) Growth of HCT116- WT , HCT116- ATP2C1 , HCT116- DUSP5 , and HCT116- FERMT2 in the presence of control medium or medium supplemented with JQ1 using the IncuCyte ZOOM live cell imaging system over 132 h. Cell growth is displayed as phase object confluence (percent) analyzed with the IncuCyte ZOOM Basic Analyzer. (B) Dose response of JQ1 on cell proliferation of HCT116- WT and HCT116- ATP2C1 cells. Cell number is measured using the CellTiter-Glo luminescence assay. Luminescence values are normalized to 100% in control medium to enable comparison between different cell lines. Mean ± S.D. (n = 3). (C) Dose response of Paclitaxel on cell proliferation of HCT116- WT , HCT116- ATP2C1 , HCT116- DUSP5 , and HCT116- FERMT2 cells. Luminescence values are normalized to 100% in control medium to enable comparison between different cell lines. Mean ± S.D. (n = 3)

Article Snippet: Rabbit polyclonal anti-ATP2C1 , Invitrogen , Cat#PA5-109430.

Techniques: Live Cell Imaging, Luminescence Assay

Journal: iScience

Article Title: Genome-wide CRISPR-Cas9 screens identify mechanisms of BET bromodomain inhibitor sensitivity

doi: 10.1016/j.isci.2021.103323

Figure Lengend Snippet: Manganese modulates cellular sensitivity to JQ1 (A) Genome wide CRISPR-Cas9 screen profiles highlighting ATP2C1 and TMEM165, in HCT116 -WT cells. The left panel shows the relative abundance of ATP2C1 and TMEM165 sgRNAs compared with the initial library. The next two panels show the relative abundance of ATP2C1 and TMEM165 sgRNAs upon JQ1 treatment at IC 20 (0.2 μM) (middle panel) and IC 50 (1 μM) (right panel) compared with untreated cells. (B) Dose response effects of JQ1, JQ1 (−), or OTX015 on cell proliferation of HCT116- WT cells in the presence of different concentrations of CaCl 2 . Luminescence values are normalized to 100% in control medium to enable comparison between different cell lines. Mean ± S.D. (n = 3). (C) Dose response of JQ1, JQ1 (−), or OTX015 on cell proliferation of HCT116- WT cells in the presence of different concentrations of MnCl 2 . Luminescence values are normalized to 100% in control medium to enable comparison between different cell lines. Mean ± S.D. (n = 3). (D) Dose response of JQ1 on cell proliferation of HT-29 cells in the presence of different concentrations of MnCl 2 . Luminescence values are normalized to 100% in control medium to enable comparison between different cell lines. Mean ± S.D. (n = 3). (E) Dose response of JQ1 on cell proliferation of SUM159PT cells in the presence of different concentrations of MnCl 2 . Luminescence values are normalized to 100% in control medium to enable comparison between different cell lines. Mean ± S.D. (n = 3). (F) Dose response of JQ1, JQ1 (−), and OTX015 on cell proliferation of HCT116- ATP2C1 cells in the presence of different concentrations of MnCl 2 . (Luminescence values are normalized to 100% in control medium to enable comparison between different cell lines. Mean ± S.D. (n = 3). (G) Dose response of JQ1, JQ1 (−), and OTX015 on cell proliferation of HCT116- ATP2C1 cells in the presence of different concentrations of CaCl 2 . (Luminescence values are normalized to 100% in control medium to enable comparison between different cell lines. Mean ± S.D. (n = 3).

Article Snippet: Rabbit polyclonal anti-ATP2C1 , Invitrogen , Cat#PA5-109430.

Techniques: Genome Wide, CRISPR

Journal: iScience

Article Title: Genome-wide CRISPR-Cas9 screens identify mechanisms of BET bromodomain inhibitor sensitivity

doi: 10.1016/j.isci.2021.103323

Figure Lengend Snippet: BET bromodomain inhibitors increase intracellular manganese levels (A) Intracellular levels of manganese measured with the Fura-2 assay in HCT116- WT cells grown in either control medium or with an additional 50 μM MnCl 2 . Dose response effects of JQ1, JQ1 (−), the inactive enantiomer of JQ1, OTX015, I-BET762, and I-BET151. Values are normalized to control medium without compound (two-way ANOVA with Dunnett's multiple comparisons test). Mean ± S.D., n = 8. ∗ p = 0.0151, ∗∗ p = 0.0058, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (B) Intracellular levels of manganese measured with the Fura-2 assay in Caco-2 cells grown in either control medium or with an additional 50 μM MnCl 2 . Dose response effects of JQ1 or I-BET151. Values are normalized to control medium without compound (two-way ANOVA with Dunnett's multiple comparisons test) Mean ± S.D., n = 8, ∗p = 0.0208, ∗∗p = 0.0056, ∗∗∗∗p < 0.0001. (C) Intracellular levels of manganese measured with the Fura-2 assay in HCT116- WT compared with HCT116- ATP2C1 cells in 50 μM MnCl 2 . Dose response effects of JQ1. Values are normalized to HCT116- WT in control medium without compound (two-way ANOVA with Dunnett's multiple comparisons test). Mean ± S.D., n = 8. #### p < 0.0001, ∗ p = 0.0136, ∗∗∗∗ p < 0.0001. (D) Metal ion analysis using ICP-MS (Inductively Coupled Plasma Mass Spectrometry) was performed on pellets of HCT116- WT cells after overnight exposure to either DMSO, 1 μM JQ1, or 1 μM JQ1 (−), the inactive enantiomer (two-way ANOVA with Dunnett's multiple comparisons test). Mean ± S.D., n = 3. ∗∗∗∗ p < 0.0001.

Article Snippet: Rabbit polyclonal anti-ATP2C1 , Invitrogen , Cat#PA5-109430.

Techniques: Mass Spectrometry

Journal: iScience

Article Title: Genome-wide CRISPR-Cas9 screens identify mechanisms of BET bromodomain inhibitor sensitivity

doi: 10.1016/j.isci.2021.103323

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-ATP2C1 , Invitrogen , Cat#PA5-109430.

Techniques: Recombinant, Synthesized, Cell Viability Assay, dsDNA Assay, Bicinchoninic Acid Protein Assay, CRISPR, Sequencing, Software

Journal: Scientific Reports

Article Title: Production of norovirus-, rotavirus-, and enterovirus-like particles in insect cells is simplified by plasmid-based expression

doi: 10.1038/s41598-024-65316-6

Figure Lengend Snippet: Characterisation of CVB3-VLPs produced by co-transfection of 3CD and P1. ( A ) SDS-PAGE and Western blot analyses of purified VLPs. The top panel shows the stain-free total protein staining of the purified CVB3-VLPs. The bottom panel shows VP0, VP1 and VP3 capsid protein detection by Western blot using an in-house produced rabbit anti-CVB1–6 polyclonal antibody. 2 µg of each purified CVB3-VLP was loaded per well recovered from cultures produced as follows (1) 2% 3CD/98% P1, 5 days production, (2) 2% 3CD/98% P1, 8 days production (3) 5% 3CD/95% P1, 5 days production, (4) 5% 3CD/95% P1, 8 days production (5) 10% 3CD/90% P1, 5 days production, 6) 10% 3CD/90% P1, 8 days production. The gel and blot were cropped for clarity. For full photos, see Supplementary Fig. 5. ( B ) Dynamic light scattering analysis of the purified CVB3-VLPs. Transmission electron microscopy images of the sucrose cushion ultracentrifugation purified CVB3-VLPs: ( C ) 2% 3CD/98% P1, 5 days production, ( D ) 2% 3CD/98% P1, 8 days production ( E ) 5% 3CD/95% P1, 5 days production, ( F ) 5% 3CD/95% P1, 8 days production ( G ) 10% 3CD/90% P1, 5 days production, ( H ) 10% 3CD/90% P1, 8 days production Scale bar 200 nm, 50,000 × magnification.

Article Snippet: VP1 and VP3 proteins were detected by Western blotting using in-house produced rabbit anti-CVB1-6 polyclonal antibody and IRDye-labelled secondary antibody (LI-COR Biosciences).

Techniques: Produced, Cotransfection, SDS Page, Western Blot, Purification, Staining, Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: Production of norovirus-, rotavirus-, and enterovirus-like particles in insect cells is simplified by plasmid-based expression

doi: 10.1038/s41598-024-65316-6

Figure Lengend Snippet: Characterisation of CVB3-VLP after pilot-scale production and ion-exchange purification. ( A ) SDS-PAGE and Western blot analyses of the purified VLPs. The left panel shows the total protein staining of the purified CVB3-VLPs with stain-free method. The right panel shows VP0, VP1 and VP3 capsid protein detection by Western blot using an in-house produced rabbit anti-CVB1-6 polyclonal antibody. 2 µg purified CVB3-VLP was loaded per well produced as follows (1) 10% 3CD/90% P1, 8 days production, (2) BEVS, 5 days production. The gel and blot were cropped for clarity. For full photos, see Supplementary Fig. 6. ( B ) Dynamic light scattering analysis of the purified CVB3-VLPs from BEVS or plasmid-based production followed by tangential flow filtration and ion exchange chromatography purification ( C ) representative transmission electron microscopy image of purified CVB3-VLP from plasmid-based expression system. 50,000 × magnification, scale bar 200 nm.

Article Snippet: VP1 and VP3 proteins were detected by Western blotting using in-house produced rabbit anti-CVB1-6 polyclonal antibody and IRDye-labelled secondary antibody (LI-COR Biosciences).

Techniques: Purification, SDS Page, Western Blot, Staining, Produced, Plasmid Preparation, Filtration, Ion Exchange Chromatography, Transmission Assay, Electron Microscopy, Expressing