Journal: mBio

Article Title: Murine AKAP7 Has a 2′,5′-Phosphodiesterase Domain That Can Complement an Inactive Murine Coronavirus ns2 Gene

doi: 10.1128/mBio.01312-14

Figure Lengend Snippet: Cellular localization of the AKAP7s in different cell types. 17Cl-1 cells (A) and B6 BMM (B) were infected with chimeric MHV viruses expressing full-length AKAP7, N-terminally truncated AKAP7 (AKAP7 ΔNTD ), the CD of AKAP7 (AKAP7 CD ), or its mutant (AKAP7 CD H185R ) and stained for the expression of ns2 and AKAP7 with monoclonal antibodies against ns2 or against murine AKAP7 CD peptide A. DAPI was used to visualize nuclei. Magnification for panels A and B, ×60. (C) 293T cells expressed AKAP7 or AKAP CD from lentivirus vectors (indicated). Western blot detection of AKAP7 and the AKAP7 CD by immunoblotting with anti-AKAP7 CD antibody (peptide B) and with antibody against β-actin as a loading and transfer control is shown. (D) 293T cell lines expressing AKAP7 or the AKAP7 CD (as in panel C) were stained with a polyclonal antiserum against murine AKAP7 CD peptide A. Nuclei were visualized with DAPI staining. Magnification, ×63.

Article Snippet: For 293T stable cells lines, the lentivirus vector pLentiCMV-Puro-DEST, from Eric Campeau , pCMV-VSV-G, expressing vesicular stomatitis virus G envelope protein, and the pCMV-dR8.2 packaging plasmid, from Robert Weinberg , were obtained from Addgene.

Techniques: Infection, Expressing, Mutagenesis, Staining, Bioprocessing, Western Blot, Control

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

Article Title: Iroquois Homeobox 1 Acts as a True Tumor Suppressor in Multiple Organs by Regulating Cell Cycle Progression 1

doi: 10.1016/j.neo.2019.08.001

Figure Lengend Snippet: Transduction of human IRX1 gene into human cholangiocarcinoma cell lines. (A) Western blot experiment showed Irx1 expression in four different cholangiocarcinoma cell lines. Among these, HuCCT1 (high Irx1 expression cell line) and SNU1196 (low Irx1 expression cell line) were selected for overexpression study. (B) Lentiviral transduction of IRX1-GFP fusion gene (right). Note the nuclear localization of IRX1-GFP in Irx1 transduced cells whereas GFP-alone transduced cell showed diffused expression (left). (C) Western blot experiment confirmed the transduced protein, tGFP. Fused IRX1-tGFP protein bands are seen with larger size. (D) Cell proliferation assay. The results showed that IRX1 overexpression resulted in decreased cellular proliferation in both HuCCT1 and SNU1196 cell lines. (E) Flow cytometry analysis for cell death induced by H 2 O 2 treatment showing increased susceptibility to H 2 O 2 in the Irx1-expressing cells. (F) Flow cytometry for cell cycle change. IRX1-expressing cells showed decreased mitotic fraction measured by phosphohistone H3 (pHH3).

Article Snippet: These cells were lentivirally transformed with the human IRX1 gene fused with IRX1-GFP (pLentiCMV:Irx1-GFP, Origene, RG218767) or GFP (pLentiCMV:GFP, Origene, PS100019) alone as a control.

Techniques: Transduction, Western Blot, Expressing, Over Expression, Proliferation Assay, Flow Cytometry

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

Article Title: Iroquois Homeobox 1 Acts as a True Tumor Suppressor in Multiple Organs by Regulating Cell Cycle Progression 1

doi: 10.1016/j.neo.2019.08.001

Figure Lengend Snippet: Cell cycle synchronization study. (A and B) Cell synchronization was obtained by treatment with hydroxyuria and nocodazole, respectively, for G1/S and G2/M phase arrest. Arrest was then released, detecting the cell cycle progression. Dotted red lines indicate 2N and 4N cells. The result showed that mitotic exit is markedly delayed by IRX1 overexpression, while S phase progression is slightly delayed. Numbers in upper and lower lines indicate G1/S and G2/M fractions, respectively. (C) The mitosis marked by pHH3. G2/M phase-blocked cells were released and stained for pHH3 at indicated time points. Yellow to white cells (red arrowheads) represent pHH3- and GFP-positive cells. Numbers of percentage indicate fractions of the pHH3-positive cells. At 3 and 6 hours after release, higher cell fractions are still positive for pHH3 in IRX1-expressing cells. (D) Western blot analysis against cyclins. The result showed that levels of various cyclin expression are decreased by IRX1 expression.

Article Snippet: These cells were lentivirally transformed with the human IRX1 gene fused with IRX1-GFP (pLentiCMV:Irx1-GFP, Origene, RG218767) or GFP (pLentiCMV:GFP, Origene, PS100019) alone as a control.

Techniques: Over Expression, Staining, Expressing, Western Blot