Journal: Frontiers in Oncology

Article Title: Overexpression of miRNA-3613-3p Enhances the Sensitivity of Triple Negative Breast Cancer to CDK4/6 Inhibitor Palbociclib

doi: 10.3389/fonc.2020.590813

Figure Lengend Snippet: Overexpression of miR-3613-3p suppresses the proliferation, migration, and clonogenic ability induces G0/G1 cell-cycle arrest in TNBC cells. Effects of miR-3613-3p on TNBC cell growth and migration in vitro . After generation of miR-3613-3p stable overexpression in MDA-MB-231. (A) Cell vitality was evaluated by MTT assay, stably transfected with miR-3613-3p or NC lentivirus. (B, C) Colonigenic ability of different MDA-MB-231 cells were tested; generation of miR-3613-3p stable overexpression, the numbers of colony were counted and compared in the diagrams. (D, E) Cell migration of different MDA-MB-231 cells was tested using monolayer wound healing assay, generation of miR-3613-3p stable overexpression; the percent of wound closure was counted and compared in the diagrams. (F, G) Cell cycle of MDA-MB-231 cells stably transfected with miR-3613-3p or NC lentivirus was analyzed by flow cytometry assay; the percentage of cells in G0/G1, S, and G2/M phase is annotated in each column. (H, I) EdU assay of relative DAPI stained cells and EdU add-in cells. MDA-MB-231 cells were stably transfected with miR-3613-3p or NC lentivirus. At least 200 cells were counted per well. (J) Western blot analysis of positive cell cycle regulators CCND1, CCND2, CCND3, pRb, p-pRb, c-MYC, CDK4, and CDK6 protein in MDA-MB-231 cells transfected with transfected with miR-3613-3p lentivirus or vector control. P values were determined by two-tailed t-test, **P < 0.01; ****p < 0.0001.

Article Snippet: Primary antibodies were used as follows: anti-c-MYC (1:1,000, Proteintech), anti-EZH2, anti-SMAD2, anti-RB, anti-CDK4, anti-CDK6, anti-CCND1, anti-CCND2 and anti-CCND3 (1:1,000, Cell Signaling Technology) and anti-Actin (1:3,000, Sigma).

Techniques: Over Expression, Migration, In Vitro, MTT Assay, Stable Transfection, Transfection, Wound Healing Assay, Cytometry, EdU Assay, Staining, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: Theranostics

Article Title: A SCID mouse-human lung xenograft model of SARS-CoV-2 infection

doi: 10.7150/thno.58321

Figure Lengend Snippet: Subcutaneous transplantation of human fetal lung tissues into SCID mice to develop the human lung xenograft mouse model. (A) Scheme of construction of the human lung xenograft mouse model. Small fragments (~10 mm 3 ) of human fetal lung tissues were transplanted subcutaneously (s.c.) into the right flank of SCID mice. After 8 weeks, the lung xenografts can grow and develop mature lung structures. (B) Gross view and (C) histological analysis of the human lung xenograft at 8 weeks post-transplantation. (D) Immunohistochemistry staining of lung marker expression in the human lung xenograft. Sections of human lung xenografts at 8 weeks post-transplantation were immunostained for human CK-19 (hCK-19), human vimentin (hVimentin), human PDPN (hPDPN), human SP-C (hSP-C), human CC10 (hCC10) to visualize the lung epithelium and mesenchyme, type I and type II pneumocytes, and bronchiolar Clara cells, respectively. Tissue sections were also stained for human ACE2 (hACE2) expression. The lower panels represent higher magnification images of the areas outlined by the dotted lines in the upper panels. Scale bars in the left and right panels in (C) represent 1 mm and 200 µm, respectively. Scale bars for 100 × and 400 × magnification in (D) represent 200 µm and 50 µm, respectively. Staining was performed on tissue sections from 4 lung xenografts obtained from two donors, and representative images are shown.

Article Snippet: Then, sections were incubated with primary mouse monoclonal antibodies of anti-SARS-CoV-2 N protein (clone 15A7, unpublished data), anti-human cytokeratin 19 (CK-19) (Abcam, ab7754) and anti-human vimentin (Abcam, ab8069) or rabbit polyclonal antibodies of anti-human angiotensin-converting enzyme 2 (ACE2) (Proteintech, 21115-1-AP), anti-human Clara cell 10 kDa protein (CC10) (Proteintech, 10490-1-AP), anti-human surfactant protein C (SP-C) (Proteintech, 10774-1-AP) and anti-human podoplanin (PDPN) (Proteintech, 11629-1-AP).

Techniques: Transplantation Assay, Immunohistochemistry, Staining, Marker, Expressing

Journal: Theranostics

Article Title: A SCID mouse-human lung xenograft model of SARS-CoV-2 infection

doi: 10.7150/thno.58321

Figure Lengend Snippet: SARS-CoV-2 tropism in human lung xenografts. Sections of SARS-CoV-2-infected lung xenografts harvested at 6 dpi were subjected to multiplex immunofluorescence assay for (A) co-staining of SARS-CoV-2 N protein (N, green), human ACE2 (hACE2, yellow), human CK-19 (hCK-19, red) and human vimentin (hVimentin, magenta), or for (B) co-staining of SARS-CoV-2 N protein (green), human ACE2 (hACE2, yellow), human SP-C (hSP-C, red) and human PDPN (hPDPN, grey). Nuclei were stained with DAPI (blue). White frame was magnified on the right and bottom panels. Solid white arrows indicate the SARS-CoV-2+/hACE2+ cells. Scale bars in the upper left panels in (A) and (B) represent 50 µm, while those in other panels represent 20 µm. Staining was performed on tissue sections from two lung xenografts obtained from two donors, and representative images are shown.

Article Snippet: Then, sections were incubated with primary mouse monoclonal antibodies of anti-SARS-CoV-2 N protein (clone 15A7, unpublished data), anti-human cytokeratin 19 (CK-19) (Abcam, ab7754) and anti-human vimentin (Abcam, ab8069) or rabbit polyclonal antibodies of anti-human angiotensin-converting enzyme 2 (ACE2) (Proteintech, 21115-1-AP), anti-human Clara cell 10 kDa protein (CC10) (Proteintech, 10490-1-AP), anti-human surfactant protein C (SP-C) (Proteintech, 10774-1-AP) and anti-human podoplanin (PDPN) (Proteintech, 11629-1-AP).

Techniques: Infection, Multiplex Assay, Immunofluorescence, Staining

Journal: Cell stem cell

Article Title: Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation.

doi: 10.1016/j.stem.2009.04.020

Figure Lengend Snippet: Figure 2. Analysis of Downstream Signaling of H-RasV12 in GSCs (A) Real-time PCR analysis of H-RasV12-GSCs (n = 3). Cells were cultured on laminin for 6 days under the indicated conditions. The values were normal- ized to Hprt1 expression, with expression levels in WT GSCs cultured with cytokines. (B) The patterns of cyclin expression during culture on laminin. The values were normalized to Hprt1 expression, with expression levels of cyclin D1. (C) Induction of cyclin expression in WT GSCs on laminin. Cells were stimu- lated with the indicated cytokines for 24 hr. The values were normalized to Hprt1 expression, with expression levels in cells cultured without cytokines. (D) Western blot analysis of WT and H-RasV12-GSCs. The cells were starved on laminin for 4 days, and then WT GSCs were either treated or not treated with the indicated cytokines. Treated cells were recovered 30 min after treatment. E, EGF; F, bFGF; G, GDNF. Error bars indicate SEM.

Article Snippet: The following primary antibodies were used: polyclonal rabbit anti-mouse Akt, polyclonal rabbit anti-mouse Akt-P (Ser 473), polyclonal rabbit anti-mouse p27, polyclonal rabbit anti-human cdk2, polyclonal rabbit anti-human cdk2-P (Thr 160), polyclonal rabbit anti-human Erk1/2-P, polyclonal rabbit anti-human cyclin D2 (Cell Signaling, Danvers, MA), mouse anti-human p27-P (Thr 187) (Invitrogen), and mouse anti-Ras (Thermo Scientific).

Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Expressing, Western Blot

Journal: Cell stem cell

Article Title: Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation.

doi: 10.1016/j.stem.2009.04.020

Figure Lengend Snippet: Figure 3. GSC Proliferation by Cyclin Trans- fection (A) RT-PCR analysis of cyclin expression in cyclin D-transfected cells. The cells were cultured on laminin for 24 hr under the indicated conditions. (B) Effect of cytokines on cyclin-transfected GSC growth. WT GSCs were transfected with cyclin D genes. The cells were cultured under the indicated conditions on MEFs for 6 days. (C) Increased expression of cyclin E after addi- tional cyclin E transfection (n = 6). The cells were cultured on laminin for 24 hr without cytokines. Values were normalized to Hprt1 expression, with expression levels in WT GSCs. (D) Appearance of cyclin-transfected cells cultured without cytokines on MEFs for 6 days. Only cyD2E-GSCs formed germ cell colonies. (E) Effect of cytokines on GSCs that were trans- fected with cyclin D and E. While H-RasV12- GSCs and cyD2E-GSCs grew without cytokines, cycD1E-GSCs and cyD3E-GSCs grew when they were supplemented with EGF and bFGF. The cells were cultured on MEFs for 6 days. (F and G) Real-time PCR analysis of cyclin-trans- fected GSCs (n = 6). Cells were cultured with (F) or without (G) cytokines on laminin for 24 hr. Values were normalized to Hprt1 expression, with expression levels in WT GSCs. Scale bar, 100 mm (D). E, EGF; F, bFGF; G, GDNF. Error bars indicate SEM.

Article Snippet: The following primary antibodies were used: polyclonal rabbit anti-mouse Akt, polyclonal rabbit anti-mouse Akt-P (Ser 473), polyclonal rabbit anti-mouse p27, polyclonal rabbit anti-human cdk2, polyclonal rabbit anti-human cdk2-P (Thr 160), polyclonal rabbit anti-human Erk1/2-P, polyclonal rabbit anti-human cyclin D2 (Cell Signaling, Danvers, MA), mouse anti-human p27-P (Thr 187) (Invitrogen), and mouse anti-Ras (Thermo Scientific).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Cell stem cell

Article Title: Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation.

doi: 10.1016/j.stem.2009.04.020

Figure Lengend Snippet: Figure 4. Phenotypic Characterization of H-RasV12-GSCs and cyclin-Transfected Cells (A) Cell-cycle distribution. Significantly more cells are in the G2/M phase in the H-RasV12-GSCs and cyD2E-GSCs. (B) Characterization of cell surface antigens by flow cytometry. Note the weaker expression of EpCAM and a6- and b1-integrin in cyD1E-GSCs and cyD3E-GSCs. Red line, specific antibody; black line, unstained control. Values indicate mean fluorescence intensity. (C) Statistically significant reduction in laminin binding of cyD1E- and cyD3E-GSCs. (D) RT-PCR analysis. Neurog3 expression was weaker in both cyD2E- and cyD3E-GSCs. (E) Immunocytochemistry of p27 and cyclin D2 in transfected cells. The transfectants were cultured on laminin without cytokines for 6 days and stained with anti-cyclin D2 (top) or p27 (bottom) antibody. Cyclin D2 was strongly expressed in both WT and H-RasV12-GSCs. p27 staining was predominantly found in the cytoplasm of H-RasV12-GSCs and cyD2E-GSCs, whereas nuclear staining was found in cyD1E-GSCs and D3E-GSCs. Counterstained by DAPI. (F) COBRA. Open arrows indicate the sizes of the methylated DNA, whereas closed arrows indicate the size of the unmethylated DNA. Percent methylation, as estimated by the intensity of each band, is indicated below the gels. U, uncleaved; C, cleaved. Scale bar, 10 mm (E). E, EGF; F, bFGF; G, GDNF. Error bars indicate SEM.

Article Snippet: The following primary antibodies were used: polyclonal rabbit anti-mouse Akt, polyclonal rabbit anti-mouse Akt-P (Ser 473), polyclonal rabbit anti-mouse p27, polyclonal rabbit anti-human cdk2, polyclonal rabbit anti-human cdk2-P (Thr 160), polyclonal rabbit anti-human Erk1/2-P, polyclonal rabbit anti-human cyclin D2 (Cell Signaling, Danvers, MA), mouse anti-human p27-P (Thr 187) (Invitrogen), and mouse anti-Ras (Thermo Scientific).

Techniques: Transfection, Cytometry, Expressing, Control, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Immunocytochemistry, Cell Culture, Staining, Combined Bisulfite Restriction Analysis Assay, Methylation

Journal: Cell stem cell

Article Title: Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation.

doi: 10.1016/j.stem.2009.04.020

Figure Lengend Snippet: Figure 6. A Model for SSC Self-Renewal Growth signals are converted to Ras activation via Src family molecules. Ras transmits signals to activate the PI3K-Akt pathway as well as other unknown pathways that run parallel to it. Cyclins D2 and E coordinate to drive SSC self-renewing division by eliminating p27 from the nucleus and upregulating b1-integrin, whereas strong cyclin D1 expression may induce differentiation.

Article Snippet: The following primary antibodies were used: polyclonal rabbit anti-mouse Akt, polyclonal rabbit anti-mouse Akt-P (Ser 473), polyclonal rabbit anti-mouse p27, polyclonal rabbit anti-human cdk2, polyclonal rabbit anti-human cdk2-P (Thr 160), polyclonal rabbit anti-human Erk1/2-P, polyclonal rabbit anti-human cyclin D2 (Cell Signaling, Danvers, MA), mouse anti-human p27-P (Thr 187) (Invitrogen), and mouse anti-Ras (Thermo Scientific).

Techniques: Activation Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 3. P53 levels are suppressed by overexpression of JNK1 and in- creased by expression of a dominant- negative JNK1 construct. A, Western blots showing the levels of JNK1, pJNK, JNK2, FAK, pFAK (phosphorylated at Tyr397), and p53 in primary human fibro- blasts transfected with JNK1 or vector control or mock transfected and treated with the VH FN fragment for 1, 3, and 7 h. C, untreated control. Actin served as a loading control. B, Western blots of pri- mary human fibroblasts transfected (Transf) with a dominant negative con- struct of JNK (JNK1 dn) or a vector con- trol or mock transfected and treated as described in A.

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Over Expression, Expressing, Dominant Negative Mutation, Construct, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 5. Overexpression of JNK2 does not suppress p53 protein levels. Western blots showing the levels of JNK2, pJNK, JNK1, FAK, pFAK (phosphorylated at Tyr397), and p53 in primary human fibro- blasts transfected with JNK2 or vector control or mock transfected and treated with the VH FN fragment for 1, 3, and 7 h. C, untreated controls. Actin was used as a loading control.

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 6. Antisense JNK2 treatment decreases p53 protein levels. A, Western blots showing the levels of JNK2, FAK, pFAK (phosphoryl- ated at Tyr397), pJNK, and p53 in primary human fibroblasts trans- fected with an antisense JNK2 oligonucleotide (AS JNK2) or a scram- bled JNK2 oligonucleotide (Scr JNK2) or mock transfected and treated with the VH FN fragment for 1, 3, and 7 h. C, untreated controls. Actin served as a loading control. B, Western blots showing JNK2, pJNK, and p53 levels in primary human fibroblasts transfected with a different antisense JNK2 oligonucleotide (AS JNK2*) or a scrambled JNK2 oligonucleotide (Scr JNK2) or mock transfected and treated with the VH fragment for 1, 3, and 7 h. Actin served as a loading control.

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Western Blot, Transfection, Control

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 7. P53 expression is suppressed in JNK2-deficient cells. A, Western blots showing the levels of FAK, pFAK (phosphorylated at Tyr397), pJNK, JNK2, and p53 in jnk2-deficient or control (CTL) cells treated with the VH FN fragment for 1, 3, or 7 h. C, untreated controls. Actin served as a loading control. B, Western blot showing p53 levels in jnk2-deficient cells from two different sources and in wild-type controls. Equal protein was loaded in each sample analyzed. C, Western blots showing the levels of FAK, pFAK (phosphorylated at Tyr397), JNK1, JNK2, and pJNK in mouse fibroblasts derived from jnk1/jnk2 double knock-outs or control cells treated with the VH fragment for 1, 3, and 7 h. Actin served as a loading control. D, Western blot showing p53 levels in mouse fibroblasts derived from jnk1/jnk2 double knock- outs and wild-type controls. Equal protein was loaded in each sample analyzed.

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Expressing, Western Blot, Control, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 8. P53 can feed back on pJNK to decrease pJNK protein levels in this pathway. Overexpressing p53 leads to a greater de- crease in pJNK levels, whereas knocking out the p53 gene leads to greater increases in pJNK protein levels in this pathway. A, Western blots showing the levels of p53, pJNK, JNK1, JNK2, FAK, and pFAK (phosphorylated at Tyr397) in primary human fibroblasts transfected with p53 or vector control or mock transfected and treated with the VH fragment for 1, 3, and 7 h. C, untreated controls. Actin was used as a loading control. B, Western blots showing the levels of FAK, pFAK (phosphorylated at Tyr397), pJNK, JNK2, JNK1, and p53 in p53 knock- out cells and wild-type controls treated with the VH FN fragment for 1, 3, and 7 h. Actin served as a loading control.

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Knock-Out

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 9. Model of fibronectin fragment-mediated apoptosis sig- naling. In this model, fibronectin fragments interact with cell surface receptors, which prior studies (Kapila et al. (4)) indicate are likely to be integrin and proteoglycan receptors. This interaction initiates the sig- nal transduction pathway that involves dephosphorylation of FAK, likely involving tyrosine 397. FAK dephosphorylation leads to an in- crease in JNK1 phosphorylation and a likely suppression (dashed ar- row) in JNK2. JNK1 triggers a decrease in p53 protein levels (negative regulator), and p53 can feed back on JNK1 to negatively regulate JNK1 phosphorylation (double-ended arrow). JNK2 positively regulates p53. JNK and p53 relocalize (red arrows) from the nucleus to the cell mem- brane to interact with FAK to transmit the apoptotic signal.

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Transduction, De-Phosphorylation Assay, Phospho-proteomics