Journal: International Journal of Biological Sciences

Article Title: Epithelial-mesenchymal reprogramming by KLF4-regulated Rictor expression contributes to metastasis of non-small cell lung cancer cells

doi: 10.7150/ijbs.73548

Figure Lengend Snippet: Expressions of Rictor in human non-small cell lung cancer. A , Western blot analysis was performed to examine Rictor expression in human NSCLC cell lines and immortalized HBE cells. β-actin was as a loading control ( upper panel ). The density of Rictor protein was shown ( lower panel ). * p <0.05; ** p <0.01; *** p <0.001; ns, not statistically significant. B , expression of Rictor mRNA in human NSCLC cell lines and HBE cells was analyzed by RT-qPCR. ** p <0.01; *** p <0.001; ns, not statistically significant. C , Rictor protein level in six representative NSCLC cases was assessed by Western blotting. β-actin was as a loading control. N, normal adjacent tissue; T, tumor ( upper panel ). Western blotting determined Rictor protein levels in the malignant and the corresponding normal adjacent tissues of 22 NSCLC patients (lower panel) . The intensity was evaluated using Image J (NIH) computer software. ** p <0.01. D , expression of Rictor mRNA in the malignant and the corresponding normal adjacent tissues of 22 NSCLC patients was analyzed by RT-qPCR. *** p <0.001. E , representative figures of IHC staining for Rictor were detected on NSCLC tissues and the corresponding normal adjacent samples.

Article Snippet: Lentivirus plasmids containing pLKO.1-shRictor (#1853 and #1854) , pLKO.1-shGFP (#30323), the lentiviral packaging plasmid psPAX2 (#12260), the envelope plasmid pMD2.G (#12259) and the Rictor expression construct myc-Rictor (#1860) were obtained from Addgene (Watertown, MA).

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, Software, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: Epithelial-mesenchymal reprogramming by KLF4-regulated Rictor expression contributes to metastasis of non-small cell lung cancer cells

doi: 10.7150/ijbs.73548

Figure Lengend Snippet: Rictor correlates with the tumorigenic properties of NSCLC cells. A , knockdown of Rictor in NCSLC cell lines and cell lysates were analyzed by Western blotting. β-actin was as a loading control. B , knockdown of Rictor inhibited the proliferation of NSCLC cells. WST-1 assays were performed. Data represent mean ± SD from three independent experiments. *** p <0.001, significant difference compared with the shGFP cells. C , knockdown of Rictor attenuated anchorage-independent growth of NSCLC cells. Soft agar assays were performed. Data represent mean ± SD from two independent experiments. *** p <0.001, significant difference compared with the shGFP cells. D , Kaplan-Meier survival analysis for the relationship between survival time and Rictor signature in lung cancer was performed using the online tool ( http://kmplot.com/analysis/ ). OS, Overall Survival. p <0.05 was considered to be a statistically significant difference.

Article Snippet: Lentivirus plasmids containing pLKO.1-shRictor (#1853 and #1854) , pLKO.1-shGFP (#30323), the lentiviral packaging plasmid psPAX2 (#12260), the envelope plasmid pMD2.G (#12259) and the Rictor expression construct myc-Rictor (#1860) were obtained from Addgene (Watertown, MA).

Techniques: Knockdown, Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: Epithelial-mesenchymal reprogramming by KLF4-regulated Rictor expression contributes to metastasis of non-small cell lung cancer cells

doi: 10.7150/ijbs.73548

Figure Lengend Snippet: Rictor involves in the regulation of migratory and invasive abilities of NSCLC cells. A , knockdown of Rictor attenuated the migratory ability of H358 and H1299 NSCLC cells. Stable Rictor knocking down H358 and H1299 cells were subjected to wound healing assays. Images were taken at 0, 24 h ( left panel ). Knockdown of Rictor on the migratory ability of H358 and H1299 cells was evaluated by measuring the width of the wound area at each time point ( right panel ). Data represent mean ± SD from three fields per treatment for three independent experiments. *** p <0.001, significant difference compared with the shGFP cells. B , knockdown of Rictor attenuated the invasive ability of H358 and H1299 NSCLC cells. Stable Rictor knocking down H358 and H1299 cells were subjected to transwell invasion assays. The invaded cells were then photographed under a light microscope ( left panel ) and quantified ( right panel ). Data represent mean ± SD from three fields per treatment for two independent experiments. ** p <0.01, *** p <0.001, significant difference compared with the shGFP cells. C , stable knockdown of Rictor in H358 and H1299 cells and the level of mesenchymal and epithelial markers were examined by Western blotting with specific antibodies. β-actin was as a loading control.

Article Snippet: Lentivirus plasmids containing pLKO.1-shRictor (#1853 and #1854) , pLKO.1-shGFP (#30323), the lentiviral packaging plasmid psPAX2 (#12260), the envelope plasmid pMD2.G (#12259) and the Rictor expression construct myc-Rictor (#1860) were obtained from Addgene (Watertown, MA).

Techniques: Knockdown, Light Microscopy, Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: Epithelial-mesenchymal reprogramming by KLF4-regulated Rictor expression contributes to metastasis of non-small cell lung cancer cells

doi: 10.7150/ijbs.73548

Figure Lengend Snippet: KLF4 regulates the transcription activity of the human Rictor promoter. A , H358, H1299, H125 and H23 cells were transfected with various human Rictor promoter constructs and subjected to dual-luciferase reporter assays. Luciferase activities were normalized to a co-expressed Renilla luminescent signal and were shown as relative folds against control samples. B , H23, H125 and H1299 cells were co-transfected a pGL3-37/+96 with various transcription factor expression constructs and subjected to dual-luciferase reporter assays. Luciferase activities were normalized to a co-expressed Renilla luminescent signal and were shown as relative folds against control samples. C , expression of KLF4 level in NSCLC cell lines was examined by Western blot analysis with specific antibodies. β-actin was as a loading control. D , H358, H1299, H125 and H23 cells were transfected with the indicated constructs and subjected to dual-luciferase reporter assays. The positions of the putative KLF4 binding site identified with MatInspector in the Rictor promoter were indicated. Luciferase activities were normalized to a co-expressed Renilla luminescent signal and were shown as relative folds against control samples. E , KLF4 is directly bound to the promoter region of the Rictor gene. H125, H358 and H1299 cells were subjected to ChIP assays with a KLF4 antibody. The size of the PCR product is 151 bp. F , knockdown of KLF4 inhibited the Rictor promoter activity and protein expression. H358-shGFP, H358-shKLF4, H1299-shGFP and H1299-shKLF4 cells were transfected with pGL3-2000/+96 or pGL3-37/+96 construct and subjected to dual-luciferase reporter assays. Luciferase activities were normalized to a co-expressed Renilla luminescent signal and were shown as relative folds against control samples. * p <0.05, significant difference compared with the pGL3-2000/+96 -transfected shGFP cells. # p <0.05, a significant difference compared with the pGL3-37/+96- transfected shGFP cells ( left and middle panels ). Effect of KLF4 knocking down on Rictor protein level in H358 and H1299 cells was examined by Western blotting with specific antibodies. β-actin was as a loading control ( right panel ). G , overexpression of KLF4 increased the Rictor promoter activity. A549 cells were co-transfected pGL3-2000/+96 or pGL3-37/+96 construct with a KLF4 expression construct and subjected to Western blot analysis ( left panel ) and dual-luciferase reporter assays ( right panel ). Luciferase activities were normalized to a co-expressed Renilla luminescent signal and were shown as relative folds against control samples. * p <0.05, ** p <0.01, significant difference compared with the pGL3-2000/+96 and vector -transfected A549 cells. # p <0.05, ## p <0.01, significant difference compared with the pGL3-37/+96 and vector- transfected A549 cells.

Article Snippet: Lentivirus plasmids containing pLKO.1-shRictor (#1853 and #1854) , pLKO.1-shGFP (#30323), the lentiviral packaging plasmid psPAX2 (#12260), the envelope plasmid pMD2.G (#12259) and the Rictor expression construct myc-Rictor (#1860) were obtained from Addgene (Watertown, MA).

Techniques: Activity Assay, Transfection, Construct, Luciferase, Control, Expressing, Western Blot, Binding Assay, Knockdown, Over Expression, Plasmid Preparation

Journal: International Journal of Biological Sciences

Article Title: Epithelial-mesenchymal reprogramming by KLF4-regulated Rictor expression contributes to metastasis of non-small cell lung cancer cells

doi: 10.7150/ijbs.73548

Figure Lengend Snippet: Knockdown of KLF4 prevents Rictor-mediated EMT, mTOR/Rictor interaction and NSCLC cell migration and invasion. A , knockdown of KLF4 in H358 and H1299 cells and the levels of Rictor, mesenchymal and epithelial markers were examined by Western blotting with specific antibodies. β-actin was as a loading control. B and C , re-introduction of Rictor rescues the inhibition of cell migration caused by KLF4 knocking down. Stable H358-shKLF4 (B) and H1299-shKLF4 (C) cells were transfected with a Rictor expression construct and subjected to a wound healing assay. ** p <0.01, *** p <0.001, significant difference compared with the shGFP cells. ### p <0.001, a significant difference compared with the shKLF4 cells. D , re-introduction of Rictor attenuates the inhibition of cell invasion caused by KLF4 knocking down. Stable H358-shKLF4 and H1299-shKLF4 cells were transfected with a Rictor expression construct and subjected to transwell invasion assays. ** p <0.01, a significant difference compared with the shGFP cells. # p <0.05, a significant difference compared with the shKLF4 cells. E , re-introduction of Rictor affects a series of characteristic changes of MET. Stable H358-shKLF4 and H1299-shKLF4 cells were transfected with a Rictor expression construct and KLF4, Rictor, mesenchymal and epithelial markers were examined by Western blotting with specific antibodies. β-actin was as a loading control. F and G , knockdown of KLF4 impedes mTOR/Rictor interaction in NSCLC cells. H358-shGFP, H358-shKLF4, H1299-shGFP and H1299-shKLF4 cells were lysed and immunoprecipitated with a Rictor, mTOR, or normal IgG antibody. The immune complexes and input were analyzed by immunoblotting with an mTOR (F) or Rictor (G) antibody.

Article Snippet: Lentivirus plasmids containing pLKO.1-shRictor (#1853 and #1854) , pLKO.1-shGFP (#30323), the lentiviral packaging plasmid psPAX2 (#12260), the envelope plasmid pMD2.G (#12259) and the Rictor expression construct myc-Rictor (#1860) were obtained from Addgene (Watertown, MA).

Techniques: Knockdown, Migration, Western Blot, Control, Inhibition, Transfection, Expressing, Construct, Wound Healing Assay, Immunoprecipitation

Journal: Cancers

Article Title: Molecular Interplay between Dormant Bone Marrow-Resident Cells (BMRCs) and CTCs in Breast Cancer

doi: 10.3390/cancers12061626

Figure Lengend Snippet: Inhibition of mTORC2 RICTOR decreases CTC proliferation markers. ( A ) High-definition immunofluorescence on MCF-10A cells showing differential expression of Ki67 proliferation marker, along with high pNDRG1 expression, indicative of active mTORC2 signaling (scale bar = 10μm). ( B ) Western blotting analyses of ShRNA RICTOR knockdowns of MCF10A cells showing that RICTOR knockdown resulted in decreased pNDRG1 expression, while p4EBP1 status remained unchanged (red boxes). Control denotes non-targeting scrambled control, and numbers 1 and 2 (below sh-RICTOR) denote two distinct lentiviral shRNA constructs against RICTOR. ( C ) (Top) Significant decrease of total BMRCs collected from ex vivo experiments using MCF-10A-shRICTOR knockdowns injected in NSG mice (1.0 × 10 5 cells/mouse; N = 11). Conversely, no change of PanCK+ or CD44+/CD24− BMRC cell populations was detected. ( D ) (Bottom) Real-time PCR of ex vivo MCF-10A shRICTOR BMRCs exhibit increased gene expression for PCNA (proliferation marker), along with decreased CDKN1A (quiescence status) and increased BBC3 (PUMA) expression, consistent with a pro-apoptotic response ( N = 11).

Article Snippet: Lentiviral pLKO.1 shRICTOR (plasmid #1853 and #1854) and scrambled shRNA (plasmid #1864) constructs were obtained from AddGene (Cambridge, MA, USA), packaged in HEK293T cells using 3 rd generation lentiviral packaging system, and transduced into MCF10A cells [ ].

Techniques: Inhibition, Immunofluorescence, Quantitative Proteomics, Marker, Expressing, Western Blot, shRNA, Knockdown, Control, Construct, Ex Vivo, Injection, Real-time Polymerase Chain Reaction, Gene Expression