Journal: Oncotarget

Article Title: Rapamycin sensitizes cancer cells to growth inhibition by the PARP inhibitor olaparib

doi: 10.18632/oncotarget.19667

Figure Lengend Snippet: (a) The growth inhibition by rapamycin (○), olaparib (Δ), or the combination of rapamycin and olaparib (×). NSCLC and TNBC cells were treated with drugs at the indicated concentrations for 48 h (A549, H157) or 72 h (H522, H1155, HCC1937 and MDA-MB-436). Bars, S.D. (b) The computer-simulated Fa-CI curves displayed synergism (CI<1), additive effects (CI=1), or antagonism (CI>1) for the entire spectrum of effect levels with the combination of rapamycin and olaparib. ○ The actual Fa-CI plot based on the experimental values. (c) A549, H157 or HCC1937 cells were treated with 0.5% DMSO, 1 nM rapamycin, olaparib (20 μM for A549 and 5 μM for H157 and HCC1937), or the combination every 3 days for 9 days. The colony counts are relative to DMSO-treated cells. Rap, rapamycin; Olap, olaparib; bars, S.D.

Article Snippet: Six-week-old female athymic NCr-nu/nu mice (Charles River Labs, Frederick, MD, USA) were subcutaneously injected in both rear flanks with 5 × 10 [ ] H157 or HCC1937 cells in 50 μ l PBS and 50 μ l BD Matrigel Basement Membrane Matrix (BD Biosciences).

Techniques: Inhibition

Journal: Oncotarget

Article Title: Rapamycin sensitizes cancer cells to growth inhibition by the PARP inhibitor olaparib

doi: 10.18632/oncotarget.19667

Figure Lengend Snippet: (a) Olaparib inhibits the PAR activity enhanced by rapamycin. A549, H157 and HCC1937 cells were treated with either DMSO, 1 nM rapamycin, 50 μM olaparib, or the combination of rapamycin and olaparib for 4h. Immunoblotting was performed. (b) The inhibition of Rad51 foci by rapamycin. Cells were treated with the drugs at the concentration described in (a) for 48 h. Rad51 focus formation was assessed based on immunofluorescence. Arrows, Rad51 focus-positive cells. Representative nucleus images are shown in the boxes. (c) Rad51 focus-positive cells were counted. Columns, the mean count from at least five high-power fields. Bars, S.D. * p <0.001.

Article Snippet: Six-week-old female athymic NCr-nu/nu mice (Charles River Labs, Frederick, MD, USA) were subcutaneously injected in both rear flanks with 5 × 10 [ ] H157 or HCC1937 cells in 50 μ l PBS and 50 μ l BD Matrigel Basement Membrane Matrix (BD Biosciences).

Techniques: Activity Assay, Western Blot, Inhibition, Concentration Assay, Immunofluorescence

Journal: Oncotarget

Article Title: Rapamycin sensitizes cancer cells to growth inhibition by the PARP inhibitor olaparib

doi: 10.18632/oncotarget.19667

Figure Lengend Snippet: (a) H157 cells (left) and HCC1937 cells (right) were grown as xenografts in athymic NCr-nu/nu. Bars, S.D. * p <0.001. (b) Representative photos of H157 xenograft tumors after 25 days of either treatment. Arrows, H157 tumors. (c) Rad51 focus formation in vivo . H157 xenograft tumors after 5 days of treatment were excised and prepared for immunofluorescence microscopy as described in the Materials and Methods. (d) Rad51 focus-positive cells were counted. Columns, the mean from all four mice examined in H157 xenografts. Bars, S.D. * p <0.001.

Article Snippet: Six-week-old female athymic NCr-nu/nu mice (Charles River Labs, Frederick, MD, USA) were subcutaneously injected in both rear flanks with 5 × 10 [ ] H157 or HCC1937 cells in 50 μ l PBS and 50 μ l BD Matrigel Basement Membrane Matrix (BD Biosciences).

Techniques: In Vivo, Immunofluorescence, Microscopy

Journal: Scientific Reports

Article Title: USP10 promotes cell proliferation, migration, and invasion in NSCLC through deubiquitination and stabilization of EIF4G1

doi: 10.1038/s41598-024-74490-6

Figure Lengend Snippet: Regulation of EIG4G1 expression by USP10 . A Two shRNAs downregulated the expression of USP10 in NSCLC cell lines H1299, H157, and H23. EIF4G1 levels were detected using western blotting, with β-actin as the internal reference. B Overexpression of USP10 . Western blotting for the protein levels of EIF4G1, with β-actin as the internal reference. C Transfection of a USP10-overexpressing plasmid in USP10 knockdown H1299 cells; EIF4G1 protein levels were detected using western blotting, with β-actin as the internal reference. D Downregulation of USP10 expression. qRT-PCR detection of the mRNA levels of EIF4G1 , with GAPDH as the internal reference. E Overexpression of USP10 . qRT-PCR detection of the mRNA levels of EIF4G1 , with GAPDH as the internal reference. Mean ± SD, n = 3. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Original blots are presented in Supplementary Fig. 2.

Article Snippet: NSCLC cell lines H157 and H23 were obtained from iCellBioscience Inc. (Shanghai, China).

Techniques: Expressing, Western Blot, Over Expression, Transfection, Plasmid Preparation, Knockdown, Quantitative RT-PCR

Journal: PLoS Genetics

Article Title: Cancer-Associated Splicing Variant of Tumor Suppressor AIMP2/p38: Pathological Implication in Tumorigenesis

doi: 10.1371/journal.pgen.1001351

Figure Lengend Snippet: (A) The AIMP2/p38 structural gene consists of four exons encoding a total of 320 aa (NM_006303) with three introns. The 312 aa version of AIMP2 is also reported as U24169. Both forms appear to work similarly for most of our experiments although we do not know the reason for the existence of the two forms at this point. The sequences of exon 1, 2 and 3, and the primers, JTV13, JTV5, JTV11 and DX2-S2 are shown in . (B) Expression of AIMP2-F and –DX2 was examined in different lung cells (Normal: WI-26, NL-20; tumorigenic: A549, H322, H460 and H157) by RT-PCR using their specific primers shown above. The pair of the primers, JTV13 and JTV11, generates the two PCR products, one from the transcript of AIMP2-full length (upper band) and the other from the exon 2-lacking transcript (lower band). RT-PCR with the primers, DX2-S2, (specific to the junction site of exon 1 and 3, ) and JTV5, generates the amplicon only from AIMP2-DX2 transcript. Actin was used as the control. (C) Expression of AIMP2-F and AIMP2-DX2 was also determined in different types of human lung cancer tissues and their normal counterparts by RT-PCR. The primer pairs of JTV-13/JTV-5, and DX2-S2/JTV-5 were used for RT-PCR of AIMP2-F and –DX2, respectively. (D) The expression of AIMP2-F and –DX2 were determined in adenocarcinoma (n = 14) and normal (n = 11) lung tissues by quantitative real-time RT-PCR. The cancer regions were obtained by laser microdissection system from archival formalin-fixed paraffin-embedded (FFPE) patient tissues for RT-PCR as described in experimental procedures. PAPOLA was chosen as the endogenous reference gene for quantitative RT-PCR. The expression results were analyzed by Mann-Whitney test and statistical analyses were achieved using SPSS software (SPSS, Chicago, IL). Each dot represents the expression values of AIMP2-F and -DX2 calibrated to the expression level of PAPOLA. The mean values are shown as lines. **, P <0.01 (E) The larger and smaller amplicons that were generated by RT-PCR with the primers JTV13 and JTV11 were cloned and expressed in AIMP2-deficient MEFs and their expression was determined by Western blotting with monoclonal anti-AIMP2 antibody (#324) that recognizes both forms of AIMP2. Small interference RNAs targeting AIMP2-F and –DX2 were designed and introduced into A549 cells, and their specific effect on the expression of the corresponding transcripts was determined by Western blotting with anti-AIMP2 antibody (right panel).

Article Snippet: Lung carcinoma cell lines A549 and NCI-H460 were obtained from ATCC, and H322 and H157 from KCLB.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Laser Capture Microdissection, Formalin-fixed Paraffin-Embedded, MANN-WHITNEY, Software, Generated, Clone Assay, Western Blot

Journal: Virologica Sinica

Article Title: Prion Protein Protects Cancer Cells against Endoplasmic Reticulum Stress Induced Apoptosis

doi: 10.1007/s12250-019-00107-2

Figure Lengend Snippet: Cancer cells express different levels of PrP. A Immunoblotting with PrP specific mAb 4H2 showed that A549, H157, H1299, and BxPC-3 cells expressed PrP. A residual level of PrP was detected in SPC-A1 cell lysate. PRNP null BxPC-3 cells were used as negative control. β-actin was used a loading control. B Confocal immunofluorescence staining with 4H2 revealed that A549, H157, H1299, and BxPC-3 cells expressed PrP, and most PrP was cell surface bound. On the contrary, no signal of PrP was detected in SPC-A1 and PRNP null BxPC-3 cells. Nuclei were counter stained with DAPI. The experiments were repeated three times with similar results.

Article Snippet: Non-small-cell lung carcinoma cell lines A549, H157, H1299, and SPC-A1 were obtained from China Center for Type Culture Collection.

Techniques: Western Blot, Negative Control, Immunofluorescence, Staining

Journal: Molecular Cancer

Article Title: Combined targeting of TGF-β1 and integrin β3 impairs lymph node metastasis in a mouse model of non-small-cell lung cancer

doi: 10.1186/1476-4598-13-112

Figure Lengend Snippet: TGF-β exposure enhances NSCLC cell adhesion and transmigration across lymphatic endothelial cells. (A) Adhesion of H157 cells to LEC monolayers after treatment for 5 days with TGF-β (2 ng/ml), and in the presence or absence of the TGF-βRI inhibitor SB 431542 (**p < 0.001, Student’s t -test). (B) Time-lapse microphotographs of the movement of H157 NSCLC cells, treated as in A, across LEC monolayers (63× water objective). Dots indicate the initial position of the cell and arrows indicate the same cell in motion. (C) Quantification of the number of H157 cells in transit over LEC monolayers, expressed as a percentage of the total number of cells counted in a single XY plane. (D) Quantification of NSCLC cell transmigration across monolayers of primary human LECs in the presence or absence of TGF-β. Data are presented as fold-change with respect to untreated control H157 cells (**p < 0.001, Student’s t- test).

Article Snippet: Female athymic nude mice (5-6 weeks old) were purchased from Harlan Laboratories and GFP-H157 cells (1 × 10 6 ) in PBS containing 10 μg of Matrigel (BD Biosciences, San José, CA, USA) were injected in a total volume of 20 μl into the left lung of these nude mice as described previously [ ].

Techniques: Transmigration Assay

Journal: Molecular Cancer

Article Title: Combined targeting of TGF-β1 and integrin β3 impairs lymph node metastasis in a mouse model of non-small-cell lung cancer

doi: 10.1186/1476-4598-13-112

Figure Lengend Snippet: TGF-β treatment induces integrin expression, FAK phosphorylation, β3 integrin-dependent adhesion and transmigration of H157 NSCLC cells across LEC monolayers. (A) mRNA expression of several integrins in H157 cells following treatment with TGF-β and its inhibitors (fold-change with respect to untreated cells) and confirmation by real-time PCR of the differential expression of β3 and β5 integrins after exposure to TGF-β. (B) FAK phosphorylation after TGF-β treatment of β3 integrin-deficient (shRNAβ3) and β3 integrin-competent H157 NSCLC cells. (C) Adhesion of TGF-β-treated H157 cells to LEC monolayers in the presence or absence of the FAK inhibitor, PF-573228 (**p < 0.001, Student’s t- test). (D) Quantification of H157 cell transmigration across LEC monolayers in the presence of the TGF-βRI inhibitor SB431542, the FAK inhibitor PF-573228, a blocking mAb against β3 integrin (Anti-β3) and that of β3 integrin-deficient H157 clones (β3). Samples were pretreated with or without TGF-β as described in the Materials and methods (**p < 0,001 compared against non-treated cells, ## p < 0,001 compared against TGFβ-treated cells, Mann–Whitney U -test).

Article Snippet: Female athymic nude mice (5-6 weeks old) were purchased from Harlan Laboratories and GFP-H157 cells (1 × 10 6 ) in PBS containing 10 μg of Matrigel (BD Biosciences, San José, CA, USA) were injected in a total volume of 20 μl into the left lung of these nude mice as described previously [ ].

Techniques: Expressing, Transmigration Assay, Real-time Polymerase Chain Reaction, Blocking Assay, Clone Assay, MANN-WHITNEY

Journal: Molecular Cancer

Article Title: Combined targeting of TGF-β1 and integrin β3 impairs lymph node metastasis in a mouse model of non-small-cell lung cancer

doi: 10.1186/1476-4598-13-112

Figure Lengend Snippet: Integrin β3 silencing in H157 cells significantly diminishes cell movement across LEC monolayers. (A) Microphotographs showing the adhesion of TGF-β-pretreated H157 cells (integrin-competent and integrin-deficient) to LEC monolayers (63× water objective). (B) Average distance covered and speed of TGF-β-pretreated H157 cells (integrin-competent and integrin-deficient): ** p = 0.001, one-way ANOVA with Dunnett’s multiple comparison test. (C) Representation of cell directionality for one representative cell from each group. A minimum of 30 cells were analyzed per group.

Article Snippet: Female athymic nude mice (5-6 weeks old) were purchased from Harlan Laboratories and GFP-H157 cells (1 × 10 6 ) in PBS containing 10 μg of Matrigel (BD Biosciences, San José, CA, USA) were injected in a total volume of 20 μl into the left lung of these nude mice as described previously [ ].

Techniques:

Journal: Molecular Cancer

Article Title: Combined targeting of TGF-β1 and integrin β3 impairs lymph node metastasis in a mouse model of non-small-cell lung cancer

doi: 10.1186/1476-4598-13-112

Figure Lengend Snippet: H157 cell transmigration across endothelial monolayers is inhibited by blocking antibodies against the β3 integrin ligands L1CAM and CD31. Quantification of the number of TGF-β-pretreated β3 integrin-competent (A) and β3 integrin-deficient (B) H157 cells transmigrating across LEC monolayers in the presence of blocking antibodies against the L1CAM RGD binding region (L1.35.9 mAb), the L1CAM homotypic binding region (L1 9.3 mAb) and CD31: **p = 0.001, one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: Female athymic nude mice (5-6 weeks old) were purchased from Harlan Laboratories and GFP-H157 cells (1 × 10 6 ) in PBS containing 10 μg of Matrigel (BD Biosciences, San José, CA, USA) were injected in a total volume of 20 μl into the left lung of these nude mice as described previously [ ].

Techniques: Transmigration Assay, Blocking Assay, Binding Assay

Journal: Molecular Cancer

Article Title: Combined targeting of TGF-β1 and integrin β3 impairs lymph node metastasis in a mouse model of non-small-cell lung cancer

doi: 10.1186/1476-4598-13-112

Figure Lengend Snippet: Integrin β3 silencing and inhibition of stromal TGF-β improves survival and attenuates tumor cell growth in an orthotopic mouse model of lung carcinoma. (A) Kaplan-Meier survival curves for mice injected with β3 integrin-deficient or β3 integrin-competent cells that were pretreated with or without TGF-β. Mice were injected intraperitoneally with 200 μg of the TGF-β inhibitor peptide P144 as described in Materials and methods. (B) Tumor size determined from microphotographs obtained from tumor sections and expressed in cm 2 (maximum diameter). Image analyses were performed using fiji software. Data were analyzed using a one-way ANOVA followed by a Dunnett’s multiple comparison test (** p < 0.001: H157 TGF-β PBS vs H157 control PBS; §§ p = 0.001: shRNA β3 TGF-β PBS vs H157 control PBS; ## p = 0.004: H157 TGF-β PBS vs H157 TGF-β P144). (C) Micro-CT analyses of mouse lungs before sacrifice. A representative image from each group is shown.

Article Snippet: Female athymic nude mice (5-6 weeks old) were purchased from Harlan Laboratories and GFP-H157 cells (1 × 10 6 ) in PBS containing 10 μg of Matrigel (BD Biosciences, San José, CA, USA) were injected in a total volume of 20 μl into the left lung of these nude mice as described previously [ ].

Techniques: Inhibition, Injection, Software, shRNA, Micro-CT

Journal: Molecular Cancer

Article Title: Combined targeting of TGF-β1 and integrin β3 impairs lymph node metastasis in a mouse model of non-small-cell lung cancer

doi: 10.1186/1476-4598-13-112

Figure Lengend Snippet: Combined targeting of TGF-β and integrin β3 attenuates lymph node metastasis of H157 NSCLC cells in the lungs of immunodeficient mice. (A) Percentage of affected lymph nodes (axillary and brachial) in the different experimental groups analyzed. Statistical analyses were performed by means of Mann–Whitney U- test. Significant values are depicted with double asterisks and correspond to p values of 0.009 for both comparisons (B) Representative microphotograph of GFP tumor detection in the histological samples obtained from lungs (upper row) and lymph nodes (lower row). (C) Immunohistochemical detection of integrin β3 in tumors obtained from mice.

Article Snippet: Female athymic nude mice (5-6 weeks old) were purchased from Harlan Laboratories and GFP-H157 cells (1 × 10 6 ) in PBS containing 10 μg of Matrigel (BD Biosciences, San José, CA, USA) were injected in a total volume of 20 μl into the left lung of these nude mice as described previously [ ].

Techniques: MANN-WHITNEY, Immunohistochemical staining