Journal: Scientific Reports

Article Title: GFP-complementation assay to detect functional CPP and protein delivery into living cells

doi: 10.1038/srep18329

Figure Lengend Snippet: ( 3a,b) SEE components are validated by co-transfection of CHO-K1 cells with hGFP1-10 and various Cargo_S11 fusion constructs. All S11 fusions complemented hGFP1-10, measured by increasing % fluorescent cells ( 3a ) or increasing MFI ( 3b ) in the viable single-cell population. In all experiments the “S11 only” control is a fusion of the [GSSG]x2 linker and S11 sequence, and does not generate a detectable GFP signal, nor do empty vector and “GFP1-10 only” controls (% GFP-positive cells <0.65 or MFI <25). Error bars represent standard error of the mean between technical replicates; data is representative of three independent experiments. ( 3c) Fluorescence microscopy detects GFP complementation (FITC channel) in HCC827/hGFP1-10 cell populations transfected with complementing β-ACTIN_S11 or TRX_S11 constructs, compared to empty vector control. Cells are also counter-stained for endogenous β-Actin (TRITC) and nuclei (DAPI) before visualizing. Bar scale is 50 μm.

Article Snippet: CHO-K1 and HCC827 monoclonal cell lines stably expressing hGFP1-10 were made through limiting dilution by Genecopoeia (USA), and were maintained in Zeocin-supplemented parental cell line medium with 200 ug/ml and 250 ug/ml Zeocin, respectively.

Techniques: Cotransfection, Construct, Control, Sequencing, Plasmid Preparation, Fluorescence, Microscopy, Transfection, Staining

Journal: Scientific Reports

Article Title: GFP-complementation assay to detect functional CPP and protein delivery into living cells

doi: 10.1038/srep18329

Figure Lengend Snippet: ( 4a,b) CHO-K1 cells are co-transfected with hGFP1-10 and various Cargo-linker combinations. All_S11 fusions complemented hGFP1-10 to various extents, measured by increasing % fluorescent cells ( 4a ) or increasing MFI ( 4b ) in the viable single-cell population. In all experiments the “S11 only” control is a fusion of the [GSSG]x2 linker and S11 sequence, and does not generate a detectable GFP signal, nor do empty vector and “GFP1-10 only” controls (% GFP-positive cells <0.65 or MFI <25). Error bars represent standard error of the mean between technical replicates; data is representative of three independent experiments. ( 4c–f) Transfection experiments validating CPP components were repeated in a monoclonal stable cell line: HCC827 cells expressing hGFP1-10. HCC827/hGFP1-10 cells are transfected with different Cargo_S11 fusion constructs ( 4c,d ) or various Cargo-linker combinations ( 4e,f ). All Cargo_S11 proteins and various Cargo-linker combinations complemented hGFP1-10, as measured by increasing % GFP-positive cells ( 4c,e ) or increasing MFI ( 4d,f ) in the viable single-cell population and compared to background (no GFP1-10 and/or empty vector). In all experiments the “S11 only” control is expressed [GSSG]x2 linker_S11 fusion and does not generate a detectable increase in GFP signal, nor do empty vector and “GFP1-10 only” controls (% GFP-positive cells <5%, or MFI <110). Error bars represent standard error of the mean between technical replicates; data is representative of three independent experiments.

Article Snippet: CHO-K1 and HCC827 monoclonal cell lines stably expressing hGFP1-10 were made through limiting dilution by Genecopoeia (USA), and were maintained in Zeocin-supplemented parental cell line medium with 200 ug/ml and 250 ug/ml Zeocin, respectively.

Techniques: Transfection, Control, Sequencing, Plasmid Preparation, Stable Transfection, Expressing, Construct

Journal: Scientific Reports

Article Title: GFP-complementation assay to detect functional CPP and protein delivery into living cells

doi: 10.1038/srep18329

Figure Lengend Snippet: CPP_TRX_S11 proteins were added to CHO-K1 ( 5a,b ) and HCC827 ( 5c,d ) monoclonal cell lines stably expressing hGFP1-10. Protein titration shows dose-dependent uptake for TAT, Penetratin (PEN), Penetratin-Arginine (PenArg) and R9 fusions read as GFP complementation measured by both % fluorescent cells ( 5a,c ) and fold change in MFI ( 5b,d ). The seven other canonical CPPs showed only minimal GFP complementation signal at the highest doses. Control TRX_S11 fusion proteins “No CPP” and “PYC35” (a peptide with no CPP activity) show negligible effect on GFP complementation signal over cell-line background (“HisMBP”). Error bars represent standard error of the mean between technical replicates; data is representative of two independent experiments per cell line. (*R9 and PenArg fusions were not sufficiently concentrated for testing at 40 μM.)

Article Snippet: CHO-K1 and HCC827 monoclonal cell lines stably expressing hGFP1-10 were made through limiting dilution by Genecopoeia (USA), and were maintained in Zeocin-supplemented parental cell line medium with 200 ug/ml and 250 ug/ml Zeocin, respectively.

Techniques: Stable Transfection, Expressing, Titration, Control, Activity Assay

Journal: Scientific Reports

Article Title: GFP-complementation assay to detect functional CPP and protein delivery into living cells

doi: 10.1038/srep18329

Figure Lengend Snippet: Recombinant proteins are added to CHO-K1 and HCC827 cells transiently transfected 24-hours prior with hGFP1-10 and left overnight to recover and express the protein. GFP complementation was measured as both % fluorescent cells ( 8a ) and fold change in MFI ( 8b ). A dose-dependent fluorescent signal is detected for the TAT fusion protein compared to controls illustrating CPP-dependent internalization in transfected cells. “No CPP” control is the TRX_S11 protein with no CPP moiety added. “PYC35” control is an unrelated peptide (known to have no CPP activity) instead of a CPP moiety expressed as part of the fusion protein. Data is representative of more than 10 independent experiments. Error bars represent standard error of the mean between technical replicates.

Article Snippet: CHO-K1 and HCC827 monoclonal cell lines stably expressing hGFP1-10 were made through limiting dilution by Genecopoeia (USA), and were maintained in Zeocin-supplemented parental cell line medium with 200 ug/ml and 250 ug/ml Zeocin, respectively.

Techniques: Recombinant, Transfection, Control, Activity Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Quercetin Inhibits the Proliferation and Metastasis of Human Non-Small Cell Lung Cancer Cell Line: The Key Role of Src-Mediated Fibroblast Growth Factor-Inducible 14 (Fn14)/Nuclear Factor kappa B (NF-κB) pathway

doi: 10.12659/MSM.920537

Figure Lengend Snippet: Administration of Que suppressed the proliferation potential of HCC827 cells. For MTT assays, cells were incubated with Que of 25 μM (Que L), 50 μM (Que M), and 100 μM (Que H) for 96 hours. Every 24 hours, cells were subjected to the testing. For colony formation assays, cells were cultured in medium containing 100 μM Que for 2 weeks. ( A ) Detection results of MTT assays. ( B ) Detection results of colony formation assays. * P <0.05 versus the Control group.

Article Snippet: Human NSCLC cell lines HCC827 (7-1150) and NCI-H1650 (7-1031) were obtained from Chi Scientific (MA, USA).

Techniques: Incubation, Cell Culture, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Quercetin Inhibits the Proliferation and Metastasis of Human Non-Small Cell Lung Cancer Cell Line: The Key Role of Src-Mediated Fibroblast Growth Factor-Inducible 14 (Fn14)/Nuclear Factor kappa B (NF-κB) pathway

doi: 10.12659/MSM.920537

Figure Lengend Snippet: Administration of Que inhibited the invasion and migration potentials, and the signaling transduction of Src/Fn14/NF-κB pathway in HCC827 cells. For scratch assays, cells were incubated with Que of 100 μM (Que H) for 48 hours. For Transwell assays, cells were incubated with Que of 100 μM (Que H) for 24 hours. For western blotting assays, cells were incubated with Que of 100 μM (Que H) for 24 hours. ( A ) Detection results of scratch assays. ( B ) Detection results of Transwell assays. ( C ) Detection results of western blotting assays. * P <0.05 versus the Control group.

Article Snippet: Human NSCLC cell lines HCC827 (7-1150) and NCI-H1650 (7-1031) were obtained from Chi Scientific (MA, USA).

Techniques: Migration, Transduction, Incubation, Western Blot, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Quercetin Inhibits the Proliferation and Metastasis of Human Non-Small Cell Lung Cancer Cell Line: The Key Role of Src-Mediated Fibroblast Growth Factor-Inducible 14 (Fn14)/Nuclear Factor kappa B (NF-κB) pathway

doi: 10.12659/MSM.920537

Figure Lengend Snippet: Overexpression of Src blocked the anti-NSCLC function of Que in HCC827 cells. Cells transfected with negative control vector (NC) or Src expression vector (Src) were treated with Que of 100 μM (Que H) and subjected to MTT assay for 96 hours (cells were collected every 24 hours), and colony formation was assay at 2 weeks, Transwell assay at 24 hours, and scratch assay at 48 hours. ( A ) Detection results of Src level. ( B ) Detection results of MTT assays. ( C ) Detection results of colony formation assay. ( D ) Detection results of Transwell assay. ( E ) Detection results of scratch assay. # P <0.05 versus Que H group.

Article Snippet: Human NSCLC cell lines HCC827 (7-1150) and NCI-H1650 (7-1031) were obtained from Chi Scientific (MA, USA).

Techniques: Over Expression, Transfection, Negative Control, Plasmid Preparation, Expressing, MTT Assay, Transwell Assay, Wound Healing Assay, Colony Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Quercetin Inhibits the Proliferation and Metastasis of Human Non-Small Cell Lung Cancer Cell Line: The Key Role of Src-Mediated Fibroblast Growth Factor-Inducible 14 (Fn14)/Nuclear Factor kappa B (NF-κB) pathway

doi: 10.12659/MSM.920537

Figure Lengend Snippet: Que inhibited the growth and metastasis potential of NSCLC in vivo in by inhibiting Src signaling. Mice were injected with different HCC827 cells and administrated with Que of 100 mg/kg body weight for 3 weeks. ( A ) Detection results of tumor volume. ( B ) Detection results of hematoxylin and eosin detection of tumor tissue. ( C ) Detection results of western blotting detection of E-cadherin and N-cadherin. * P <0.05 versus Que H+Src group. Scale bar, 100 μm.

Article Snippet: Human NSCLC cell lines HCC827 (7-1150) and NCI-H1650 (7-1031) were obtained from Chi Scientific (MA, USA).

Techniques: In Vivo, Injection, Western Blot

Journal: Cancers

Article Title: Early On-Treatment Prediction of the Mechanisms of Acquired Resistance to EGFR Tyrosine Kinase Inhibitors

doi: 10.3390/cancers14061512

Figure Lengend Snippet: Selection of clones harboring resistant gene alteration in short term EGFR-TKI treatment. EGFR-TKI-resistant cells were spiked into parental EGFR-TKI-sensitive cells at the indicated percentages, and these cell mixtures were treated with DMSO, gefitinib (0.1 µM), for 48 h. ( A ) PC9GR cells were mixed with PC9 cells at the indicated percentages. ddPCR assays were performed to evaluate the allele frequency of EGFR T790M mutations. ( B ) HCC827GR cells were mixed with HCC827 cells at the indicated percentages. ddPCR assays were performed to evaluate the C-MET gene copy number. In all graphs, the error bars represent the mean ± SEM. Statistical analysis was performed using t -test. ** p ≤ 0.05; NS, not significant.

Article Snippet: HCC827 and PC9 cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea) and RIKEN BioResource Center cell bank (Ibaraki, Japan), respectively.

Techniques: Selection, Clone Assay

Journal: Cancers

Article Title: Early On-Treatment Prediction of the Mechanisms of Acquired Resistance to EGFR Tyrosine Kinase Inhibitors

doi: 10.3390/cancers14061512

Figure Lengend Snippet: Expression of specific gene alterations related to EGFR-TKI resistant mechanism in different cell lines. In the parental cell lines (PC9 and HCC827), ( A ) EGFR T790M mutation levels and ( B ) C-MET mRNA expression were measured after 48 h of treatment with DMSO, gefitinib (0.1 µM), or paclitaxel (0.1 µM). The allele frequency of EGFR T790M mutation was calculated using the ddPCR assay. C-MET mRNA expression was measured by RT-PCR. ( C ) PC9 and HCC827 cells were sorted according to the expression levels of annexin-V-FITC and MET-Alexa 647 after 48 h of treatment with DMSO, gefitinib (0.1 µM), or paclitaxel (0.1 µM). The copy number of C-MET gene in each group was measured using the ddPCR assay. ( D , E ) The amount of EGFR T790M mutation and C-MET expression was also measured using nanowire-based (NW) colorimetric assays. ( F , G ) In H4006 cells, the mRNA and protein expression levels of E-CADHERIN and VIMENTIN were measured by RT-PCR and Western blotting after 48 h of treatment with gefitinib (0.1 µM). In all graphs, the error bars represent the mean ± SEM. Statistical analysis was performed using t -test. ** p ≤ 0.05; NS, not significant.

Article Snippet: HCC827 and PC9 cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea) and RIKEN BioResource Center cell bank (Ibaraki, Japan), respectively.

Techniques: Expressing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Cancers

Article Title: Early On-Treatment Prediction of the Mechanisms of Acquired Resistance to EGFR Tyrosine Kinase Inhibitors

doi: 10.3390/cancers14061512

Figure Lengend Snippet: Expression of specific gene alterations related to EGFR-TKI resistant mechanism in vivo. ( A ) Schematic diagram of xenograft experiments. ( B , C ) EGFR T790M mutation changes in each sub-cutaneous PC9 or HCC827 xenograft tumor after gefitinib or paclitaxel treatment. The T790M mutation allele frequency was measured using the ddPCR assay. The dotted line indicates the threshold for EGFR T790M mutation detection (0.05%). ( D ) Change in the C-MET mRNA expression level in each subcutaneous PC9 or HCC827 xenograft tumor after gefitinib or paclitaxel treatment. C-MET mRNA expression was measured by RT-PCR. ( E ) The levels of EGFR T790M mutation and C-MET expression were measured using the nanowire-based (NW) colorimetric assay. Error bars represent the mean ± SEM. Statistical analysis was performed using t-test. ** p ≤ 0.05; NS, not significant.

Article Snippet: HCC827 and PC9 cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea) and RIKEN BioResource Center cell bank (Ibaraki, Japan), respectively.

Techniques: Expressing, In Vivo, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Colorimetric Assay

Journal: Frontiers in Oncology

Article Title: “Sandwich” Strategy to Intensify EGFR Blockade by Concurrent Tyrosine Kinase Inhibitor and Monoclonal Antibody Treatment in Highly Selected Patients

doi: 10.3389/fonc.2022.952939

Figure Lengend Snippet: Preclinical data evaluating the “sandwich” strategy.

Article Snippet: 2019 Hasegawa ( ) , Italy , Osimertinib+cetuximab , HCC827 (E746-A759del/T790M), H1975 (L858R/T790M), PC9-T790M (E746-A759del/T790M) cell lines and xenografts , tumor regressions and survival periods.

Techniques: Expressing