Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: Characterization of A549 and A549/DDP cells. (A) A549 and A549/DDP cells were exposed to different concentrations of DDP (1–32 µg/ml) for 24 h, and cell viability was determined by Cell Counting Kit-8 assay. The protein expression levels of (B) E-cadherin, vimentin, α-SMA and (C) CIP2A, p-AKT, AKT, p-mTOR were measured using western blotting; E, epithelial; SMA, smooth muscle actin; p-mTOR, phosphorylated mammalian target of rapamycin; AKT, protein kinase B; DDP, cisplatin; CIP2A, cancerous inhibitor of protein phosphatase 2A; IC 50 , half maximal inhibitory concentration.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Cell Counting, Expressing, Western Blot, Concentration Assay

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII induce cytotoxicity in A549 and A549/DDP cells. (A) Chemical structure of PPI and PPVII. (B) A549 and A549/DDP cells were treated with different dose of PPI (0.5–8 µg/ml) and PPVII (0.5–8 µg/ml) for 24 h, respectively. In A549 cells, the cell inhibition rate of 0.5 µg/ml PPI was significantly different from other concentration groups (**P<0.01); 1.0 µg/ml was significantly different from 4.0 and 8.0 µg/ml (**P<0.01), and 2.0 µg/ml was significantly different from 8.0 µg/ml (**P<0.01). As for A549/DDP cells, the cell inhibition rates of 0.5 µg/ml PPI and 1.0 µg/ml were both significantly different from other concentration groups ( ## P<0.01), and 2.0 µg/ml was significantly different from 8.0 µg/ml ( ## P<0.01). In A549 cells, there were significant differences in cell inhibition rates of PPVII among different concentration groups (**P<0.01); as for A549/DDP cells, there were significant differences among different concentration groups except for 4.0 and 8.0 µg/ml ( ## P<0.01). PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Inhibition, Concentration Assay

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII possess chemo-sensitizing effects on A549/DDP cells. (A) A549/DDP cells treated with indicated doses of DDP, or DDP plus either 0.375 µg/ml PPI or 0.75 µg/ml PPI, respectively for 24 h. (B) A549/DDP cells treated with indicated doses of DDP, or DDP plus either 0.375 µg/ml PPVII or 0.75 µg/ml PPVII, respectively for 24 h, cell viability was measured with Cell Counting Kit-8 assay. Data were presented as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001 vs. DDP treated group. # P<0.05 and ## P<0.01. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Cell Counting, Standard Deviation

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII reduce the IC 50 value of cisplatin in A549/DDP cells

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques:

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII enhance DDP-induced apoptosis. A549/DDP cells were treated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, respectively. Apoptosis of cells was detected by Annexin V-FITC/PI staining and flow cytometry. The apoptotic percentage is shown as a bar graph. Data represents the mean ± standard deviation of three independent experiments. **P<0.01 and ***P<0.001. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Staining, Flow Cytometry, Standard Deviation

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII enhance DDP-induced apoptosis through the P53 pathway and caspases-dependent pathway. A549/DDP cells were treated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, respectively. The protein expression levels of P53, Bax, Bcl-2 (A), PARP, C-PARP, pro-Caspase-3, C-Caspase-3 (B) were measured using western blotting. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; C-PARP, cleaved-poly (ADP-ribose) polymerase 1.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Expressing, Western Blot

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII reverse EMT and suppress the CIP2A/AKT/mTOR pathway. A549/DDP cells were incubated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, then cells were harvested for further western blotting analysis. (A) The protein expression levels of E-cadherin, vimentin, α-SMA in A549/DDP cells were measured. (B) The protein expression levels of CIP2A, p-AKT, AKT, p-mTOR, m-TOR were measured. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; p-AKT, phosphorylated-protein kinase B; mTOR, mammalian target of rapamycin; CIP2A, cancerous inhibitor of protein phosphatase 2A; SMA, smooth muscle actin; E, epithelial.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Incubation, Western Blot, Expressing

Journal: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: Two-component vector system where Cas9 is integrated into the AAVS1 safe harbor locus to sustain stable expression. The second component is a lentiviral system containing a sgRNA targeting on and off-target sites downstream of an AcrIIA4 variant that is uniquely barcoded. B. Editing efficiency measured over time within cells expressing wild-type AcrIIA4 (circular points) versus those without wild-type AcrIIA4 (rectangular). C. Indel mutation profiles spanning the on- and off-target regions. Deletions are shown in blue and insertions are in orange. D. Experimental design in which AcrIIA4 variants were delivered to A549 cells expressing Cas9, which were cultured for 10 days prior to collection and sequencing of the AcrobaTx barcode. E. Structure of the AcrobaTx barcode and definitions of gene editing activity metrics derived from measuring editing outcomes within the on- and off-target sites. F. Editing precision of alanine scanning variants normalized to the wild-type control. D69A variant is highlighted as the variant with the highest precision within the alanine scan mutant group. G. Crystal structure of AcrIIA4 bound to the SpCas9-gRNA complex. The interaction between D69 and R1335, required for PAM recognition, is highlighted. H. Distribution of editing efficiency ( EE , left) and editing precision ( EP , right) across all AcrIIA4 variants. Variants that resulted in EP in the top 90 th percentile are highlighted in red. I. Deletion profiles of DNA repair outcomes from cells without AcrIIA4 (blue) versus cells that express a variant that promotes precise editing (red). J. Editing precision measured across AcrIIA4 variants at Day 10 versus at Day 7 (Pearson Correlation Coefficient r = 0.56).

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Plasmid Preparation, Expressing, Variant Assay, Mutagenesis, Cell Culture, Sequencing, Activity Assay, Derivative Assay, Control

Journal: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: A. Experimental design in which the top 2000 AcrIIA4 variants, rank-ordered by EP , were delivered to A549 and 293T cells for another round of screening. B. Distribution of EP across all sample groups in the second round of screening versus the first round, at left. C. Frequency of editing within the AcrobaTx barcode across seven lead candidate enAcr groups. Yellow rectangles indicate areas in which editing occurs frequently while violet depicts areas with low editing activity. D. Ratio of on- to off-target editing in cells expressing seven representative lead candidate enAcrs (purple) versus ratios observed from DNA repair outcomes within cells expressing previously described benchmark AcrIIA4 variants.

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Activity Assay, Expressing