Journal: Frontiers in Nutrition

Article Title: Active fractions of golden-flowered tea ( Camellia nitidissima Chi ) inhibit epidermal growth factor receptor mutated non-small cell lung cancer via multiple pathways and targets in vitro and in vivo

doi: 10.3389/fnut.2022.1014414

Figure Lengend Snippet: Inhibition of proliferation and clonogenic ability of NSCLC cell lines by four active fractions of CNC. (A) Inhibitory effects of different concentrations of CLP, CLS, CFP, and CFS (6.25, 12.5, 25, 50 μg⋅mL –1 ) on NCI-H1975, HCC827 and A549 cells, respectively. (B) IC50 of different concentrations of CLP, CLS, CFP, and CFS (6.25, 8, 10, 12.5, 15, 20, 25, and 50 μg⋅mL –1 ) on NCI-H1975, HCC827, and A549 cells, respectively. (C) Inhibitory effects of different concentrations of CLS, CLP, CFS, and CFP (1.5625, 3.125, 6.25 μg⋅mL –1 ) on colony formation of NCI-H1975 cells, respectively. *Indicates p < 0.05, **indicates p < 0.01 and *** indicates p < 0.001 relative to the control by ANOVA. The data are presented as the mean ± standard deviation ( n = 3).

Article Snippet: NCI-H1975 cells were incubated in different concentration of CLS for 24 h, the cells were cultured with 1 μM DCFH 2 -DA (Solarbio, D6470) for 30 min at 37°C.

Techniques: Inhibition, Control, Standard Deviation

Journal: Frontiers in Nutrition

Article Title: Active fractions of golden-flowered tea ( Camellia nitidissima Chi ) inhibit epidermal growth factor receptor mutated non-small cell lung cancer via multiple pathways and targets in vitro and in vivo

doi: 10.3389/fnut.2022.1014414

Figure Lengend Snippet: Inhibition of proliferation rate of NCI-H1975 cells by four active fractions of CNC. (A–D) Representative field of view of immunofluorescence assay with anti-EdU antibody is shown, where in NCI-H1975 cells, the signal of incorporating with EdU was green and the nucleus was stained blue by Hoechst. Histograms showed the mean values of cell proliferation rates after administration of different concentrations of CLS, CLP, CFS, and CFP (6.25, 12.5, 25 μg⋅mL –1 ). *Indicates p < 0.05, **indicates p < 0.01 and ***indicates p < 0.001 relative to the control by ANOVA. The data are presented as the mean ± standard deviation ( n = 3).

Article Snippet: NCI-H1975 cells were incubated in different concentration of CLS for 24 h, the cells were cultured with 1 μM DCFH 2 -DA (Solarbio, D6470) for 30 min at 37°C.

Techniques: Inhibition, Immunofluorescence, Staining, Control, Standard Deviation

Journal: Frontiers in Nutrition

Article Title: Active fractions of golden-flowered tea ( Camellia nitidissima Chi ) inhibit epidermal growth factor receptor mutated non-small cell lung cancer via multiple pathways and targets in vitro and in vivo

doi: 10.3389/fnut.2022.1014414

Figure Lengend Snippet: CLS induced programmed NSCLC death through pyroptosis. (A) TdT-mediated dUTP Nick-End Labeling (TUNEL) positive rate of NCI-H1975 cells after administration of different concentrations of CLS (6.25, 12.5, 25 μg⋅mL –1 ). (B) Reactive oxygen species (ROS) positive rate of NCI-H1975 cells after administration of different concentrations of CLS (6.25, 12.5, 25 μg⋅mL –1 ). (C) Annexin V-FITC/propidine iodide (PI) staining of NCI-H1975 cells after 48 h was performed to identify early/late apoptosis, and the data were analyzed via flow cytometry. (D) SEM was used to detect the morphological changes of NCI-H1975 cells. (E,F) Relative uperoxide dismutase (SOD) activity and relative lactate dehydrogenase (LDH) activity of NCI-H1975 cells after administration of different concentrations of CLS (6.25, 12.5, 25 μg⋅mL –1 ). (G) Relative IL-1β activity and relative LDH activity of NCI-H1975 cells after administration of different concentrations of CLS (6.25, 12.5, 25 μg⋅mL –1 ). *Indicates p < 0.05, **indicates p < 0.01 and ***indicates p < 0.001 relative to the control by ANOVA. The data are presented as the mean ± standard deviation ( n = 3).

Article Snippet: NCI-H1975 cells were incubated in different concentration of CLS for 24 h, the cells were cultured with 1 μM DCFH 2 -DA (Solarbio, D6470) for 30 min at 37°C.

Techniques: End Labeling, TUNEL Assay, Staining, Flow Cytometry, Activity Assay, Control, Standard Deviation

Journal: Frontiers in Nutrition

Article Title: Active fractions of golden-flowered tea ( Camellia nitidissima Chi ) inhibit epidermal growth factor receptor mutated non-small cell lung cancer via multiple pathways and targets in vitro and in vivo

doi: 10.3389/fnut.2022.1014414

Figure Lengend Snippet: Transcriptome analysis of NCI-H1975 cells between the control group and CLS-treated group. (A) Volcano plot with 1,077 DEGs up-regulated and 1,008 DEGs down-regulated. (B) GO enrichment analysis of up-regulated and down-regulated DEGs. (C) KEGG enrichment analysis of up-regulated and down-regulated DEGs. (D) The expression heatmap of 22 DEGs related to Cytokine-cytokine receptor interaction and Rheumatoid arthritis signaling pathway in each sequencing sample. (E) The expression heatmap of 23 DEGs related to PIK-Akt signaling pathway in each sequencing sample. (F) The expression heatmap of 16 DEGs related to MAPK signaling pathway in each sequencing sample. (H,I) Interaction relationships of DEGs on four signaling pathways, including TGF-β signaling pathway, TNF signaling pathway, PIK-Akt signaling pathway and MAPK signaling pathway.

Article Snippet: NCI-H1975 cells were incubated in different concentration of CLS for 24 h, the cells were cultured with 1 μM DCFH 2 -DA (Solarbio, D6470) for 30 min at 37°C.

Techniques: Control, Expressing, Sequencing, Protein-Protein interactions

Journal: Frontiers in Nutrition

Article Title: Active fractions of golden-flowered tea ( Camellia nitidissima Chi ) inhibit epidermal growth factor receptor mutated non-small cell lung cancer via multiple pathways and targets in vitro and in vivo

doi: 10.3389/fnut.2022.1014414

Figure Lengend Snippet: Comparative analysis of quantitative real-time PCR (RT-qPCR) data and Western Blot (WB) data. (A) Relative mRNA levels of nine genes including TGFB2, INHBB, TNFRSF10C, PIK3R3, ITGB8, EIF4E1B, NTRK, CACN1D, MEF2C. (B) Protein levels of INHBB, PIK3R3, TGFB2, ITGB8, TrkB, CACNA1D detected in CLS-treated NCI-H1975 cells were detected by Western blot with quantification of each protein, n = 3 biologically independent embryos. *Indicates p < 0.05, **indicates p < 0.01 and ***indicates p < 0.001 relative to the control by ANOVA. The data are presented as the mean ± standard deviation ( n = 3).

Article Snippet: NCI-H1975 cells were incubated in different concentration of CLS for 24 h, the cells were cultured with 1 μM DCFH 2 -DA (Solarbio, D6470) for 30 min at 37°C.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Control, Standard Deviation

Journal: British Journal of Pharmacology

Article Title: Luteolin is effective in the non-small cell lung cancer model with L858R/T790M EGF receptor mutation and erlotinib resistance

doi: 10.1111/bph.12610

Figure Lengend Snippet: Luteolin reduces the viability of NCI-H1975 cells. (A and B) A549, HCC827 and NCI-H1975 cells were treated with a range of concentrations of erlotinib (A) or luteolin (B) for 48 h. Cell viability is shown as relative viability compared to the untreated control. (C) NCI-H1975 cells were treated with luteolin at different concentrations for 48 h. Cell morphology was imaged under a Nikon microscope (200×). (D) L02, H9c2 and HEK293 cells were treated with the indicated concentrations of luteolin for 48 h. Cell viability was expressed as % of surviving cells compared to the control group. Each bar represents the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01.

Article Snippet: Confocal microscopy NCI-H1975 cells were seeded onto coverslips (BD Biosciences, San Jose, CA, USA) and then were treated with luteolin (50 μmol·L −1 ) or 17-DMAG (1 μmol·L −1 ) for 24 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100.

Techniques: Microscopy

Journal: British Journal of Pharmacology

Article Title: Luteolin is effective in the non-small cell lung cancer model with L858R/T790M EGF receptor mutation and erlotinib resistance

doi: 10.1111/bph.12610

Figure Lengend Snippet: Luteolin induces apoptosis of NCI-H1975 cells. (A and B) NCI-H1975 cells were treated with luteolin 0–100 μmol·L−1 for 24 h. Cell lysates were prepared and subjected to immunoblotting with anti-PARP, caspase-3 (A), Bcl-2 and BAX (B) antibodies respectively. β-Actin was used as an internal control. (C and D) NCI-H1975 cells were treated with the indicated concentrations of luteolin (C) or erlotinib (D) for 48 h. The apoptotic cells were assessed by annexin V-FITC/PI detection. The four quadrants denote non-apoptotic live cells (red), early apoptotic stage cells (blue), late apoptotic cells (green) and dead cells (purple). Similar results were obtained from three experiments.

Article Snippet: Confocal microscopy NCI-H1975 cells were seeded onto coverslips (BD Biosciences, San Jose, CA, USA) and then were treated with luteolin (50 μmol·L −1 ) or 17-DMAG (1 μmol·L −1 ) for 24 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100.

Techniques: Western Blot

Journal: British Journal of Pharmacology

Article Title: Luteolin is effective in the non-small cell lung cancer model with L858R/T790M EGF receptor mutation and erlotinib resistance

doi: 10.1111/bph.12610

Figure Lengend Snippet: Luteolin down-regulates the mutant EGF receptor protein. (A and B) NCI-H1975 cells were treated with the indicated concentrations of luteolin (A) or erlotinib (B) for 24 h. Cell lysates were prepared and subjected to immunoblotting to measure the protein levels of EGF receptors and phospho-EGF receptors (Y1068) with respective antibodies and equal protein loading was confirmed by β-actin. (C) NCI-H820 cells were treated with the indicated concentrations of luteolin for 24 h. Cell lysates were prepared and subjected to immunoblotting with anti-EGF receptor and β-actin. (D) NCI-H1975 cells were treated with luteolin (50 μmol·L−1) or 17-DMAG (1 μmol·L−1) for 24 h. Cells were then fixed, permeabilized and incubated with anti-EGF receptor monoclonal primary antibody, and then stained with Texas Red conjugated goat anti-mouse IgG. Nuclei were stained with DAPI. Cells were observed under a confocal microscope (red: EGF receptor, blue: nuclei, Scale bar: 10 μm). (E) NCI-H1975 cells were treated with luteolin (0–100 μmol·L−1) for 24 h. Total RNA was isolated and EGF receptor mRNA was detected by RT-PCR. GAPDH was used as a control. (F) ErbB2 (HER2) protein levels were measured by immunoblotting after different concentrations of luteolin treatment for 24 h. Results shown are representative of three experiments.

Article Snippet: Confocal microscopy NCI-H1975 cells were seeded onto coverslips (BD Biosciences, San Jose, CA, USA) and then were treated with luteolin (50 μmol·L −1 ) or 17-DMAG (1 μmol·L −1 ) for 24 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100.

Techniques: Mutagenesis, Western Blot, Incubation, Staining, Microscopy, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: British Journal of Pharmacology

Article Title: Luteolin is effective in the non-small cell lung cancer model with L858R/T790M EGF receptor mutation and erlotinib resistance

doi: 10.1111/bph.12610

Figure Lengend Snippet: Luteolin promotes lysosome-dependent degradation of the EGF by inhibiting its interaction with Hsp90. (A) NCI-H1975 cells were cultured with 1 μg·mL−1 CHX in the presence or absence of 50 μmol·L−1 luteolin for the indicated time. EGF receptor and β-actin protein levels were assessed by immunoblot analysis. (B) NCI-H1975 cells were co-treated with 50 μmol·L−1 luteolin and NH4Cl (10 mmol·L−1) or MG132 (5 μmol·L−1) for 24 h. EGF receptor protein level was detected by immunoblotting. (C) NCI-H1975 cells were co-treated with 50 μmol·L−1 of luteolin and MG132 for 0–8 h. Cell lysates were subjected to immunoprecipitation with anti-EGF receptor antibody and analysed by immunoblotting with anti-ubiquitination antibody or anti-EGF receptor antibody. Results shown are representative of three experiments. (D) NCI-H1975 cells were treated with 50 μmol·L−1 luteolin for 24 h in presence of NH4Cl to block the lysosomal degradation of the EGF receptor. Cell lysates were subjected to immunoprecipitation with anti-EGF receptor antibody and analysed by immunoblotting with anti-Hsp90 antibody or anti-EGF receptor antibody.

Article Snippet: Confocal microscopy NCI-H1975 cells were seeded onto coverslips (BD Biosciences, San Jose, CA, USA) and then were treated with luteolin (50 μmol·L −1 ) or 17-DMAG (1 μmol·L −1 ) for 24 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100.

Techniques: Cell Culture, Western Blot, Immunoprecipitation, Blocking Assay

Journal: British Journal of Pharmacology

Article Title: Luteolin is effective in the non-small cell lung cancer model with L858R/T790M EGF receptor mutation and erlotinib resistance

doi: 10.1111/bph.12610

Figure Lengend Snippet: Luteolin prevents EGF receptor/PI3K/Akt/mTOR signalling in NCI-H1975 and inhibits cell migration. (A and B) Exponentially growing NCI-H1975 cells were treated with the indicated concentrations of luteolin for 24 h, and lysates were subjected to immunoblotting with the indicated antibodies. (C) HCC827 and NCI-H1975 cells were treated with a range of concentrations of erlotinib for 24 h. Cell lysates were subjected to immunoblotting by using the antibody to Akt, ERK1/2, p-Akt and p-ERK1/2. Equal loading of protein was confirmed by β-actin. (D) Migration of NCI-H1975 cells was evaluated using the scratch wound-healing assay in the absence and presence of 50 μmol·L−1 luteolin. Images of the results were obtained using a Nikon microscope (200×). (E) NCI-H1975 cells were stained with FITC-phalloidin, a substance that specifically detects F-actin. In the control group, stress fibres are present (white arrows). Results shown are representative of three experiments. Scale bar: 10 μm.

Article Snippet: Confocal microscopy NCI-H1975 cells were seeded onto coverslips (BD Biosciences, San Jose, CA, USA) and then were treated with luteolin (50 μmol·L −1 ) or 17-DMAG (1 μmol·L −1 ) for 24 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100.

Techniques: Migration, Western Blot, Wound Healing Assay, Microscopy, Staining

Journal: Journal of Cancer

Article Title: Antitumor activity of histone deacetylase inhibitor chidamide alone or in combination with epidermal growth factor receptor tyrosine kinase inhibitor icotinib in NSCLC

doi: 10.7150/jca.28570

Figure Lengend Snippet: The IC 50 values of chidamide and icotinib in NSCLC cell lines

Article Snippet: One × 10 7 HCC827IR and H1975 cells were re-suspended in 100μL of serum-free RPMI 1640 medium and inoculated into the subcutaneous tissue of the right flank of 4-week-old female Bagg Albino (BALB/c) athymic nude mice (Beijing HFK Bioscience Co., Ltd, China).

Techniques: Mutagenesis

Journal: Journal of Cancer

Article Title: Antitumor activity of histone deacetylase inhibitor chidamide alone or in combination with epidermal growth factor receptor tyrosine kinase inhibitor icotinib in NSCLC

doi: 10.7150/jca.28570

Figure Lengend Snippet: Effects of chidamide or combined with icotinib on the apoptosis in NSCLC cells. A, percentage of Annexin V-/PI- cells after treatment of serial dilutions of chidamide in HCC827, HCC827IR, A549 and H1975 cells (48h); B-E, expression and activation level of Caspase-3 and PARP after treatment of serial dilutions of chidamide in HCC827, HCC827IR, A549 and H1975 cells (48h); F, percentage of Annexin V-/PI- cells after treatment of chidamide or combined with icotinib in H1975 cells (48h). G, expression and activation level of Caspase-3 and PARP after treatment of chidamide or combined with icotinb in H1975 cells (48h). Experiment was repeated at least thrice. Control: DMSO.

Article Snippet: One × 10 7 HCC827IR and H1975 cells were re-suspended in 100μL of serum-free RPMI 1640 medium and inoculated into the subcutaneous tissue of the right flank of 4-week-old female Bagg Albino (BALB/c) athymic nude mice (Beijing HFK Bioscience Co., Ltd, China).

Techniques: Expressing, Activation Assay, Control

Journal: Journal of Cancer

Article Title: Antitumor activity of histone deacetylase inhibitor chidamide alone or in combination with epidermal growth factor receptor tyrosine kinase inhibitor icotinib in NSCLC

doi: 10.7150/jca.28570

Figure Lengend Snippet: Effects of chidamide or combined with icotinib on the receptor tyrosine kinase (PTK) signaling molecules in NSCLC cells. A-D, chidamide inhibition of PTK signaling molecules in HCC827, HCC827IR, A549 and H1975 cells (48h). E, chidamide or combined with icotinib inhibition of PTK signaling molecules in H1975 cells (48h). Experiment was repeated at least thrice. Control: DMSO. GAPDH was used as a loading control.

Article Snippet: One × 10 7 HCC827IR and H1975 cells were re-suspended in 100μL of serum-free RPMI 1640 medium and inoculated into the subcutaneous tissue of the right flank of 4-week-old female Bagg Albino (BALB/c) athymic nude mice (Beijing HFK Bioscience Co., Ltd, China).

Techniques: Inhibition, Control

Journal: Journal of Cancer

Article Title: Antitumor activity of histone deacetylase inhibitor chidamide alone or in combination with epidermal growth factor receptor tyrosine kinase inhibitor icotinib in NSCLC

doi: 10.7150/jca.28570

Figure Lengend Snippet: Effect of chidamide or combined with icotinib on E-cadherin and β-catenin expression in NSCLC cells. A, expression level of E-cadherin protein in 10 NSCLC cell lines; B, relative expression level of E-cadherin mRNA in 10 NSCLC cell lines (relative to GAPDH); C-F, expression of E-cadherin and β-catenin protein in HCC827, HCC827IR, A549 and H1975 cells after treatment of serial dilutions of chidamide (48h); G, expression of E-cadherin mRNA in H1975 cells after treatment of serial dilutions of chidamide (24h); H, expression of E-cadherin and β-catenin protein in H1975 cells after treatment of chidamide or combined with icotinib (48h). Experiment was repeated at least thrice. Control: DMSO.

Article Snippet: One × 10 7 HCC827IR and H1975 cells were re-suspended in 100μL of serum-free RPMI 1640 medium and inoculated into the subcutaneous tissue of the right flank of 4-week-old female Bagg Albino (BALB/c) athymic nude mice (Beijing HFK Bioscience Co., Ltd, China).

Techniques: Expressing, Control

Journal: Journal of Cancer

Article Title: Antitumor activity of histone deacetylase inhibitor chidamide alone or in combination with epidermal growth factor receptor tyrosine kinase inhibitor icotinib in NSCLC

doi: 10.7150/jca.28570

Figure Lengend Snippet: Antitumor activity in vivo and putative mechanisms of chidamide or in combination with icotinib in NSCLC. A and C, tumor growth curves showed that chidamide could significantly inhibit tumor growth in HCC827IR xenograft models compared to vehicle and icotinib; and chidamide plus icotinib could significantly inhibit tumor growth in H1975 xenograft models compared to vehicle, icotinib and chidamide, respectively; B and D, nude mice well tolerated the treatment; E, schematic illustration of the antitumor mechanisms of chidamide or combinated with icotinib in NSCLC. *** P <0.001.

Article Snippet: One × 10 7 HCC827IR and H1975 cells were re-suspended in 100μL of serum-free RPMI 1640 medium and inoculated into the subcutaneous tissue of the right flank of 4-week-old female Bagg Albino (BALB/c) athymic nude mice (Beijing HFK Bioscience Co., Ltd, China).

Techniques: Activity Assay, In Vivo

Journal: Clinical and Translational Medicine

Article Title: Tumor‐derived exosomal miR‐19b‐3p facilitates M2 macrophage polarization and exosomal LINC00273 secretion to promote lung adenocarcinoma metastasis via Hippo pathway

doi: 10.1002/ctm2.478

Figure Lengend Snippet: LUAD cells induces M2 macrophage polarization by secreting exosomes. (A) Schematic illustration of the co‐culturing model for macrophages and LUAD cells using a transwell chamber. After PMA induction, THP1 (Mφ) were mono‐cultured (NC) or co‐cultured with untreated LUAD cells (A549 or H1975), LUAD cells treated with DMSO (A549/DMSO or H1975/DMSO), or LUAD cells treated with 5 μM of exosome secretion inhibitor GW4869 (A549/GW4869 or H1975/GW4869). (B) qRT‐PCR was adopted to detect the levels of M2 markers (ARG1, CD206, TGFβ and IL10) in THP1 (Mφ) in each group of co‐culturing system. (C) Flow cytometry was applied to measure macrophage marker (CD11b) and M2 macrophage‐related phenotypic marker (CD206) on the surfaces of macrophages in each group. The ratio of CD206‐positive plus CD11b‐positive (CD206+CD11b+) macrophages of each group were quantitated. (D) TEM images and NTA analysis results of A549‐Exo or H1975‐Exo. Scale bar: 100 nm. (E) Western blot of exosome markers CD9 and CD81 in extracted exosomes from LUAD cells treated without or with DMSO or GW4869. (F) Fluorescence microscopy images illustrated the process of LUAD cell‐derived exosomes transmitted to macrophages. A549 and H1975 cell‐derived exosomes were labeled by DiO (green). TRITC‐Phalloidin (red) was adopted for labeling cytoskeleton of THP1 (Mφ) cells. Scale bar: 10μm. (G) Flow cytometry was applied to measure CD206+CD11+ macrophages in each group without (NC) or with LUAD cell‐derived exosome treatment. THP1 (Mφ), macrophages induced from THP1 cells. A549‐Exo or H1975‐Exo, exosomes derived from A549 or H1975 cells. Data are presented as mean ± SD of at least three independent experiments. **p < 0.01

Article Snippet: Human LUAD cells H1975 were bought from the European Collection of Authenticated Cell Cultures (ECACC; Salisbury, UK).

Techniques: Cell Culture, Quantitative RT-PCR, Flow Cytometry, Marker, Western Blot, Fluorescence, Microscopy, Derivative Assay, Labeling

Journal: Clinical and Translational Medicine

Article Title: Tumor‐derived exosomal miR‐19b‐3p facilitates M2 macrophage polarization and exosomal LINC00273 secretion to promote lung adenocarcinoma metastasis via Hippo pathway

doi: 10.1002/ctm2.478

Figure Lengend Snippet: miR‐19b‐3p knockdown in LUAD cells inhibits M2 polarization of co‐cultured macrophages. (A) Schematic illustration of the co‐culturing model for macrophages and LUAD cells transfected with Cy3‐labeled miR‐19b‐3p. (B) Fluorescence microscopy images of Cy3‐miR‐19b‐3p fluorescence in THP1 (Mφ) cells cultured without (Control) or with A549 LUAD cells transfected with Cy3‐miR‐19b‐3p. FITC‐Phalloidin (green) and Cy3 (red) were adopted for labeling cytoskeleton and miR‐19b‐3p, respectively. Scale bar: 10μm. (C) THP1 (Mφ) cells were co‐cultured with untreated LUAD cells (A549 or H1975) or LUAD cells transfected with NC inhibitors (A549/NC inhibitors or H1975/NC inhibitors) or miR‐19b‐3p inhibitors (A549/miR‐19b‐3p inhibitors or H1975/miR‐19b‐3p inhibitors), or without co‐culturing (NC). qRT‐PCR was adopted to detect the levels of M2 markers in THP1 (Mφ) cells of each group of co‐culturing system. (D) Flow cytometry was applied to measure CD206 +CD11b + macrophages in each group of co‐culturing system. THP1 (Mφ), macrophages induced from THP1 cells. Data are presented as mean ± SD of at least three independent experiments. **p < 0.01

Article Snippet: Human LUAD cells H1975 were bought from the European Collection of Authenticated Cell Cultures (ECACC; Salisbury, UK).

Techniques: Knockdown, Cell Culture, Transfection, Labeling, Fluorescence, Microscopy, Control, Quantitative RT-PCR, Flow Cytometry

Journal: Clinical and Translational Medicine

Article Title: Tumor‐derived exosomal miR‐19b‐3p facilitates M2 macrophage polarization and exosomal LINC00273 secretion to promote lung adenocarcinoma metastasis via Hippo pathway

doi: 10.1002/ctm2.478

Figure Lengend Snippet: Exosomes from miR‐19b‐3p‐induced macrophages regulates Hippo signaling pathway by inducing LATS2 ubiquitination in LUAD cells. (A) A549 and H1975 cells were cultured with the CM from THP1 (Mφ) treated with NC mimics, miR‐19b‐3p mimics, miR‐19b‐3p mimics + DMSO, or miR‐19b‐3p mimics + GW4869. miR‐19b‐3p level in LUAD cell‐derived exosomes of each group was detected using qRT‐PCR. (B) A549 and H1975 cells were treated with THP1 (Mφ)/NC mimics‐Exo or THP1 (Mφ)/miR‐19b‐3p mimics‐Exo. miR‐19b‐3p level in LUAD cell‐derived exosomes of each group was detected using qRT‐PCR. (C) A series of qRT‐PCR analyses illustrated the fold changes of exosomal miR‐19b‐3p levels from A549 and H1975 cells treated with various small‐molecule inhibitors. DMSO treatment was regarded as a control. (D) Luciferase reporter assay detected YAP signaling in A549 and H1975 cells treated with THP1 (Mφ)/NC mimics‐Exo or THP1 (Mφ)/miR‐19b‐3p mimics‐Exo using 8xGTIIC‐luciferase YAP/TAZ reporters. (E) The effects of rising dose of THP1 (Mφ)/miR‐19b‐3p mimics‐Exo on the levels of p‐YAP, YAP, LATS2 and MST1 in LUAD cells were evaluated using western blot. (F) The effects of rising dose of THP1 (Mφ)/miR‐19b‐3p mimics‐Exo on the levels of nuclear or cytoplasmic YAP in LUAD cells were evaluated using western blot. (G) The effects of rising dose of THP1 (Mφ)/miR‐19b‐3p mimics‐Exo on the levels of YAP downstream genes in LUAD cells were evaluated using western blot. (H) CHX chase assay evaluated the stability of LATS2 protein in LUAD cells cultured with the exosomes of each group. Western blot was used to detect LATS2 expression in LUAD cells of each group after CHX treatment at different time points. (I) Ubiquitination assay evaluated the ubiquitination level of LATS2 protein in LUAD cells cultured with the exosomes of each group. THP1 (Mφ), macrophages induced from THP1 cells. THP1 (Mφ)/NC mimics‐Exo or THP1 (Mφ)/miR‐19b‐3p mimics‐Exo, exosomes derived from THP1 (Mφ) transfected with NC mimics or miR‐19b‐3p mimics. Data are presented as mean ± SD of at least three independent experiments. **p < 0.01

Article Snippet: Human LUAD cells H1975 were bought from the European Collection of Authenticated Cell Cultures (ECACC; Salisbury, UK).

Techniques: Ubiquitin Proteomics, Cell Culture, Derivative Assay, Quantitative RT-PCR, Control, Luciferase, Reporter Assay, Western Blot, Expressing, Transfection

Journal: Clinical and Translational Medicine

Article Title: Tumor‐derived exosomal miR‐19b‐3p facilitates M2 macrophage polarization and exosomal LINC00273 secretion to promote lung adenocarcinoma metastasis via Hippo pathway

doi: 10.1002/ctm2.478

Figure Lengend Snippet: Macrophage‐transferred exosomal LINC00273 regulates Hippo signaling pathway by recruiting NEDD4 in LUAD cells. (A) Twenty‐four highly expressed lncRNAs (log 2 FC > 1) in lung cancer were searched out from BBCancer database. After the transfection of NC mimics or miR‐19b‐3p mimics, exosomal lncRNA levels from THP1 (Mφ) were detected, and 7 significantly up‐regulated lncRNAs by miR‐19b‐3p mimics were selected. (B) A series of luciferase reporter assay illustrated the fold change of YAP signaling in A549 and H1975 cells were treated with THP1 (Mφ)‐derived exosomes and were subjected to the knockdown of each lncRNA selected above or sh‐NC. (C) THP1 (Mφ) cells were transfected with Cy3‐LINC00273 (red) and the exosomes derived from the transfected THP1 (Mφ) cells were added to the medium of LUAD cells for incubation. Fluorescence microscopy images illustrated the transportation of THP1 (Mφ)‐derived exosomal LINC00273 to LUAD cells. FITC Phalloidin (green) and Cy3 (red) were adopted for labeling cytoskeleton of LUAD cells and LINC00273, respectively. Scale bar: 10μm. (D) LATS2, p‐YAP and YAP levels in A549 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273 were evaluated using western blot. (E) Nuclear and cytoplasmic YAP levels in A549 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273 were evaluated using western blot. (F) The levels of YAP downstream genes in A549 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273 were evaluated using western blot. (G) CHX chase assay evaluated the stability of LATS2 protein in A549 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273. (H) Ubiquitination assay evaluated the ubiquitination level of LATS2 protein in A549 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273. (I) Proteins recruited by LINC00273 in A549 cells were assessed by mass spectrometric analyses following RNA pull‐down assay. The protein band corresponding to NEDD4 was indicated in the image of SDS‐PAGE gel. (J) The pull‐down enrichment product of Bio‐LINC00273‐Sense or Antisense probe was subjected to western blot to determine the existence of NEDD4. (K) The binding capacity between NEDD4 and LINC00273 was confirmed by RIP assay in A549 cells with LINC00273 overexpression. U1 RNA co‐immunoprecipitated with SNRNP70 was adopted as the positive control. (L) The combination of immunofluorescence and FISH illustrated the overlapped distribution of NEDD4 (green) and LINC00273 (red) in cytoplasm of A549 cells with LINC00273 overexpression. Scale bar: 10μm. (M) Co‐IP assay detected the level of LATS2 or NEDD4 co‐immunoprecipitated with anti‐NEDD4 (anti‐IgG as negative control) in A549 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273. THP1 (Mφ), macrophages induced from THP1 cells. Data are presented as mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Human LUAD cells H1975 were bought from the European Collection of Authenticated Cell Cultures (ECACC; Salisbury, UK).

Techniques: Transfection, Luciferase, Reporter Assay, Derivative Assay, Knockdown, Incubation, Fluorescence, Microscopy, Labeling, Western Blot, Ubiquitin Proteomics, Pull Down Assay, SDS Page, Binding Assay, Over Expression, Immunoprecipitation, Positive Control, Immunofluorescence, Co-Immunoprecipitation Assay, Negative Control

Journal: Clinical and Translational Medicine

Article Title: Tumor‐derived exosomal miR‐19b‐3p facilitates M2 macrophage polarization and exosomal LINC00273 secretion to promote lung adenocarcinoma metastasis via Hippo pathway

doi: 10.1002/ctm2.478

Figure Lengend Snippet: LINC00273 facilitates exosomal miR‐19b‐3p secretion via up‐regulating RBMX. (A) Exosomes were extracted from A549 cells transfected with Cy3‐miR‐19b‐3p and pcDNA3.1 or pcDNA3.1/LINC00273. Fluorescence microscopy images detected the Cy3‐miR‐19b‐3p fluorescence in THP1 (Mφ) cells incubated with A549‐derived exosomes of each group. FITC‐ Phalloidin (green) and Cy3 (red) were adopted for labeling cytoskeleton and miR‐19b‐3p, respectively. The percentages of Cy3‐positive macrophages were measured after certain hours of co‐culturing. Scale bar: 10 μm. (B) qRT‐PCR assessed miR‐19b‐3p level in A549 or H1975 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273. (C) qRT‐PCR assessed exosomal miR‐19b‐3p level from A549 or H1975 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273. (D) qRT‐PCR following RNA pull‐down assay evaluated the binding between miR‐19b‐3p and Bio‐LINC00273‐Sense or Antisense probe. (E) RBMX, the putative RBP for miR‐19b‐3p, was predicted by RBPDB. The nucleotides highlighted in green stand for the presumed binding site for RBMX in miR‐19b‐3p sequence or mutant binding site designed for RNA pull‐down assay. (F) Western blot following RNA pull‐down assay evaluated the enrichment of RBMX in the pulldown product of Bio‐NC, Bio‐miR‐19b‐3p‐WT or Bio‐miR‐19b‐3p‐Mut probe in LUAD cells or their exosomes. (G) RIP assay detected the relative input recovery of miR‐19b‐3p with anti‐RBMX or anti‐IgG in LUAD cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273 or their exosomes. (H) qRT‐PCR assessed RBMX level in A549 or H1975 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273. (I) Western blot assessed exosomal RBMX and RBMX expression of LUAD cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273. (J) Exosomes were extracted from A549 cells transfected with sh‐NC or sh‐RBMX#1. Fluorescence microscopy images illustrates the Cy3 fluorescence (red) in THP1 (Mφ) cells incubated with A549‐derived exosomes of each group. The percentages of Cy3‐positive macrophages were measured after certain hours. Scale bar: 10 μm. THP1 (Mφ), macrophages induced from THP1 cells. A549‐Exo or H1975‐Exo, exosomes derived from A549 or H1975 cells. Data are presented as mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., no significance

Article Snippet: Human LUAD cells H1975 were bought from the European Collection of Authenticated Cell Cultures (ECACC; Salisbury, UK).

Techniques: Transfection, Fluorescence, Microscopy, Incubation, Derivative Assay, Labeling, Quantitative RT-PCR, Pull Down Assay, Binding Assay, Sequencing, Mutagenesis, Western Blot, Expressing

Journal: Journal of Translational Medicine

Article Title: Potential benefit of osismertinib plus bevacizumab in leptomeningeal metastasis with EGFR mutant non-small-cell lung cancer

doi: 10.1186/s12967-022-03331-9

Figure Lengend Snippet: Construction of LM model and short-term oral absorption test in vivo. A Flow chart of LM model. H1975-luc cells were injected through the right ventricle. Representative images of B IVIS imaging, C MRI at week 2 after injection tumor cells, and E the leptomeningeal tissue of normal group and LM group was compared by H&E staining. D Concentration of osimertinib in the mouse brain tissue in 1,6,12,24 h after single dose of osimertinib with or without bevacizumab (n = 3, * means p < 0.05, *** means p < 0.001)

Article Snippet: H1975 cells (EGFR exon20 T790M and exon21 L858R) were purchased from Pro-cell in 2020 (Wuhan, China, was identified by STR) and maintained in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum and 1% antibiotic solution in a humidified incubator with 5% CO 2 .

Techniques: In Vivo, Injection, Imaging, Staining, Concentration Assay

Journal: Journal of Translational Medicine

Article Title: Potential benefit of osismertinib plus bevacizumab in leptomeningeal metastasis with EGFR mutant non-small-cell lung cancer

doi: 10.1186/s12967-022-03331-9

Figure Lengend Snippet: Patient characteristics (n = 27)

Article Snippet: H1975 cells (EGFR exon20 T790M and exon21 L858R) were purchased from Pro-cell in 2020 (Wuhan, China, was identified by STR) and maintained in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum and 1% antibiotic solution in a humidified incubator with 5% CO 2 .

Techniques: Mutagenesis