h1975  (ATCC)


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    Structured Review

    ATCC h1975
    Cytotoxic effect of MnTnHex combined with cisplatin in A549 and <t>H1975</t> cells. Cells were simultaneously treated with ( A ) MnTnHex (0.5 and 1 μM) and cisplatin (1 and 2 μM) in A549 cells and ( B ) MnTnHex (0.5 and 1 μM) and cisplatin (1 and 5 μM) in H1975 cells, for 72 h. This effect was evaluated using a CV assay. ( C ) A549 cell morphology upon treatment with MnTnHex and/or cisplatin. ( D ) H1975 cell morphology upon treatment with MnTnHex and/or cisplatin. Values represent mean ± SD (n = 4) and are expressed as percentages relative to control cells. * p
    H1975, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "MnTnHex-2-PyP5+ Displays Anticancer Properties and Enhances Cisplatin Effects in Non-Small Cell Lung Cancer Cells"

    Article Title: MnTnHex-2-PyP5+ Displays Anticancer Properties and Enhances Cisplatin Effects in Non-Small Cell Lung Cancer Cells

    Journal: Antioxidants

    doi: 10.3390/antiox11112198

    Cytotoxic effect of MnTnHex combined with cisplatin in A549 and H1975 cells. Cells were simultaneously treated with ( A ) MnTnHex (0.5 and 1 μM) and cisplatin (1 and 2 μM) in A549 cells and ( B ) MnTnHex (0.5 and 1 μM) and cisplatin (1 and 5 μM) in H1975 cells, for 72 h. This effect was evaluated using a CV assay. ( C ) A549 cell morphology upon treatment with MnTnHex and/or cisplatin. ( D ) H1975 cell morphology upon treatment with MnTnHex and/or cisplatin. Values represent mean ± SD (n = 4) and are expressed as percentages relative to control cells. * p
    Figure Legend Snippet: Cytotoxic effect of MnTnHex combined with cisplatin in A549 and H1975 cells. Cells were simultaneously treated with ( A ) MnTnHex (0.5 and 1 μM) and cisplatin (1 and 2 μM) in A549 cells and ( B ) MnTnHex (0.5 and 1 μM) and cisplatin (1 and 5 μM) in H1975 cells, for 72 h. This effect was evaluated using a CV assay. ( C ) A549 cell morphology upon treatment with MnTnHex and/or cisplatin. ( D ) H1975 cell morphology upon treatment with MnTnHex and/or cisplatin. Values represent mean ± SD (n = 4) and are expressed as percentages relative to control cells. * p

    Techniques Used:

    GC-MS-based metabolomics analysis of the extracellular media of H1975 cells exposed to MnTnHex and cisplatin, alone and combined. ( A ) Volcano plot of the GC-MS data of H1975 cells exposed to MnTnHex (1 μM) 72 h compared with controls. ( B ) Volcano plot of GC-MS data of H1975 cells co-exposed to Cisplatin (1 μM) and MnTnHex (1 μM) compared with controls, indicating the statistical significance ( p -value) vs. the magnitude of change (fold change). Statistical significance was assessed using the Mann-Whitney test and p -values
    Figure Legend Snippet: GC-MS-based metabolomics analysis of the extracellular media of H1975 cells exposed to MnTnHex and cisplatin, alone and combined. ( A ) Volcano plot of the GC-MS data of H1975 cells exposed to MnTnHex (1 μM) 72 h compared with controls. ( B ) Volcano plot of GC-MS data of H1975 cells co-exposed to Cisplatin (1 μM) and MnTnHex (1 μM) compared with controls, indicating the statistical significance ( p -value) vs. the magnitude of change (fold change). Statistical significance was assessed using the Mann-Whitney test and p -values

    Techniques Used: Gas Chromatography-Mass Spectrometry, MANN-WHITNEY

    Effect of MnTnHex alone or combined with cisplatin on the collective migration of NSCLC cells. Cell migration was evaluated by wound-healing assay using MnTnHex (5 μM) and/or cisplatin (0.5 μM) in A549 cells at ( A ) 24 h and ( B ) 32 h. ( C ) Representative microscopy images of the wound-healing assay of A549 cells. Cell migration was also evaluated in H1975 cells at ( D ) 24 h and ( E ) 32 h using MnTnHex (0.5 μM) and/or cisplatin (1 μM). ( F ) Representative microscopy images of the wound-healing assay of H1975 cells. Values for the wound-healing assay represent mean ± SD ( n = 3–4) and are expressed as percentages relative to control cells for each time point. The statistical analysis was performed for each time point, comparing each condition with the control cells; * p
    Figure Legend Snippet: Effect of MnTnHex alone or combined with cisplatin on the collective migration of NSCLC cells. Cell migration was evaluated by wound-healing assay using MnTnHex (5 μM) and/or cisplatin (0.5 μM) in A549 cells at ( A ) 24 h and ( B ) 32 h. ( C ) Representative microscopy images of the wound-healing assay of A549 cells. Cell migration was also evaluated in H1975 cells at ( D ) 24 h and ( E ) 32 h using MnTnHex (0.5 μM) and/or cisplatin (1 μM). ( F ) Representative microscopy images of the wound-healing assay of H1975 cells. Values for the wound-healing assay represent mean ± SD ( n = 3–4) and are expressed as percentages relative to control cells for each time point. The statistical analysis was performed for each time point, comparing each condition with the control cells; * p

    Techniques Used: Migration, Wound Healing Assay, Microscopy

    Cytotoxic effects of MnTnHex (0.5–25 μM) in A549 and H1975 cells. The decrease in cell viability upon exposure to MnTnHex, for 72 h, was assessed by CV and MTS assays in ( A ) A549 and ( B ) H1975 cells. Values represent mean ± SD ( n = 3–4) and are expressed as percentages of the vehicle-treated control cells. ( C ) A549 cell morphology upon treatment with MnTnHex. ( D ) H1975 cell morphology upon treatment with MnTnHex. Scale bar = 100 μm.
    Figure Legend Snippet: Cytotoxic effects of MnTnHex (0.5–25 μM) in A549 and H1975 cells. The decrease in cell viability upon exposure to MnTnHex, for 72 h, was assessed by CV and MTS assays in ( A ) A549 and ( B ) H1975 cells. Values represent mean ± SD ( n = 3–4) and are expressed as percentages of the vehicle-treated control cells. ( C ) A549 cell morphology upon treatment with MnTnHex. ( D ) H1975 cell morphology upon treatment with MnTnHex. Scale bar = 100 μm.

    Techniques Used:

    Effect of MnTnHex combined with cisplatin in A549 and H1975 cells on cell cycle distribution. Cells were treated with the indicated concentrations of MnTnHex and cisplatin (1 µM) for 72 h and DNA content was assessed on fixed cells by flow cytometry. ( A ) Representative flow cytometry histograms of PI-stained cells. Summary data of cell cycle populations for ( B ) A549 and ( C ) H1975 cells. Individual populations for ( D – F ) A549 and ( G – I ) H1975 cells. Values represent mean ± SD ( n = 3–4) and are expressed as percentages of total cells. * p
    Figure Legend Snippet: Effect of MnTnHex combined with cisplatin in A549 and H1975 cells on cell cycle distribution. Cells were treated with the indicated concentrations of MnTnHex and cisplatin (1 µM) for 72 h and DNA content was assessed on fixed cells by flow cytometry. ( A ) Representative flow cytometry histograms of PI-stained cells. Summary data of cell cycle populations for ( B ) A549 and ( C ) H1975 cells. Individual populations for ( D – F ) A549 and ( G – I ) H1975 cells. Values represent mean ± SD ( n = 3–4) and are expressed as percentages of total cells. * p

    Techniques Used: Flow Cytometry, Staining

    2) Product Images from "Polyclonal antibody-induced downregulation of HER1/EGFR and HER2 surpasses the effect of combinations of specific registered antibodies"

    Article Title: Polyclonal antibody-induced downregulation of HER1/EGFR and HER2 surpasses the effect of combinations of specific registered antibodies

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2022.951267

    Promotion of the endocytosis and degradation of HER1 and HER2 by the PAbs. (A) H292, H1975, and PC9ER cells were treated for 24 h with PAbs-containing immune sera or pre-immune sera as negative control (1/100). After washing with acidic buffer (glycine 100 mM, pH 3.0), cells were incubated with antibodies against HER1 and HER2 tagged to fluorophores, and surface expression of the receptors was analyzed using flow cytometry. Death cells were excluded with DAPI. Density plots show the distribution of the cell population according to recognition of HER1 ( y -axis) and HER2 ( x -axis), and the percentage of cells expressing both receptors is specified. Data are representative of two independent experiments. (B) H292 and (C) H1975 cells were seeded in coverslips and treated as in (A) for 24 h. Cells were washed in acidic buffer, fixed in paraformaldehyde (4%), and incubated with specific primary antibodies, followed by an Alexa Fluor 555‐conjugated secondary antibody. Images were captured using a confocal microscope (40× magnification) and quantification was performed using ImageJ software. In the graphs, data are means ± SD of triplicates in a representative experiment of three conducted. Means were compared using Kruskal–Wallis test followed by a Games–Howell post-test. Significant differences among control and PAbs-treated cells are represented * p
    Figure Legend Snippet: Promotion of the endocytosis and degradation of HER1 and HER2 by the PAbs. (A) H292, H1975, and PC9ER cells were treated for 24 h with PAbs-containing immune sera or pre-immune sera as negative control (1/100). After washing with acidic buffer (glycine 100 mM, pH 3.0), cells were incubated with antibodies against HER1 and HER2 tagged to fluorophores, and surface expression of the receptors was analyzed using flow cytometry. Death cells were excluded with DAPI. Density plots show the distribution of the cell population according to recognition of HER1 ( y -axis) and HER2 ( x -axis), and the percentage of cells expressing both receptors is specified. Data are representative of two independent experiments. (B) H292 and (C) H1975 cells were seeded in coverslips and treated as in (A) for 24 h. Cells were washed in acidic buffer, fixed in paraformaldehyde (4%), and incubated with specific primary antibodies, followed by an Alexa Fluor 555‐conjugated secondary antibody. Images were captured using a confocal microscope (40× magnification) and quantification was performed using ImageJ software. In the graphs, data are means ± SD of triplicates in a representative experiment of three conducted. Means were compared using Kruskal–Wallis test followed by a Games–Howell post-test. Significant differences among control and PAbs-treated cells are represented * p

    Techniques Used: Negative Control, Incubation, Expressing, Flow Cytometry, Microscopy, Software

    Inhibition of tumor cell viability. Cells were treated with PAbs (10 µg/ml) or with the combination of cetuximab (cetu, 10 µg/ml) or nimotuzumab (nimo, 10 µg/ml) with trastuzumab (trastuz, 1 µg/ml). Unspecific PAbs (CTR, 10 µg/ml) or specific TKI AG1478 (in the case of H292 and A549 cells, 10 µM), lapatinib (in the case of SKBR3 20 µM), or osimertinib (for HER1-mutated lines cells,1 µM) was included as negative and positive controls, respectively. After 72 h of treatment, the viability of (A) H292, (B) SKBR3, (C) H3255 (L835R), (D) H1975 (L835R/T790M), (E) PC9 (exon19 deletion), and (F) PC9ER cells (exon 19 deletion and T790M mutation) was evaluated by MTT. In the graphs, data are means ± SD of triplicates in one experiment representative of at least two conducted, for each cell line. Differences among means, when normality and variance homogeneity were confirmed, were analyzed using one-way ANOVA, and Tukey test was used for multiple comparisons. Alternatively, non-parametric Kruskal–Wallis test was conducted, followed by Games–Howell post-test. Significant differences among negative control PAbs and the treatments as well as among specific PAbs and MAbs combinations are represented * p
    Figure Legend Snippet: Inhibition of tumor cell viability. Cells were treated with PAbs (10 µg/ml) or with the combination of cetuximab (cetu, 10 µg/ml) or nimotuzumab (nimo, 10 µg/ml) with trastuzumab (trastuz, 1 µg/ml). Unspecific PAbs (CTR, 10 µg/ml) or specific TKI AG1478 (in the case of H292 and A549 cells, 10 µM), lapatinib (in the case of SKBR3 20 µM), or osimertinib (for HER1-mutated lines cells,1 µM) was included as negative and positive controls, respectively. After 72 h of treatment, the viability of (A) H292, (B) SKBR3, (C) H3255 (L835R), (D) H1975 (L835R/T790M), (E) PC9 (exon19 deletion), and (F) PC9ER cells (exon 19 deletion and T790M mutation) was evaluated by MTT. In the graphs, data are means ± SD of triplicates in one experiment representative of at least two conducted, for each cell line. Differences among means, when normality and variance homogeneity were confirmed, were analyzed using one-way ANOVA, and Tukey test was used for multiple comparisons. Alternatively, non-parametric Kruskal–Wallis test was conducted, followed by Games–Howell post-test. Significant differences among negative control PAbs and the treatments as well as among specific PAbs and MAbs combinations are represented * p

    Techniques Used: Inhibition, Mutagenesis, MTT Assay, Negative Control

    3) Product Images from "Targeting SPHK1/PBX1 Axis Induced Cell Cycle Arrest in Non-Small Cell Lung Cancer"

    Article Title: Targeting SPHK1/PBX1 Axis Induced Cell Cycle Arrest in Non-Small Cell Lung Cancer

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms232112741

    PBX1-mediated SPHK1 inhibition induced G1/S stage cell cycle arrest. ( A ) Western blot of PBX1 protein in cultured NSCLC cell lines (A549, H520, H1299, H460, H226, H1792 and H1975) and in normal epithelial cells (BEAS-2B), β-actin was used as a loading control. ( B ) Western blot detected the change in SPHK1, PBX1, CDK4, CDK2 and CyclinD1 in H460 cells with SPHK1 or PBX1 silence (siRNA 50 nM) in the presence or the absence of S1P (5 μM). ( C ) Flow cytometry analyzed cell cycle distribution in H460 cells with PBX1 silence (siRNA 50 nM) in the presence or the absence of S1P (5 μM). ( D ) PBX1 -overexpressing efficiency was validated on PBX1 gene in H460 sh NC and H460 sh SPHK1 cells by RT-qPCR. **** p
    Figure Legend Snippet: PBX1-mediated SPHK1 inhibition induced G1/S stage cell cycle arrest. ( A ) Western blot of PBX1 protein in cultured NSCLC cell lines (A549, H520, H1299, H460, H226, H1792 and H1975) and in normal epithelial cells (BEAS-2B), β-actin was used as a loading control. ( B ) Western blot detected the change in SPHK1, PBX1, CDK4, CDK2 and CyclinD1 in H460 cells with SPHK1 or PBX1 silence (siRNA 50 nM) in the presence or the absence of S1P (5 μM). ( C ) Flow cytometry analyzed cell cycle distribution in H460 cells with PBX1 silence (siRNA 50 nM) in the presence or the absence of S1P (5 μM). ( D ) PBX1 -overexpressing efficiency was validated on PBX1 gene in H460 sh NC and H460 sh SPHK1 cells by RT-qPCR. **** p

    Techniques Used: Inhibition, Western Blot, Cell Culture, Flow Cytometry, Quantitative RT-PCR

    4) Product Images from "Exosomes in malignant pleural effusion from lung cancer patients impaired the cytotoxicity of double-negative T cells"

    Article Title: Exosomes in malignant pleural effusion from lung cancer patients impaired the cytotoxicity of double-negative T cells

    Journal: Translational Oncology

    doi: 10.1016/j.tranon.2022.101564

    MPE exosomes decreased the cytotoxicity of DNT cells. (A) MPE-derived exosomes were detected by transmission electron microscopy. (B) The surface markers (CD63 and CD81) expressed on MPE-derived exosomes were confirmed by Western blot. (C–E) MPE-derived exosomes (0, 5, 15, 25, 50 μg/ml) were added to DNT cell cultures. Twenty-four hours post-treatment, DNT cells were used to kill lung cancer cells for 24 h in a 1:1 ratio. The cytotoxicity of DNT cells to lung cancer cells was calculated using the CCK-8 method. The cytotoxicity of DNT cells to A549 cells, H1975 cells or SPCA-1 cells was decreased ( n = 3). *, p
    Figure Legend Snippet: MPE exosomes decreased the cytotoxicity of DNT cells. (A) MPE-derived exosomes were detected by transmission electron microscopy. (B) The surface markers (CD63 and CD81) expressed on MPE-derived exosomes were confirmed by Western blot. (C–E) MPE-derived exosomes (0, 5, 15, 25, 50 μg/ml) were added to DNT cell cultures. Twenty-four hours post-treatment, DNT cells were used to kill lung cancer cells for 24 h in a 1:1 ratio. The cytotoxicity of DNT cells to lung cancer cells was calculated using the CCK-8 method. The cytotoxicity of DNT cells to A549 cells, H1975 cells or SPCA-1 cells was decreased ( n = 3). *, p

    Techniques Used: Derivative Assay, Transmission Assay, Electron Microscopy, Western Blot, CCK-8 Assay

    5) Product Images from "Erdafitinib Inhibits Tumorigenesis of Human Lung Adenocarcinoma A549 by Inducing S-Phase Cell-Cycle Arrest as a CDK2 Inhibitor"

    Article Title: Erdafitinib Inhibits Tumorigenesis of Human Lung Adenocarcinoma A549 by Inducing S-Phase Cell-Cycle Arrest as a CDK2 Inhibitor

    Journal: Molecules

    doi: 10.3390/molecules27196733

    Erdafitinib inhibited cell viability of human lung adenocarcinoma A549 cells by inducing S-phage cell-cycle arrest and cell apoptosis. ( A ) The chemical structure of erdafitinib. ( B ) The effects of erdafitinib (0, 3.125, 6.25, 12.5, 25 and 50 μM, 24 h) on human lung adenocarcinoma cell lines including A549, H1975, H2009, Calu-3 and PC-9 were assessed by MTT assay. ( C ) The effects of erdafitinib (0, 3.125, 6.25, 12.5, 25 and 50 μM, 24 h) on human lung adenocarcinoma A549 cells and human embryonic lung fibroblast MRC-5 cells were assessed by MTT assay. ( D ) The effects of erdafitinib (0, 6.25, and 12.5 μM, 12, 24 and 48 h) on human lung adenocarcinoma A549 cells were assessed by MTT assay. ( E ) A549 cells were treated with erdafitinib (0, 6.25 and 12.5 μM, 24 h) and cell cycle was analyzed by flow cytometry and statistically analyzed. ( F ) A549 cells were treated with erdafitinib (0, 6.25 and 12.5 μM, 24 h) and cell apoptosis was analyzed by flow cytometry and statistically analyzed. The Annexin V+/PI- and Annexin V+/PI+ cells were considered as early and late apoptotic cells, respectively, and the sum of the above two was calculated as apoptotic cells. Data were represented as means ± SD ( n = 3). * p
    Figure Legend Snippet: Erdafitinib inhibited cell viability of human lung adenocarcinoma A549 cells by inducing S-phage cell-cycle arrest and cell apoptosis. ( A ) The chemical structure of erdafitinib. ( B ) The effects of erdafitinib (0, 3.125, 6.25, 12.5, 25 and 50 μM, 24 h) on human lung adenocarcinoma cell lines including A549, H1975, H2009, Calu-3 and PC-9 were assessed by MTT assay. ( C ) The effects of erdafitinib (0, 3.125, 6.25, 12.5, 25 and 50 μM, 24 h) on human lung adenocarcinoma A549 cells and human embryonic lung fibroblast MRC-5 cells were assessed by MTT assay. ( D ) The effects of erdafitinib (0, 6.25, and 12.5 μM, 12, 24 and 48 h) on human lung adenocarcinoma A549 cells were assessed by MTT assay. ( E ) A549 cells were treated with erdafitinib (0, 6.25 and 12.5 μM, 24 h) and cell cycle was analyzed by flow cytometry and statistically analyzed. ( F ) A549 cells were treated with erdafitinib (0, 6.25 and 12.5 μM, 24 h) and cell apoptosis was analyzed by flow cytometry and statistically analyzed. The Annexin V+/PI- and Annexin V+/PI+ cells were considered as early and late apoptotic cells, respectively, and the sum of the above two was calculated as apoptotic cells. Data were represented as means ± SD ( n = 3). * p

    Techniques Used: MTT Assay, Flow Cytometry

    6) Product Images from "Capicua suppresses YAP1 to limit tumorigenesis and maintain drug sensitivity in human cancer"

    Article Title: Capicua suppresses YAP1 to limit tumorigenesis and maintain drug sensitivity in human cancer

    Journal: Cell reports

    doi: 10.1016/j.celrep.2022.111443

    YAP in CIC-deficient human lung cancer drives tumor progression and resistance to MAPK inhibitors (A, F, and K) CTG viability curves in HCC1359 (A), H1975 (F), and HCC364 (K) following treatment with trametinib (tram), Osimertinib (osi), or vemurafenib (vem), respectively. (B, G, and L) Relative cell number of HCC1359 (B), H1975 (G), and HCC364 (L) cells expressing sgCtrl, sgCIC1, or sgCIC2 and treated (6 d) with tram, osi, or vem, respectively. **p
    Figure Legend Snippet: YAP in CIC-deficient human lung cancer drives tumor progression and resistance to MAPK inhibitors (A, F, and K) CTG viability curves in HCC1359 (A), H1975 (F), and HCC364 (K) following treatment with trametinib (tram), Osimertinib (osi), or vemurafenib (vem), respectively. (B, G, and L) Relative cell number of HCC1359 (B), H1975 (G), and HCC364 (L) cells expressing sgCtrl, sgCIC1, or sgCIC2 and treated (6 d) with tram, osi, or vem, respectively. **p

    Techniques Used: Expressing

    MAPK inhibitor resistance and tumor growth in CIC-deficient lung cancer are overcome through YAP1 inhibition in vivo (A) H1975 xenografts comparing vehicle or osimertinib-treated H1975, H1975 with CIC KO, or H1975 with CIC KO + YAP1 KD (n = 10). (B) Tumor weights from mice harboring H1975, H1975 CIC KO, or H1975 CIC KO + YAP1 KD ± osimertinib. (C) Immunoblots of CIC, YAP, and HSP90 expression in H1975 xenografts derived from tumors of different groups in (A). (D) Representative figures of parental, CIC KO, or CIC KO + YAP1 KD in HCC1359 tumor-bearing mice. (E) Bar graph comparing the incidence of HCC1359 (n = 2/20), HCC1359 CIC KO (n = 13/20), or HCC1359 CIC KO + YAP1 KD (n = 1/20) tumor formation in mice. (F) Relative growth of HCC1359 CIC KO tumors treated with vehicle or verteporfin. (G) Correlation plot between CIC and YAP expression from LC specimens (tau = −0.33, p = 0.0002, n = 100). (H) Representative images of CIC and YAP-stained LC tissue. Scale bar, 50 μm. (I) Model of CIC-mediated repression of YAP1 and phenotypes. Error bars represent SD.
    Figure Legend Snippet: MAPK inhibitor resistance and tumor growth in CIC-deficient lung cancer are overcome through YAP1 inhibition in vivo (A) H1975 xenografts comparing vehicle or osimertinib-treated H1975, H1975 with CIC KO, or H1975 with CIC KO + YAP1 KD (n = 10). (B) Tumor weights from mice harboring H1975, H1975 CIC KO, or H1975 CIC KO + YAP1 KD ± osimertinib. (C) Immunoblots of CIC, YAP, and HSP90 expression in H1975 xenografts derived from tumors of different groups in (A). (D) Representative figures of parental, CIC KO, or CIC KO + YAP1 KD in HCC1359 tumor-bearing mice. (E) Bar graph comparing the incidence of HCC1359 (n = 2/20), HCC1359 CIC KO (n = 13/20), or HCC1359 CIC KO + YAP1 KD (n = 1/20) tumor formation in mice. (F) Relative growth of HCC1359 CIC KO tumors treated with vehicle or verteporfin. (G) Correlation plot between CIC and YAP expression from LC specimens (tau = −0.33, p = 0.0002, n = 100). (H) Representative images of CIC and YAP-stained LC tissue. Scale bar, 50 μm. (I) Model of CIC-mediated repression of YAP1 and phenotypes. Error bars represent SD.

    Techniques Used: Inhibition, In Vivo, Mouse Assay, Western Blot, Expressing, Derivative Assay, Staining

    7) Product Images from "Inhibiting ALK-TOPK signaling pathway promotes cell apoptosis of ALK-positive NSCLC"

    Article Title: Inhibiting ALK-TOPK signaling pathway promotes cell apoptosis of ALK-positive NSCLC

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-05260-3

    ALK phosphorylates TOPK at Y74 ex vivo and enhances the stability of TOPK. A TOPK bound with endogenous ALK of H2228 cells by pull-down assay. B H2228 cells were treated with Alectinib (0 μΜ, 0.01 μΜ, 0.1 μΜ, or 1 μΜ) for 24 h or 0.1 μM Alectinib for 0 h, 6 h, 12 h, or 24 h, and the samples were analyzed by western blotting. C The stable shMock and shALK in H2228 cells were established and the samples were analyzed by western blotting. D Flag-ALK (v3a/v3b) was cotransfected in HEK293T cells with HA-TOPK-WT or HA-TOPK 74F, and the samples were analyzed by western blotting. E The expression of ALK and TOPK in various lung cancer cells. F The stable ALK (v3a) or (v3b)-overexpression in H1299 and H1975 cells were established and the samples were analyzed by western blotting. G The cells were treated with CHX (100 μg/ml) for 0 h, 4 h, 8 h, and 12 h, and the samples were analyzed by western blotting. Data are representative of results from triplicate experiments.
    Figure Legend Snippet: ALK phosphorylates TOPK at Y74 ex vivo and enhances the stability of TOPK. A TOPK bound with endogenous ALK of H2228 cells by pull-down assay. B H2228 cells were treated with Alectinib (0 μΜ, 0.01 μΜ, 0.1 μΜ, or 1 μΜ) for 24 h or 0.1 μM Alectinib for 0 h, 6 h, 12 h, or 24 h, and the samples were analyzed by western blotting. C The stable shMock and shALK in H2228 cells were established and the samples were analyzed by western blotting. D Flag-ALK (v3a/v3b) was cotransfected in HEK293T cells with HA-TOPK-WT or HA-TOPK 74F, and the samples were analyzed by western blotting. E The expression of ALK and TOPK in various lung cancer cells. F The stable ALK (v3a) or (v3b)-overexpression in H1299 and H1975 cells were established and the samples were analyzed by western blotting. G The cells were treated with CHX (100 μg/ml) for 0 h, 4 h, 8 h, and 12 h, and the samples were analyzed by western blotting. Data are representative of results from triplicate experiments.

    Techniques Used: Ex Vivo, Pull Down Assay, Western Blot, Expressing, Over Expression

    8) Product Images from "The regulation of ISG20 expression on SARS-CoV-2 infection in cancer patients and healthy individuals"

    Article Title: The regulation of ISG20 expression on SARS-CoV-2 infection in cancer patients and healthy individuals

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2022.958898

    Cordycepin (CD) and N6, N6-dimethyladenosine (m 6 2 A) increase ISG20 expression in various cancer cell lines. (A, B) CD increases ISG20 expression in the H1975 lung cancer cell line. (C, D) CD increases ISG20 expression in the 22RV1 prostate cancer cell line. (E, F) m 6 a 2 A increase ISG20 expression in the HepG2 live cancer cell line.
    Figure Legend Snippet: Cordycepin (CD) and N6, N6-dimethyladenosine (m 6 2 A) increase ISG20 expression in various cancer cell lines. (A, B) CD increases ISG20 expression in the H1975 lung cancer cell line. (C, D) CD increases ISG20 expression in the 22RV1 prostate cancer cell line. (E, F) m 6 a 2 A increase ISG20 expression in the HepG2 live cancer cell line.

    Techniques Used: Expressing

    9) Product Images from "The HDAC inhibitor GCJ-490A suppresses c-Met expression through IKKα and overcomes gefitinib resistance in non-small cell lung cancer"

    Article Title: The HDAC inhibitor GCJ-490A suppresses c-Met expression through IKKα and overcomes gefitinib resistance in non-small cell lung cancer

    Journal: Cancer Biology & Medicine

    doi: 10.20892/j.issn.2095-3941.2021.0130

    GCJ-490A inhibits NSCLC cell proliferation in vitro and in vivo. (A) IC 50 values for exposure of NSCLC cell lines to GCJ-490A for 72 h. (B) Assessment of apoptosis by FACS analysis. NSCLC cells were treated with GCJ-490A for 48 h. The sum of annexin V + /PI - and annexin V + /PI + labeled cells was counted as the total apoptotic cells. (C) The levels of apoptosis-related proteins were detected in A549, H460, H1975, and HCC827/GR6 cells treated with GCJ-490A for 48 h. (D) Antitumor activity of GCJ-490A in vivo . Tumor growth curve graphs show relative tumor volume (RTV) over time in each treatment group. (E) Body weights of animals in each treatment group over time. All in vitro data are presented as the mean ± SD from at least 3 independent experiments, and were analyzed by 2-tailed Student’s t -test, * P
    Figure Legend Snippet: GCJ-490A inhibits NSCLC cell proliferation in vitro and in vivo. (A) IC 50 values for exposure of NSCLC cell lines to GCJ-490A for 72 h. (B) Assessment of apoptosis by FACS analysis. NSCLC cells were treated with GCJ-490A for 48 h. The sum of annexin V + /PI - and annexin V + /PI + labeled cells was counted as the total apoptotic cells. (C) The levels of apoptosis-related proteins were detected in A549, H460, H1975, and HCC827/GR6 cells treated with GCJ-490A for 48 h. (D) Antitumor activity of GCJ-490A in vivo . Tumor growth curve graphs show relative tumor volume (RTV) over time in each treatment group. (E) Body weights of animals in each treatment group over time. All in vitro data are presented as the mean ± SD from at least 3 independent experiments, and were analyzed by 2-tailed Student’s t -test, * P

    Techniques Used: In Vitro, In Vivo, FACS, Labeling, Activity Assay

    10) Product Images from "Electronic Coupling in 1,2,3-Triazole Bridged Ferrocenes and Its Impact on Reactive Oxygen Species Generation and Deleterious Activity in Cancer Cells"

    Article Title: Electronic Coupling in 1,2,3-Triazole Bridged Ferrocenes and Its Impact on Reactive Oxygen Species Generation and Deleterious Activity in Cancer Cells

    Journal: Inorganic Chemistry

    doi: 10.1021/acs.inorgchem.2c01110

    Relative ROS amount in H1975 cells treated with 20 μM of compounds 1a and 1c and reference drugs with or without 50 μM NAC. The ROS levels were measured by a fluorimetric assay in duplicates. Data are mean ± standard deviation (SD) ( n = 3). ** p
    Figure Legend Snippet: Relative ROS amount in H1975 cells treated with 20 μM of compounds 1a and 1c and reference drugs with or without 50 μM NAC. The ROS levels were measured by a fluorimetric assay in duplicates. Data are mean ± standard deviation (SD) ( n = 3). ** p

    Techniques Used: Fluorimetry Assay, Standard Deviation

    Viability of H1975 cells treated for 72 h with 20 μM of compounds 1a and 1c and reference drugs with or without 50 μM NAC. Cell viability was measured spectrophotometrically in triplicate. Data are mean ± SD ( n = 3). ** p
    Figure Legend Snippet: Viability of H1975 cells treated for 72 h with 20 μM of compounds 1a and 1c and reference drugs with or without 50 μM NAC. Cell viability was measured spectrophotometrically in triplicate. Data are mean ± SD ( n = 3). ** p

    Techniques Used:

    11) Product Images from "Microfluidics-free single-cell genomics with templated emulsification"

    Article Title: Microfluidics-free single-cell genomics with templated emulsification

    Journal: bioRxiv

    doi: 10.1101/2022.06.10.495582

    Identification of Gefitinib-specific transcriptional responses in cancer cell lines. (a) Violin plots of median expression values for selected differentially expressed genes. (b) The expression of selected differentially expressed genes superimposed on H1975 and PC9 cell clusters.
    Figure Legend Snippet: Identification of Gefitinib-specific transcriptional responses in cancer cell lines. (a) Violin plots of median expression values for selected differentially expressed genes. (b) The expression of selected differentially expressed genes superimposed on H1975 and PC9 cell clusters.

    Techniques Used: Expressing

    Molecular signatures of drug resistant cancer phenotypes in cell lines and patient samples. (a) A two-by-two experimental study design using lung adenocarcinoma cell lines (H1975, PC9) treated with Gefitinib or DMSO. (b) Clustering of single-cell RNA-seq after drug treatment shows transcriptional perturbations in Gefitinib-sensitive PC9, but not Gefitinib- resistant H1975 cells. (c) Increased expression of TACSTD2 in PC9 challenged with Gefitinib. (d) Identification of drug-resistant H1975 cells spiked into drug-sensitive PC9 cells based on Gefitinib-induced transcriptional perturbation. (e-l) PIP-seq RNA and barcoded antibody (CITE- seq) analysis of mixed phenotype acute leukemia (MPAL). (e) Clustering of single cells for patient 65 before (left panel) and after (right panel) chemotherapy. ( f) Clustering of single cells for patient 873 before (left panel) and after (right panel) chemotherapy. (g-h) Antibody derived tag (ADT) abundance, by cluster, before (t1) and after (t2) chemotherapy. ADTs change as a function of chemotherapy but are consistent among clusters for both patients, with the exception of T cell subsets. (i-l) Analysis of transcriptional heterogeneity in MPAL samples (i) Heatmap of top differentially expressed marker genes by cluster after relapse in patient 63. (j) GSEA pre- ranked analysis comparing transcriptomic differences between clusters 1 and 7 in patient 65 using gene sets HP Acute Leukemia (M35856), Hallmark G2M Checkpoint (M5901), Hallmark Oxidative Phosphorylation (M5936), amd GOCC Ribosome (M17089) (k) Heatmap of top differentially expressed marker genes by cluster after relapse in patient 873. (l) GSEA pre-ranked analysis comparing transcriptomic differences between clusters 3 and 5 in patient 873 using gene sets HP Acute Myeloid Leukemia (M36586), GOCC Ribosome (M17089), GOBP Oxidative Phosphorylation (M17089), and Abnormal Myeloid Leukocyte Morphology (M37711).
    Figure Legend Snippet: Molecular signatures of drug resistant cancer phenotypes in cell lines and patient samples. (a) A two-by-two experimental study design using lung adenocarcinoma cell lines (H1975, PC9) treated with Gefitinib or DMSO. (b) Clustering of single-cell RNA-seq after drug treatment shows transcriptional perturbations in Gefitinib-sensitive PC9, but not Gefitinib- resistant H1975 cells. (c) Increased expression of TACSTD2 in PC9 challenged with Gefitinib. (d) Identification of drug-resistant H1975 cells spiked into drug-sensitive PC9 cells based on Gefitinib-induced transcriptional perturbation. (e-l) PIP-seq RNA and barcoded antibody (CITE- seq) analysis of mixed phenotype acute leukemia (MPAL). (e) Clustering of single cells for patient 65 before (left panel) and after (right panel) chemotherapy. ( f) Clustering of single cells for patient 873 before (left panel) and after (right panel) chemotherapy. (g-h) Antibody derived tag (ADT) abundance, by cluster, before (t1) and after (t2) chemotherapy. ADTs change as a function of chemotherapy but are consistent among clusters for both patients, with the exception of T cell subsets. (i-l) Analysis of transcriptional heterogeneity in MPAL samples (i) Heatmap of top differentially expressed marker genes by cluster after relapse in patient 63. (j) GSEA pre- ranked analysis comparing transcriptomic differences between clusters 1 and 7 in patient 65 using gene sets HP Acute Leukemia (M35856), Hallmark G2M Checkpoint (M5901), Hallmark Oxidative Phosphorylation (M5936), amd GOCC Ribosome (M17089) (k) Heatmap of top differentially expressed marker genes by cluster after relapse in patient 873. (l) GSEA pre-ranked analysis comparing transcriptomic differences between clusters 3 and 5 in patient 873 using gene sets HP Acute Myeloid Leukemia (M36586), GOCC Ribosome (M17089), GOBP Oxidative Phosphorylation (M17089), and Abnormal Myeloid Leukocyte Morphology (M37711).

    Techniques Used: RNA Sequencing Assay, Expressing, Derivative Assay, Marker

    12) Product Images from "Ring finger protein 180 suppresses cell proliferation and energy metabolism of non-small cell lung cancer through downregulating C-myc"

    Article Title: Ring finger protein 180 suppresses cell proliferation and energy metabolism of non-small cell lung cancer through downregulating C-myc

    Journal: World Journal of Surgical Oncology

    doi: 10.1186/s12957-022-02599-x

    RNF180 interacted with c-Myc and enhanced its ubiquitination in NSCLC cells. A Functional analysis indicated that RNF180 negatively correlated with C-myc. B qRT-PCR was used to examine the relative mRNA levels of C-myc in oeNC and oeRNF180 transfecting H358 and H1975 cells. C Western blot was used to examine the protein levels of C-myc in oeNC or oeRNF180 transfecting cells with or without the treatment of the proteasome inhibitor MG132. D CO-IP assay indicated that RNF180 interacted with C-myc. E Overexpression of RNF180 enhanced the ubiquitination of C-myc in NSCLC cells
    Figure Legend Snippet: RNF180 interacted with c-Myc and enhanced its ubiquitination in NSCLC cells. A Functional analysis indicated that RNF180 negatively correlated with C-myc. B qRT-PCR was used to examine the relative mRNA levels of C-myc in oeNC and oeRNF180 transfecting H358 and H1975 cells. C Western blot was used to examine the protein levels of C-myc in oeNC or oeRNF180 transfecting cells with or without the treatment of the proteasome inhibitor MG132. D CO-IP assay indicated that RNF180 interacted with C-myc. E Overexpression of RNF180 enhanced the ubiquitination of C-myc in NSCLC cells

    Techniques Used: Functional Assay, Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Over Expression

    13) Product Images from "Mediating EGFR-TKI Resistance by VEGF/VEGFR Autocrine Pathway in Non-Small Cell Lung Cancer"

    Article Title: Mediating EGFR-TKI Resistance by VEGF/VEGFR Autocrine Pathway in Non-Small Cell Lung Cancer

    Journal: Cells

    doi: 10.3390/cells11101694

    Effect of anti-VEGF treatment in NSCLC cell lines. A total of 8,000 H2170-ER, H358-ER, H1975, and H3255 NSCLC cells were treated with diluent (control), 10 µM Erlotinib treatment, 200 ng/mL Avastin treatment, and a combination of 10 µM Erlotinib and 200 ng/mL Avastin. Cell viability was assessed with an MTT cell viability assay to study the efficacy of Erlotinib in conjunction with the VEGF inhibitor, Avastin, relative to the control. In ( A ) H2170-ER cells, ( B ) H358-ER cells, ( C ) H1975 cells, and ( D ) H3255 cells, cell viability was measured. Six duplicates from each cell line were used for each treatment group. Absorbance was recorded, and percent viability was calculated using appropriate controls for each sample. Statistical analysis was performed, and the results were found to be statistically significant (* = p
    Figure Legend Snippet: Effect of anti-VEGF treatment in NSCLC cell lines. A total of 8,000 H2170-ER, H358-ER, H1975, and H3255 NSCLC cells were treated with diluent (control), 10 µM Erlotinib treatment, 200 ng/mL Avastin treatment, and a combination of 10 µM Erlotinib and 200 ng/mL Avastin. Cell viability was assessed with an MTT cell viability assay to study the efficacy of Erlotinib in conjunction with the VEGF inhibitor, Avastin, relative to the control. In ( A ) H2170-ER cells, ( B ) H358-ER cells, ( C ) H1975 cells, and ( D ) H3255 cells, cell viability was measured. Six duplicates from each cell line were used for each treatment group. Absorbance was recorded, and percent viability was calculated using appropriate controls for each sample. Statistical analysis was performed, and the results were found to be statistically significant (* = p

    Techniques Used: MTT Assay, Viability Assay

    Secretion of VEGF in NSCLC cell lines. For the analysis 3.75 × 10 6 H2170-P/ER, H358-P/ER, H1975, and H3255 cells were plated in vented flasks and allowed to adhere for 24 h in RPMI medium with 10% FBS. After 24 h, medium was removed, followed by PBS washes, and then starving medium (RPMI + 0.5% BSA) was added and incubated for 24 h. The medium was then centrifuged, and the pellets were stored in −70 °C. Collection of conditioned medium was repeated for 24 h and 48 h time points. The amount of VEGF in ( A ) H2170-P/ER, ( B ) H358-P/ER, and ( C ) H1975 and H3255 NSCLC cell lines was quantified at two time points, 24 and 48 h, and the experiments were run in triplicate. Statistical analysis was performed, and the results were found to be statistically significant (* = p
    Figure Legend Snippet: Secretion of VEGF in NSCLC cell lines. For the analysis 3.75 × 10 6 H2170-P/ER, H358-P/ER, H1975, and H3255 cells were plated in vented flasks and allowed to adhere for 24 h in RPMI medium with 10% FBS. After 24 h, medium was removed, followed by PBS washes, and then starving medium (RPMI + 0.5% BSA) was added and incubated for 24 h. The medium was then centrifuged, and the pellets were stored in −70 °C. Collection of conditioned medium was repeated for 24 h and 48 h time points. The amount of VEGF in ( A ) H2170-P/ER, ( B ) H358-P/ER, and ( C ) H1975 and H3255 NSCLC cell lines was quantified at two time points, 24 and 48 h, and the experiments were run in triplicate. Statistical analysis was performed, and the results were found to be statistically significant (* = p

    Techniques Used: Incubation

    Effect of anti-VEGFR-2 treatments in resistant and mutated NSCLC cell lines. A total of 8000 H2170-ER, H358-ER, H1975, and H3255 NSCLC cells were treated with diluent (control), 5 µM Erlotinib treatment, 12 µM ZM treatment, and a combination of 5 µM Erlotinib and 12 µM ZM. Cell viability was assessed with an MTT cell viability assay to study the efficacy of Erlotinib in conjunction with ZM, a VEGFR-2 inhibitor, relative to the control. In ( A ) H2170-ER cells, ( B ) H358-ER cells, (C ) H1975-ER cells, and ( D ) H3255-ER cells, percent cell viability was measured. Six duplicates from each cell line were used for each treatment group. Absorbance was recorded and percent viability was calculated using appropriate controls for each sample. Statistical analysis was performed, and the results were found to be statistically significant (* = p
    Figure Legend Snippet: Effect of anti-VEGFR-2 treatments in resistant and mutated NSCLC cell lines. A total of 8000 H2170-ER, H358-ER, H1975, and H3255 NSCLC cells were treated with diluent (control), 5 µM Erlotinib treatment, 12 µM ZM treatment, and a combination of 5 µM Erlotinib and 12 µM ZM. Cell viability was assessed with an MTT cell viability assay to study the efficacy of Erlotinib in conjunction with ZM, a VEGFR-2 inhibitor, relative to the control. In ( A ) H2170-ER cells, ( B ) H358-ER cells, (C ) H1975-ER cells, and ( D ) H3255-ER cells, percent cell viability was measured. Six duplicates from each cell line were used for each treatment group. Absorbance was recorded and percent viability was calculated using appropriate controls for each sample. Statistical analysis was performed, and the results were found to be statistically significant (* = p

    Techniques Used: MTT Assay, Viability Assay

    Mechanisms of resistance to molecularly targeted therapy in NSCLC . In TKI-resistant NSCLC cells activated EGFR leads to activation of the MAPK, FAK-Paxillin, and PI3K-AKT-mTOR downstream pathways, resulting in increased proliferation, survival, migration, and angiogenesis. EGFR TKIs inhibit this activation; however, due to mutations (primary L858R and secondary T790M mutations), tumor cells acquire resistance against these TKIs, activating these pathways further. Hypoxia also induces upregulation of VEGF. Upregulation of VEGF, VEGFR-2, and NP-1/2 may also lead to activation of the downstream pathways mentioned above, causing TKI resistance in NSCLC.
    Figure Legend Snippet: Mechanisms of resistance to molecularly targeted therapy in NSCLC . In TKI-resistant NSCLC cells activated EGFR leads to activation of the MAPK, FAK-Paxillin, and PI3K-AKT-mTOR downstream pathways, resulting in increased proliferation, survival, migration, and angiogenesis. EGFR TKIs inhibit this activation; however, due to mutations (primary L858R and secondary T790M mutations), tumor cells acquire resistance against these TKIs, activating these pathways further. Hypoxia also induces upregulation of VEGF. Upregulation of VEGF, VEGFR-2, and NP-1/2 may also lead to activation of the downstream pathways mentioned above, causing TKI resistance in NSCLC.

    Techniques Used: Activation Assay, Migration

    Modulation of VEGFR-2 and NP-1 protein expression in NSCLC cell lines . ( A ) For the analysis, 2.5 × 10 5 H2170-P/ER, H358-P/ER, H1975, and H3255 cells were seeded in Petri-dishes. Cells were allowed to adhere and grow for 24 h, then starving media (RPMI with 0.5% BSA) was added for 24 h. Cells were then treated with Erlotinib (10 µM Erlotinib) for 24 h and/or with ligands (15 ng/mL EGF for 2.5 min in starving media) before collection of cell lysates. Immunoblotting was then performed. ( B ) The data were normalized with beta-actin, and the graphical representation is relative to protein expression of the corresponding parental or single-mutant cell line. The modulations were calculated by densitometric analysis using ImageJ software. Statistical analysis was performed and it was demonstrated that the results were statistically significant ( p
    Figure Legend Snippet: Modulation of VEGFR-2 and NP-1 protein expression in NSCLC cell lines . ( A ) For the analysis, 2.5 × 10 5 H2170-P/ER, H358-P/ER, H1975, and H3255 cells were seeded in Petri-dishes. Cells were allowed to adhere and grow for 24 h, then starving media (RPMI with 0.5% BSA) was added for 24 h. Cells were then treated with Erlotinib (10 µM Erlotinib) for 24 h and/or with ligands (15 ng/mL EGF for 2.5 min in starving media) before collection of cell lysates. Immunoblotting was then performed. ( B ) The data were normalized with beta-actin, and the graphical representation is relative to protein expression of the corresponding parental or single-mutant cell line. The modulations were calculated by densitometric analysis using ImageJ software. Statistical analysis was performed and it was demonstrated that the results were statistically significant ( p

    Techniques Used: Expressing, Mutagenesis, Software

    Immunostaining and expression of VEGFR-2 and NP-1 in NSCLC cell lines. Immunofluorescent images showing modulation in EGFR-TKI resistance-related proteins in ( A ) H2170-P/ER, ( B ) H358-P/ER, and ( C ) H1975 and H3255 cells. For the analysis, 2 × 10 4 cells per well were plated in an 8-well chamber slide, fixed, permeabilized, and probed with anti-VEGFR-2 and anti-NP-1 primary antibodies and a DyLight 488 conjugated secondary antibody for immunostaining. Nuclei were stained using DAPI, and cells were visualized using an Olympus Fluoview confocal microscope. The mean fluorescent intensities of VEGFR-2 and NP-1 receptors were graphically represented for ( D ) H2170-ER cells as compared to parental cells, ( E ) H358-ER cells as compared to H358-P cells, and ( F ) H1975 double-mutants (T790M and L858R) as compared to H3255 single-mutant (L858R) cells. Fluorescence quantifications were performed using Olympus Fluoview image analysis software, and mean fluorescent intensities are graphically depicted. Statistical analyses demonstrated that the results were statistically significant ( p
    Figure Legend Snippet: Immunostaining and expression of VEGFR-2 and NP-1 in NSCLC cell lines. Immunofluorescent images showing modulation in EGFR-TKI resistance-related proteins in ( A ) H2170-P/ER, ( B ) H358-P/ER, and ( C ) H1975 and H3255 cells. For the analysis, 2 × 10 4 cells per well were plated in an 8-well chamber slide, fixed, permeabilized, and probed with anti-VEGFR-2 and anti-NP-1 primary antibodies and a DyLight 488 conjugated secondary antibody for immunostaining. Nuclei were stained using DAPI, and cells were visualized using an Olympus Fluoview confocal microscope. The mean fluorescent intensities of VEGFR-2 and NP-1 receptors were graphically represented for ( D ) H2170-ER cells as compared to parental cells, ( E ) H358-ER cells as compared to H358-P cells, and ( F ) H1975 double-mutants (T790M and L858R) as compared to H3255 single-mutant (L858R) cells. Fluorescence quantifications were performed using Olympus Fluoview image analysis software, and mean fluorescent intensities are graphically depicted. Statistical analyses demonstrated that the results were statistically significant ( p

    Techniques Used: Immunostaining, Expressing, Staining, Microscopy, Mutagenesis, Fluorescence, Software

    Increased cell surface expression of VEGFR-2 and NP-1 receptors in Erlotinib-resistant NSCLC cell lines. For the analysis, 5 × 10 5 H2170-P/ER, H358-P/ER, H1975, and H3255 cells were seeded in a Petri dish and allowed to grow and adhere for 48 h. After the growth medium was removed, the cells were washed and detached. Cells were then collected, pelleted, re-suspended in a buffer, pelleted again, probed with antibody, washed, and re-suspended in buffer before being used for flow cytometry. Cell surface expression was compared in ( A ) H2170-ER cells compared to H2170-P cells, (B ) H358-ER cells compared to parental cells, and ( C ) T790M- and L858R-mutant H1975 cells compared to L858R-mutant H3255 cells. Statistical analyses were performed, and it was demonstrated that the results were statistically significant ( p
    Figure Legend Snippet: Increased cell surface expression of VEGFR-2 and NP-1 receptors in Erlotinib-resistant NSCLC cell lines. For the analysis, 5 × 10 5 H2170-P/ER, H358-P/ER, H1975, and H3255 cells were seeded in a Petri dish and allowed to grow and adhere for 48 h. After the growth medium was removed, the cells were washed and detached. Cells were then collected, pelleted, re-suspended in a buffer, pelleted again, probed with antibody, washed, and re-suspended in buffer before being used for flow cytometry. Cell surface expression was compared in ( A ) H2170-ER cells compared to H2170-P cells, (B ) H358-ER cells compared to parental cells, and ( C ) T790M- and L858R-mutant H1975 cells compared to L858R-mutant H3255 cells. Statistical analyses were performed, and it was demonstrated that the results were statistically significant ( p

    Techniques Used: Expressing, Flow Cytometry, Mutagenesis

    14) Product Images from "27-hydroxycholesterol linked high cholesterol diet to lung adenocarcinoma metastasis"

    Article Title: 27-hydroxycholesterol linked high cholesterol diet to lung adenocarcinoma metastasis

    Journal: Oncogene

    doi: 10.1038/s41388-022-02285-y

    27-HC promoted lung adenocarcinoma proliferation, colony formation, and invasion. A A549 and H1975 cells were exposed to different concentration of 27-HC for 24 h. The cell viability was determined using CCK-8 assay. B A549 and H1975 cells were treated with 0, 1.0, or 10 μM 27-HC for 14 days, respectively. Colony formation was detected using crystal violet staining. C A549 and H1975 cells were treated with 1 μM 27-HC (monoculture system) or cocultured with THP1-derived macrophages treated with 1 μM 27-HC for 24 h. Invasive cells was stained by crystal violet. * P
    Figure Legend Snippet: 27-HC promoted lung adenocarcinoma proliferation, colony formation, and invasion. A A549 and H1975 cells were exposed to different concentration of 27-HC for 24 h. The cell viability was determined using CCK-8 assay. B A549 and H1975 cells were treated with 0, 1.0, or 10 μM 27-HC for 14 days, respectively. Colony formation was detected using crystal violet staining. C A549 and H1975 cells were treated with 1 μM 27-HC (monoculture system) or cocultured with THP1-derived macrophages treated with 1 μM 27-HC for 24 h. Invasive cells was stained by crystal violet. * P

    Techniques Used: Concentration Assay, CCK-8 Assay, Staining, Derivative Assay

    Cyp27A1 knockdown inhibited and Cyp7B1 promoted cholesterol-induced lung adenocarcinoma cells proliferation and invasion. A , B A549 and H1975 cells were transfected with lentivirus carrying shRNAs targeting Cyp27A1 or Cyp7B1. The expression of Cyp27A1 and Cyp7B1 were measured by western blot. C After transfected with shRNAs against Cyp27A1 or Cyp7B1, cells were treated with 1 μM 27-HC or 0.8 mg/ml cholesterol in the monoculture or coculture system with THP1-derived macrophages for 72 h. CCK-8 assay was performed to determine the cell viability. D Cells were cultured in monoculture system or coculture system exposed to 1 μM 27-HC or 0.8 mg/ml cholesterol for 72 h. Flow cytometer analysis was performed to determine the cell necrosis and apoptosis. E Cells were cultured in monoculture or coculture system exposed to 1 μM 27-HC or 0.8 mg/ml cholesterol for 24 h. Transwell assay were performed to determine cell invasion. * P
    Figure Legend Snippet: Cyp27A1 knockdown inhibited and Cyp7B1 promoted cholesterol-induced lung adenocarcinoma cells proliferation and invasion. A , B A549 and H1975 cells were transfected with lentivirus carrying shRNAs targeting Cyp27A1 or Cyp7B1. The expression of Cyp27A1 and Cyp7B1 were measured by western blot. C After transfected with shRNAs against Cyp27A1 or Cyp7B1, cells were treated with 1 μM 27-HC or 0.8 mg/ml cholesterol in the monoculture or coculture system with THP1-derived macrophages for 72 h. CCK-8 assay was performed to determine the cell viability. D Cells were cultured in monoculture system or coculture system exposed to 1 μM 27-HC or 0.8 mg/ml cholesterol for 72 h. Flow cytometer analysis was performed to determine the cell necrosis and apoptosis. E Cells were cultured in monoculture or coculture system exposed to 1 μM 27-HC or 0.8 mg/ml cholesterol for 24 h. Transwell assay were performed to determine cell invasion. * P

    Techniques Used: Transfection, Expressing, Western Blot, Derivative Assay, CCK-8 Assay, Cell Culture, Flow Cytometry, Transwell Assay

    15) Product Images from "Osimertinib and anti-HER3 combination therapy engages immune dependent tumor toxicity via STING activation in trans"

    Article Title: Osimertinib and anti-HER3 combination therapy engages immune dependent tumor toxicity via STING activation in trans

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-04701-3

    Stable H1975-BioSTING inducible cells, Fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET)
    Figure Legend Snippet: Stable H1975-BioSTING inducible cells, Fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET)

    Techniques Used: Fluorescence, Imaging, Microscopy, Förster Resonance Energy Transfer

    Osimertinib increases cGAMP production in tumor cells leading to type-I IFN production in macrophages. A Quantification of H1975-derived human type-I IFN. H1975 cells pre-treated with osimertinib (200 nM, 24 h) were treated with anti-HER3 (MP-RM-1, 10 μg/ml, 1 h) and then either co-cultured with bone marrow derived macrophages (BMDMs, 24 h, ratio 10:1) or maintained for 24 h; supernatants were assessed for ELISA as described in materials and methods. Data are shown as mean ± SEM from 3 independent experiments. Non-significant (ns) differences calculated using unpaired t -test. B Quantification of macrophage-derived murine type-I IFN. Supernatants from macrophages treated as indicated (24 h) or co-cultured with H1975 cells as described for Panel A , were assessed for ELISA as described in materials and methods. Data shown as mean ± SEM from 3 independent experiments. ** P
    Figure Legend Snippet: Osimertinib increases cGAMP production in tumor cells leading to type-I IFN production in macrophages. A Quantification of H1975-derived human type-I IFN. H1975 cells pre-treated with osimertinib (200 nM, 24 h) were treated with anti-HER3 (MP-RM-1, 10 μg/ml, 1 h) and then either co-cultured with bone marrow derived macrophages (BMDMs, 24 h, ratio 10:1) or maintained for 24 h; supernatants were assessed for ELISA as described in materials and methods. Data are shown as mean ± SEM from 3 independent experiments. Non-significant (ns) differences calculated using unpaired t -test. B Quantification of macrophage-derived murine type-I IFN. Supernatants from macrophages treated as indicated (24 h) or co-cultured with H1975 cells as described for Panel A , were assessed for ELISA as described in materials and methods. Data shown as mean ± SEM from 3 independent experiments. ** P

    Techniques Used: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    Combination therapy with osimertinib and anti-HER3 antibodies triggers macrophage-mediated FcγR-dependent cell toxicity. A H1975 cells pre-treated with osimertinib (Osi, 200 nM, 24 h) were treated with anti-HER3 (MP-RM-1, 10 μg/ml, 1 h) and then co-cultured with macrophages (2 h, ratio 10: 1) pre-labelled with cell tracker. Apoptosis of H1975 cells was assessed by flow cytometry excluding bone marrow-derived macrophages (BMDM) as described in materials and methods. Dot plots are representative of 3 independent replicates. B Quantification summary from the representative data shown in panel A . H1975 cells alone or co-cultured with macrophages were assessed for apoptosis using flow cytometry with DiOC 6 (3) for mitochondrial transmembrane potential (ΔΨ m ) and Dapi for cell viability, as described in materials and methods. FcγR block was added in co-culture conditions as indicated to assess FcγR function. Results shown as mean ± SEM from three replicates; * P
    Figure Legend Snippet: Combination therapy with osimertinib and anti-HER3 antibodies triggers macrophage-mediated FcγR-dependent cell toxicity. A H1975 cells pre-treated with osimertinib (Osi, 200 nM, 24 h) were treated with anti-HER3 (MP-RM-1, 10 μg/ml, 1 h) and then co-cultured with macrophages (2 h, ratio 10: 1) pre-labelled with cell tracker. Apoptosis of H1975 cells was assessed by flow cytometry excluding bone marrow-derived macrophages (BMDM) as described in materials and methods. Dot plots are representative of 3 independent replicates. B Quantification summary from the representative data shown in panel A . H1975 cells alone or co-cultured with macrophages were assessed for apoptosis using flow cytometry with DiOC 6 (3) for mitochondrial transmembrane potential (ΔΨ m ) and Dapi for cell viability, as described in materials and methods. FcγR block was added in co-culture conditions as indicated to assess FcγR function. Results shown as mean ± SEM from three replicates; * P

    Techniques Used: Cell Culture, Flow Cytometry, Derivative Assay, Blocking Assay, Co-Culture Assay

    Osimertinib triggers apoptosis in 2D, 3D and in vivo models, leading to HER3 upregulation and macrophage tumour-infiltration. A H1975 cells were treated in 2D at the indicated osimertinib (Osi) concentrations for 24 h; apoptosis was measured using flow cytometry with DiOC 6 (3) for mitochondrial transmembrane potential (ΔΨ m ) and PI for viability, as described in materials and methods. Representative dot plots show vehicle vs. 0.1 μM osimertinib-treated cells; the graph shows mean ± SEM of three experiments. ** P
    Figure Legend Snippet: Osimertinib triggers apoptosis in 2D, 3D and in vivo models, leading to HER3 upregulation and macrophage tumour-infiltration. A H1975 cells were treated in 2D at the indicated osimertinib (Osi) concentrations for 24 h; apoptosis was measured using flow cytometry with DiOC 6 (3) for mitochondrial transmembrane potential (ΔΨ m ) and PI for viability, as described in materials and methods. Representative dot plots show vehicle vs. 0.1 μM osimertinib-treated cells; the graph shows mean ± SEM of three experiments. ** P

    Techniques Used: In Vivo, Flow Cytometry

    IRE1α is required for osimertinib to trigger HER3 upregulation in the cell surface. A Near-infrared quantitative westernblot analysis of H1975 cells treated with 200 nM osimertinib (Osi) or ER-stressor brefeldin-A (Bf-A, 0.5 μg/ml) for 24 h. Densitometry levels of HER3 or IRE1α relative to GAPDH were quantified from 3 independent replicates, and are shown under the representative blot lanes as normalized mean values (a.u.), where ** P
    Figure Legend Snippet: IRE1α is required for osimertinib to trigger HER3 upregulation in the cell surface. A Near-infrared quantitative westernblot analysis of H1975 cells treated with 200 nM osimertinib (Osi) or ER-stressor brefeldin-A (Bf-A, 0.5 μg/ml) for 24 h. Densitometry levels of HER3 or IRE1α relative to GAPDH were quantified from 3 independent replicates, and are shown under the representative blot lanes as normalized mean values (a.u.), where ** P

    Techniques Used:

    Combination therapy with osimertinib and anti-HER3 antibodies shows increased efficacy and immune engagement in vivo. A Diagram illustrating in vivo xenograft experimental design; 2.5 × 10 6 H1975 cells were injected s.c . into CD1-nude mice. Once tumor volumes reached 200 mm 3 (day 15), mice were divided into 4 groups and treatments begun as indicated, with daily osimertinib (Osi) or vehicle (hydroxyl propyl methyl cellulose) oral gavage and/or i.p . injections every 3 days (PBS or Anti-HER3, MP-RM-1). After 7 days (day 22), 6 mice per group were collected for tumor analysis, n = 3 for flow cytometry and n = 3 for immunofluorescence (IF) microscopy studies; the n = 6 remaining mice per group were kept for tumor size monitoring until day 29. B . Tumor volumes are shown for each group as indicated, mean ± SD. Statistical significance with one-way ANOVA with Tukey’s multiple group comparison test revealed * P
    Figure Legend Snippet: Combination therapy with osimertinib and anti-HER3 antibodies shows increased efficacy and immune engagement in vivo. A Diagram illustrating in vivo xenograft experimental design; 2.5 × 10 6 H1975 cells were injected s.c . into CD1-nude mice. Once tumor volumes reached 200 mm 3 (day 15), mice were divided into 4 groups and treatments begun as indicated, with daily osimertinib (Osi) or vehicle (hydroxyl propyl methyl cellulose) oral gavage and/or i.p . injections every 3 days (PBS or Anti-HER3, MP-RM-1). After 7 days (day 22), 6 mice per group were collected for tumor analysis, n = 3 for flow cytometry and n = 3 for immunofluorescence (IF) microscopy studies; the n = 6 remaining mice per group were kept for tumor size monitoring until day 29. B . Tumor volumes are shown for each group as indicated, mean ± SD. Statistical significance with one-way ANOVA with Tukey’s multiple group comparison test revealed * P

    Techniques Used: In Vivo, Injection, Mouse Assay, Flow Cytometry, Immunofluorescence, Microscopy

    Addition of a STING agonist leads to increased type-I IFN production in macrophages and enhances macrophage-mediated tumor toxicity. A H1975 cells pre-treated with osimertinib (Osi, 200 nM, 24 h) and anti-HER3 (MP-RM-1, 10 μg/ml, 1 h) were co-cultured with M1-like bone marrow derived macrophages (BMDM) or BMDMs pre-treated with STING agonist (STago) 2’3’-cGAMP as indicated in materials and methods. Apoptosis of H1975 cells was assessed by flow cytometry excluding BMDMs; cells labelled with DiOC 6 (3) for mitochondrial transmembrane potential (ΔΨ m ) and Dapi for cell viability, as described in materials and methods. Dot plots are representative of 3 independent replicates. A quantification summary is shown in the lower graph. Results shown as mean ± SEM from three replicates; *** P
    Figure Legend Snippet: Addition of a STING agonist leads to increased type-I IFN production in macrophages and enhances macrophage-mediated tumor toxicity. A H1975 cells pre-treated with osimertinib (Osi, 200 nM, 24 h) and anti-HER3 (MP-RM-1, 10 μg/ml, 1 h) were co-cultured with M1-like bone marrow derived macrophages (BMDM) or BMDMs pre-treated with STING agonist (STago) 2’3’-cGAMP as indicated in materials and methods. Apoptosis of H1975 cells was assessed by flow cytometry excluding BMDMs; cells labelled with DiOC 6 (3) for mitochondrial transmembrane potential (ΔΨ m ) and Dapi for cell viability, as described in materials and methods. Dot plots are representative of 3 independent replicates. A quantification summary is shown in the lower graph. Results shown as mean ± SEM from three replicates; *** P

    Techniques Used: Cell Culture, Derivative Assay, Flow Cytometry

    16) Product Images from "Combining EGFR-TKI With SAHA Overcomes EGFR-TKI-Acquired Resistance by Reducing the Protective Autophagy in Non-Small Cell Lung Cancer"

    Article Title: Combining EGFR-TKI With SAHA Overcomes EGFR-TKI-Acquired Resistance by Reducing the Protective Autophagy in Non-Small Cell Lung Cancer

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2022.837987

    EZH2 is a negative regulator of autophagy. (A) EGFR-TKIs can cause decreased expression of EZH2 protein in PC-9/H1975 and corresponding drug-resistant cells. (B) Western blotting revealed lower levels of EZH2 protein and higher autophagic activation in drug-resistant cells. (C) Western blotting showed enhanced autophagic activity of drug-resistant cells following transfection with siEZH2. (D) Fluorescence assay showing representative images of cells transfected with GFP-RFP-LC3 double-labeled adenovirus. Autophagic flux was increased after knockdown of EZH2 in H1975 and H1975OR. Quantitative analysis of the number of yellow autophagosomes and red autolysosomes. * p
    Figure Legend Snippet: EZH2 is a negative regulator of autophagy. (A) EGFR-TKIs can cause decreased expression of EZH2 protein in PC-9/H1975 and corresponding drug-resistant cells. (B) Western blotting revealed lower levels of EZH2 protein and higher autophagic activation in drug-resistant cells. (C) Western blotting showed enhanced autophagic activity of drug-resistant cells following transfection with siEZH2. (D) Fluorescence assay showing representative images of cells transfected with GFP-RFP-LC3 double-labeled adenovirus. Autophagic flux was increased after knockdown of EZH2 in H1975 and H1975OR. Quantitative analysis of the number of yellow autophagosomes and red autolysosomes. * p

    Techniques Used: Expressing, Western Blot, Activation Assay, Activity Assay, Transfection, Fluorescence, Labeling

    EGFR-TKIs induced protective autophagy in NSCLC cells. (A) The CCK-8 method was used to detect parental PC-9 and H1975 cells and their corresponding drug-resistant cells, and the concentration of gefitinib or osimertinib was increased and treated for 48 h. Three replicates were used for the experiments, and data are presented as mean ± SEM. (B) EGFR-TKIs treatment decreased p62 protein levels and increased LC3-II accumulation in PC-9/H1975 and corresponding drug-resistant cells. (C) Fluorescence assay showing representative images of cells transfected with GFP-RFP-LC3 double-labeled adenovirus. Autophagic flux was significantly increased in H1975 and H1975OR after 1 μM osimertinib treatment for 48 h. Quantitative analysis of the number of yellow autophagosomes and red autolysosomes. **** p
    Figure Legend Snippet: EGFR-TKIs induced protective autophagy in NSCLC cells. (A) The CCK-8 method was used to detect parental PC-9 and H1975 cells and their corresponding drug-resistant cells, and the concentration of gefitinib or osimertinib was increased and treated for 48 h. Three replicates were used for the experiments, and data are presented as mean ± SEM. (B) EGFR-TKIs treatment decreased p62 protein levels and increased LC3-II accumulation in PC-9/H1975 and corresponding drug-resistant cells. (C) Fluorescence assay showing representative images of cells transfected with GFP-RFP-LC3 double-labeled adenovirus. Autophagic flux was significantly increased in H1975 and H1975OR after 1 μM osimertinib treatment for 48 h. Quantitative analysis of the number of yellow autophagosomes and red autolysosomes. **** p

    Techniques Used: CCK-8 Assay, Concentration Assay, Fluorescence, Transfection, Labeling

    17) Product Images from "An Efficient Aequorea victoria Green Fluorescent Protein for Stimulated Emission Depletion Super-Resolution Microscopy"

    Article Title: An Efficient Aequorea victoria Green Fluorescent Protein for Stimulated Emission Depletion Super-Resolution Microscopy

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23052482

    STED performances of c3GFP. ( a ) STED and confocal images of Vim-c3GFP in a H1975 cell (scale bar: 5 μM); the inset shows a zoom of the IF enclosed in the yellow dotted square region of the main panel (scale bar: 1 μM). ( b ) Fluorescence profile of the Vim-c3GFP IF along the white line visible in the inset of panel ( a ): the FWHM obtained by Gaussian fitting is reported. ( c ) STED and ( d ) confocal zooms of the cyan dotted square region of panel ( a ); scale bars: 1 μM. ( e ) Fluorescence profiles of the Vim-c3GFP IF along the white lines visible in ( c , d ) panels; STED: red line/markers; confocal: blue line/markers; the FWHM obtained by Gaussian fitting of the second STED peak is reported.
    Figure Legend Snippet: STED performances of c3GFP. ( a ) STED and confocal images of Vim-c3GFP in a H1975 cell (scale bar: 5 μM); the inset shows a zoom of the IF enclosed in the yellow dotted square region of the main panel (scale bar: 1 μM). ( b ) Fluorescence profile of the Vim-c3GFP IF along the white line visible in the inset of panel ( a ): the FWHM obtained by Gaussian fitting is reported. ( c ) STED and ( d ) confocal zooms of the cyan dotted square region of panel ( a ); scale bars: 1 μM. ( e ) Fluorescence profiles of the Vim-c3GFP IF along the white lines visible in ( c , d ) panels; STED: red line/markers; confocal: blue line/markers; the FWHM obtained by Gaussian fitting of the second STED peak is reported.

    Techniques Used: Fluorescence

    18) Product Images from "Anti–PD-L1 and anti-CD73 combination therapy promotes T cell response to EGFR-mutated NSCLC"

    Article Title: Anti–PD-L1 and anti-CD73 combination therapy promotes T cell response to EGFR-mutated NSCLC

    Journal: JCI Insight

    doi: 10.1172/jci.insight.142843

    High CD73 expression in EGFR-mutated NSCLC. ( A ) Representative histology images of CD73 expression in EGFR WT and EGFR-mutated (MT) tumors. Scale bar: 50 μm. ( B ) Semiquantitative pathological assessment of CD73 expression in EGFR WT and MT tumor specimens. Each circle represents the data from 1 NSCLC surgical resection. ( C ) CD73 (also known as NT5E) mRNA and protein expression in EGFR WT and MT cell lines. ( D ) CD73 protein expression in EGFR WT and MT cell lines treated with EGF or osimertinib for 48 hours. ( E ) CD73 protein expression in EGFR WT and MT cell lines treated with ERK, PKC, or NF-κB inhibitor for 48 hours. ( C–E ) Data are representative of 2 independent experiments. EGFR WT cell lines were H1355, H157, and H322; EGFR MT cell lines were H1975, HCC2935, and PC9. Student’s t test ( B and C ) and ANOVA ( D and E ) were used. Bars indicate the median in B and the mean in C–E . Data are shown as the mean ± SEM. * P
    Figure Legend Snippet: High CD73 expression in EGFR-mutated NSCLC. ( A ) Representative histology images of CD73 expression in EGFR WT and EGFR-mutated (MT) tumors. Scale bar: 50 μm. ( B ) Semiquantitative pathological assessment of CD73 expression in EGFR WT and MT tumor specimens. Each circle represents the data from 1 NSCLC surgical resection. ( C ) CD73 (also known as NT5E) mRNA and protein expression in EGFR WT and MT cell lines. ( D ) CD73 protein expression in EGFR WT and MT cell lines treated with EGF or osimertinib for 48 hours. ( E ) CD73 protein expression in EGFR WT and MT cell lines treated with ERK, PKC, or NF-κB inhibitor for 48 hours. ( C–E ) Data are representative of 2 independent experiments. EGFR WT cell lines were H1355, H157, and H322; EGFR MT cell lines were H1975, HCC2935, and PC9. Student’s t test ( B and C ) and ANOVA ( D and E ) were used. Bars indicate the median in B and the mean in C–E . Data are shown as the mean ± SEM. * P

    Techniques Used: Expressing

    19) Product Images from "FGFC1 Selectively Inhibits Erlotinib-Resistant Non-Small Cell Lung Cancer via Elevation of ROS Mediated by the EGFR/PI3K/Akt/mTOR Pathway"

    Article Title: FGFC1 Selectively Inhibits Erlotinib-Resistant Non-Small Cell Lung Cancer via Elevation of ROS Mediated by the EGFR/PI3K/Akt/mTOR Pathway

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2021.764699

    FGFC1 induced apoptosis in erlotinib-resistant NSCLC cells via mitochondrial dysfunction and accumulation of ROS. (A) FGFC1 significantly downregulated the MMP level. H1975, A549 and H1299 cells were treated with FGFC1 at different concentrations (0, 10 and 20 μM) for 24 h. The treated cells were stained with JC-1 dye and then analyzed by Flow cytometry. Cell populations indicating JC-1 aggregates (red) were in the upper right quadrant and JC-1 monomers (green) were in the lower right part. (B) The treated cells stained with JC-1 were pictured by fluorescence microscope to estimate the alteration in MMP level (scale bar = 100 μm). (C) FGFC1 significantly enhanced ROS production in H1975 cells. H1975, A549 and H1299 cells were incubated with the indicated concentrations of FGFC1 (0, 5, 10, and 20 μM) for 24 h, followed by ROS measurement. Representative flow cytometry histograms displaying levels of fluorescent DCFH-DA in cells were presented. (D) Statistical analysis of the percentage of ROS generation. (E) FGFC1 induced GSH depletion in H1975 cells. The above described cells were treated with various doses of FGFC1 (0, 5, 10, and 20 μM) for 24 h, and then the cellular GSH content was measured using an Assay Kit. (F) H1975 cells were pretreated with or without NAC (5 mM) for 2 h, then treated with FGFC1 (10 μM) and NAC (5 mM) alone or in combination for 24 h. Intra cellular ROS were measured by flow cytometry after 10 μM DCFH-DA staining. (G) Statistical analysis of the percentage of ROS generation. (H) H1975 cells were pretreated with or without NAC (5 mM) for 2 h, then treated with FGFC1 (10 μM) and NAC (5 mM) alone or in combination for 24 h. Cleaved-PARP, cleaved-caspase 3, Bax, Bcl-2 and p-Akt protein expression were evaluated by Western blotting. β-actin was detected as the endogenous loading control, accordingly. (I) The statistical result of (H) . All data were represented as the means ± SD for at least three independent experiments. ( *p
    Figure Legend Snippet: FGFC1 induced apoptosis in erlotinib-resistant NSCLC cells via mitochondrial dysfunction and accumulation of ROS. (A) FGFC1 significantly downregulated the MMP level. H1975, A549 and H1299 cells were treated with FGFC1 at different concentrations (0, 10 and 20 μM) for 24 h. The treated cells were stained with JC-1 dye and then analyzed by Flow cytometry. Cell populations indicating JC-1 aggregates (red) were in the upper right quadrant and JC-1 monomers (green) were in the lower right part. (B) The treated cells stained with JC-1 were pictured by fluorescence microscope to estimate the alteration in MMP level (scale bar = 100 μm). (C) FGFC1 significantly enhanced ROS production in H1975 cells. H1975, A549 and H1299 cells were incubated with the indicated concentrations of FGFC1 (0, 5, 10, and 20 μM) for 24 h, followed by ROS measurement. Representative flow cytometry histograms displaying levels of fluorescent DCFH-DA in cells were presented. (D) Statistical analysis of the percentage of ROS generation. (E) FGFC1 induced GSH depletion in H1975 cells. The above described cells were treated with various doses of FGFC1 (0, 5, 10, and 20 μM) for 24 h, and then the cellular GSH content was measured using an Assay Kit. (F) H1975 cells were pretreated with or without NAC (5 mM) for 2 h, then treated with FGFC1 (10 μM) and NAC (5 mM) alone or in combination for 24 h. Intra cellular ROS were measured by flow cytometry after 10 μM DCFH-DA staining. (G) Statistical analysis of the percentage of ROS generation. (H) H1975 cells were pretreated with or without NAC (5 mM) for 2 h, then treated with FGFC1 (10 μM) and NAC (5 mM) alone or in combination for 24 h. Cleaved-PARP, cleaved-caspase 3, Bax, Bcl-2 and p-Akt protein expression were evaluated by Western blotting. β-actin was detected as the endogenous loading control, accordingly. (I) The statistical result of (H) . All data were represented as the means ± SD for at least three independent experiments. ( *p

    Techniques Used: Staining, Flow Cytometry, Fluorescence, Microscopy, Incubation, Expressing, Western Blot

    FGFC1 selectively induced apoptosis in erlotinib-resistant NSCLC cells. (A) FGFC1 significantly enhanced H1975 cells death. H1975, A549 and H1299 cells were incubated with the indicated concentrations of FGFC1 (0, 5, 10, and 20 μM) for 48 h, and the cell apoptosis rates were detected by the Annexin-V FITC/PI staining assay. (B) The statistical result of (A) . (C) H1975, A549 and H1299 cells were treated with different concentrations of FGFC1 (0, 5, 10, and 20 μM) for 24 h. Cleaved-PARP, cleaved-caspase 3, Bax and Bcl-2 protein levels were examined by Western blotting. β-actin were detected as the endogenous loading control, accordingly. (D) The statistical result of (C) . All data were represented as the means ± SD for at least three independent experiments ( *p
    Figure Legend Snippet: FGFC1 selectively induced apoptosis in erlotinib-resistant NSCLC cells. (A) FGFC1 significantly enhanced H1975 cells death. H1975, A549 and H1299 cells were incubated with the indicated concentrations of FGFC1 (0, 5, 10, and 20 μM) for 48 h, and the cell apoptosis rates were detected by the Annexin-V FITC/PI staining assay. (B) The statistical result of (A) . (C) H1975, A549 and H1299 cells were treated with different concentrations of FGFC1 (0, 5, 10, and 20 μM) for 24 h. Cleaved-PARP, cleaved-caspase 3, Bax and Bcl-2 protein levels were examined by Western blotting. β-actin were detected as the endogenous loading control, accordingly. (D) The statistical result of (C) . All data were represented as the means ± SD for at least three independent experiments ( *p

    Techniques Used: Incubation, Staining, Western Blot

    FGFC1 selectively inhibited the migration and invasion of H1975 NSCLC cells. (A) The migration of H1975, A549 and H1299 cells was evaluated using the scratch wound-healing assay after cells treated with indicated concentrations of FGFC1. NSCLC cells were seeded in 6-well plates and were treated with the indicated dose of FGFC1 (0, 5, and 10 μM) for 24 h. Images of the results were obtained under microscope (200×) (scale bar = 100 μm). The migrated cells were quantified manually. (B) The invasion activity of H1975, A549 and H1299 cells treated with FGFC1 were measured by transwell invasion assay. NSCLC cells were seeded in 24-well plates and were treated with the indicated concentrations of FGFC1 (0, 5, and 10 μM). After 24 h, the invasive cells were stained with crystal violet, and photos were taken under microscope (200×). The invasion activity of these cells was measured by counting the staining positive cells (scale bar = 100 μm). (C) Quantification of the data in (A) . (D) Quantification of the data in (B) . All data were represented as the means ± SD for at least three independent experiments. ( *p
    Figure Legend Snippet: FGFC1 selectively inhibited the migration and invasion of H1975 NSCLC cells. (A) The migration of H1975, A549 and H1299 cells was evaluated using the scratch wound-healing assay after cells treated with indicated concentrations of FGFC1. NSCLC cells were seeded in 6-well plates and were treated with the indicated dose of FGFC1 (0, 5, and 10 μM) for 24 h. Images of the results were obtained under microscope (200×) (scale bar = 100 μm). The migrated cells were quantified manually. (B) The invasion activity of H1975, A549 and H1299 cells treated with FGFC1 were measured by transwell invasion assay. NSCLC cells were seeded in 24-well plates and were treated with the indicated concentrations of FGFC1 (0, 5, and 10 μM). After 24 h, the invasive cells were stained with crystal violet, and photos were taken under microscope (200×). The invasion activity of these cells was measured by counting the staining positive cells (scale bar = 100 μm). (C) Quantification of the data in (A) . (D) Quantification of the data in (B) . All data were represented as the means ± SD for at least three independent experiments. ( *p

    Techniques Used: Migration, Wound Healing Assay, Microscopy, Activity Assay, Transwell Invasion Assay, Staining

    FGFC1 meditated apoptosis through the EGFR pathway against erlotinib-resistant NSCLC cells. (A) FGFC1 remarkably suppressed the phosphorylation of EGFR and its downstream targets in H1975 cells. H1975, A549 and H1299 cells were treated with the indicated concentrations of FGFC1 (0, 5, 10, and 20 μM) for 24 h. The expression of p-EGFR, EGFR, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR was examined by Western blotting. β-actin was detected as the endogenous loading control. (B) The statistical result of (A) . (C) H1975 and PC9 cells were treated with a range of concentrations of erlotinib (0, 0.01, 0.05, and 0.1 μM) for 24 h, the cell lysates were subjected to Western blotting with the indicated antibodies. (D) The statistical result of (C) . (E) H1975 cells were treated with FGFC1 (10 μM) and LY294002 (20 μM) alone or in combination for 24 h. The expression of p-EGFR, EGFR, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, mTOR, cleaved-caspase3, cleaved-PARP, Bax, and Bcl-2 was evaluated by Western blotting. β-actin was detected as the endogenous loading control, accordingly. (F) The statistical result of (E) . (G) H1975 cells were treated with FGFC1 (10 μM) and LY294002 (20 μM) alone or in combination for 24 h. Intra cellular ROS were measured by flow cytometry after 10 μM DCFH-DA staining. (H) Statistical analysis of the percentage of ROS generation. (I) Docking score of FGFC1 with EGFR T790M/L858R and EGFR WT . (J) The binding mode of FGFC1 docked into EGFR T790M/L858R and EGFR WT , respectively. Data shown were representative of three independent experiments. All data were represented as the means ± SD for at least three independent experiments. ( *p
    Figure Legend Snippet: FGFC1 meditated apoptosis through the EGFR pathway against erlotinib-resistant NSCLC cells. (A) FGFC1 remarkably suppressed the phosphorylation of EGFR and its downstream targets in H1975 cells. H1975, A549 and H1299 cells were treated with the indicated concentrations of FGFC1 (0, 5, 10, and 20 μM) for 24 h. The expression of p-EGFR, EGFR, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR was examined by Western blotting. β-actin was detected as the endogenous loading control. (B) The statistical result of (A) . (C) H1975 and PC9 cells were treated with a range of concentrations of erlotinib (0, 0.01, 0.05, and 0.1 μM) for 24 h, the cell lysates were subjected to Western blotting with the indicated antibodies. (D) The statistical result of (C) . (E) H1975 cells were treated with FGFC1 (10 μM) and LY294002 (20 μM) alone or in combination for 24 h. The expression of p-EGFR, EGFR, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, mTOR, cleaved-caspase3, cleaved-PARP, Bax, and Bcl-2 was evaluated by Western blotting. β-actin was detected as the endogenous loading control, accordingly. (F) The statistical result of (E) . (G) H1975 cells were treated with FGFC1 (10 μM) and LY294002 (20 μM) alone or in combination for 24 h. Intra cellular ROS were measured by flow cytometry after 10 μM DCFH-DA staining. (H) Statistical analysis of the percentage of ROS generation. (I) Docking score of FGFC1 with EGFR T790M/L858R and EGFR WT . (J) The binding mode of FGFC1 docked into EGFR T790M/L858R and EGFR WT , respectively. Data shown were representative of three independent experiments. All data were represented as the means ± SD for at least three independent experiments. ( *p

    Techniques Used: Expressing, Western Blot, Flow Cytometry, Staining, Binding Assay

    FGFC1 inhibited the growth of H1975 cell-derived tumor in vivo . H1975 cells were subcutaneously inoculated into the flank of nude mice. The mice were randomized into three groups. (A) Tumor volume was measured by caliper once every 3 days when the tumor reached approximately 60 mm 3 in size. (B) 7 days after H1975 cells implantation, mice were treated with vehicle (5% DMSO in PBS, ip), FGFC1 (10 mg/kg, ip) and erlotinib (10 mg/kg, ip) once a day for 21 consecutive days. The body weight was quantified in each group. (C) The representative stripped images of the tumor entity after being treated with vehicle, FGFC1 and erlotinib for 21 days. (D) The scatter plot summarized the weight of the tumors. (E) FGFC1 decreased the expression of p-EGFR, Ki-67 in vivo . The expression of p-EGFR and Ki-67 in tumor tissues from nude mice was assessed by immunohistochemistry (400×) (scale bar = 50 μm). (F) The IHC score of p-EGFR and Ki-67 was quantified by the IRS system ( n = 10 fields of view). All data were represented as the means ± SD for at least three independent experiments. ( *p
    Figure Legend Snippet: FGFC1 inhibited the growth of H1975 cell-derived tumor in vivo . H1975 cells were subcutaneously inoculated into the flank of nude mice. The mice were randomized into three groups. (A) Tumor volume was measured by caliper once every 3 days when the tumor reached approximately 60 mm 3 in size. (B) 7 days after H1975 cells implantation, mice were treated with vehicle (5% DMSO in PBS, ip), FGFC1 (10 mg/kg, ip) and erlotinib (10 mg/kg, ip) once a day for 21 consecutive days. The body weight was quantified in each group. (C) The representative stripped images of the tumor entity after being treated with vehicle, FGFC1 and erlotinib for 21 days. (D) The scatter plot summarized the weight of the tumors. (E) FGFC1 decreased the expression of p-EGFR, Ki-67 in vivo . The expression of p-EGFR and Ki-67 in tumor tissues from nude mice was assessed by immunohistochemistry (400×) (scale bar = 50 μm). (F) The IHC score of p-EGFR and Ki-67 was quantified by the IRS system ( n = 10 fields of view). All data were represented as the means ± SD for at least three independent experiments. ( *p

    Techniques Used: Derivative Assay, In Vivo, Mouse Assay, Expressing, Immunohistochemistry

    The cytotoxic effects of FGFC1 on different cancer cells. (A) Chemical structure of FGFC1 (MW = 869.4947). (B) NSCLC cell lines (PC9) were more sensitive to FGFC1 than the other four cell lines for 72 h. PC9, HCT116, HeLa, B16, and 293T cells were incubated with the indicated concentrations of DMSO or FGFC1 (0.01, 0.1, 1, 2, 5, 10, 20, 40, 80, and 100 μM) for 72 h. Cell viability was assessed using CCK-8 assay and shown as relative viability compared to the untreated control. Each test was performed in triplicate. (C) The IC 50 values of FGFC1 in PC9, HCT116, HeLa, B16, and 293T cells were assessed and expressed as the mean ± SD ( n = 3). (D) Above described cells were treated with different concentrations of FGFC1 (0, 5, and, 10 μM) for 72 h. Cell morphology was imaged under microscope (200×). Scale bar = 100 μm. (E) FGFC1 selectively inhibited the growth of H1975 erlotinib-resistant NSCLC cells in a dose-dependent manner. H1975, A549, H1299, and BEAS-2B cells were treated with FGFC1 (0.01, 0.1, 1, 2, 5, 10, 20, 40, 80, and 100 μM) for 72 h, respectively. Cell viability was examined by CCK-8 assay and shown as relative viability compared to the untreated control. Each test was performed in triplicate. (F) The IC 50 values of FGFC1, the EGFR mutations status, and the erlotinib sensitivity of these four cells were assessed and expressed as the mean ± SD ( n = 3). (G) H1975, A549, H1299, and BEAS-2B cells were treated with different concentrations of FGFC1 (0, 5, and 10 μM) for 72 h. Cell morphology was imaged under microscope (200×). Scale bar = 100 μm. (H) FGFC1 remarkably increased the LDH leakage of H1975 cells. The above described cells were incubated with the indicated concentrations of DMSO or FGFC1 (0, 5, 10, and 20 μM) for 48 h. The cytotoxicity of FGFC1 on these cells was examined by the release of LDH. (I) FGFC1 significantly inhibited colony formation ability of H1975 cells in a dose-dependent manner. Colony formation of H1975 and A549 cells was monitored after FGFC1 (0, 5, 10, and 20 μM) treatment for 14 days, and photographs of crystal violet stained colonies were depicted. (J) The statistical result of (I) . All data were represented as the means ± SD for at least three independent experiments. ( *p
    Figure Legend Snippet: The cytotoxic effects of FGFC1 on different cancer cells. (A) Chemical structure of FGFC1 (MW = 869.4947). (B) NSCLC cell lines (PC9) were more sensitive to FGFC1 than the other four cell lines for 72 h. PC9, HCT116, HeLa, B16, and 293T cells were incubated with the indicated concentrations of DMSO or FGFC1 (0.01, 0.1, 1, 2, 5, 10, 20, 40, 80, and 100 μM) for 72 h. Cell viability was assessed using CCK-8 assay and shown as relative viability compared to the untreated control. Each test was performed in triplicate. (C) The IC 50 values of FGFC1 in PC9, HCT116, HeLa, B16, and 293T cells were assessed and expressed as the mean ± SD ( n = 3). (D) Above described cells were treated with different concentrations of FGFC1 (0, 5, and, 10 μM) for 72 h. Cell morphology was imaged under microscope (200×). Scale bar = 100 μm. (E) FGFC1 selectively inhibited the growth of H1975 erlotinib-resistant NSCLC cells in a dose-dependent manner. H1975, A549, H1299, and BEAS-2B cells were treated with FGFC1 (0.01, 0.1, 1, 2, 5, 10, 20, 40, 80, and 100 μM) for 72 h, respectively. Cell viability was examined by CCK-8 assay and shown as relative viability compared to the untreated control. Each test was performed in triplicate. (F) The IC 50 values of FGFC1, the EGFR mutations status, and the erlotinib sensitivity of these four cells were assessed and expressed as the mean ± SD ( n = 3). (G) H1975, A549, H1299, and BEAS-2B cells were treated with different concentrations of FGFC1 (0, 5, and 10 μM) for 72 h. Cell morphology was imaged under microscope (200×). Scale bar = 100 μm. (H) FGFC1 remarkably increased the LDH leakage of H1975 cells. The above described cells were incubated with the indicated concentrations of DMSO or FGFC1 (0, 5, 10, and 20 μM) for 48 h. The cytotoxicity of FGFC1 on these cells was examined by the release of LDH. (I) FGFC1 significantly inhibited colony formation ability of H1975 cells in a dose-dependent manner. Colony formation of H1975 and A549 cells was monitored after FGFC1 (0, 5, 10, and 20 μM) treatment for 14 days, and photographs of crystal violet stained colonies were depicted. (J) The statistical result of (I) . All data were represented as the means ± SD for at least three independent experiments. ( *p

    Techniques Used: Incubation, CCK-8 Assay, Microscopy, Staining

    20) Product Images from "Enhanced nuclear localization of YAP1‐2 contributes to EGF‐induced EMT in NSCLC, et al. Enhanced nuclear localization of YAP1‐2 contributes to EGF‐induced EMT in NSCLC"

    Article Title: Enhanced nuclear localization of YAP1‐2 contributes to EGF‐induced EMT in NSCLC, et al. Enhanced nuclear localization of YAP1‐2 contributes to EGF‐induced EMT in NSCLC

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.17150

    EGF upregulates YAP1 and promotes lung cancer epithelial‐mesenchymal transition (EMT). (A,B) A549 cells were treated with 25 ng/ml EGF according to the time table, Western blots (A) and qPCR (B) were used to detect the expression of EMT‐related proteins. (C,E) A549, H460 and H1975 cells were pre‐treated with 25 ng/ml EGF for 72 h, the scratch healing assay (C) and trans‐well assay (E) were performed to detect the migration ability. (D,F) Statistic analysis of the scratch healing assay (D) and trans‐well assay (F). * p
    Figure Legend Snippet: EGF upregulates YAP1 and promotes lung cancer epithelial‐mesenchymal transition (EMT). (A,B) A549 cells were treated with 25 ng/ml EGF according to the time table, Western blots (A) and qPCR (B) were used to detect the expression of EMT‐related proteins. (C,E) A549, H460 and H1975 cells were pre‐treated with 25 ng/ml EGF for 72 h, the scratch healing assay (C) and trans‐well assay (E) were performed to detect the migration ability. (D,F) Statistic analysis of the scratch healing assay (D) and trans‐well assay (F). * p

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Migration

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  • 98
    ATCC nci h1975
    miR-455-5p overexpression decreases T cell apoptosis. ( A ) Representative flow cytometry histogram plot (upper panel) and quantification (lower panel) of PD-1 MFI in Jurkat cells stimulated either with ± CD3/CD28/CD2 soluble antibody complexes for 72 hours. Results are shown as fold change of MFI relative to not active cells. Data are mean ± SEM (N=4). P-value was calculated by one sample t-test. ( B and C ) <t>NCI-H1975</t> cells transfected with the indicated oligos were exposed to IFN-γ for 8 hours and then co-cultured for 72 hours with Jurkat cells in the presence of T cell activator. (B) Representative flow cytometry histogram plots (left) and quantification (right) of PD-L1 MFI at the indicated experimental conditions. Results are shown as fold change of MFI relative to control conditions. Data are mean ± SEM (N=4). P-values were calculated by one sample t-test. (C) Analysis of Jurkat apoptosis rate co-cultured with the indicated cell lines by AnnexinV/7-AAD staining. Right panels: Representative flow cytometric plots (left) and quantification (right) of apoptotic dead Jurkat cells (Annexin V+, 7-AAD+; highlighted in red). Results are shown as fold change of apoptotic dead cells relative to matched control conditions. Data are mean ± SEM (N=4). P-values were calculated by one sample t-test. ( D-E-F ) Distribution of the percentage of CD8+ cells and miR-455-5p expression, expressed as z-score, in NSCLC primary tumors from CD8-CIMA-CUN (D), CD8-CSS Cohort (E) and after pooling together the two cohorts (F). Tumors were stratified in high and low CD8-tumors based on the median value of CD8+ z-score. ( G ) Left: correlation analysis of miR-455-5p levels with PD-L1 mRNA, gene signature for exhausted CD8+ T cell (GET), IFN-γ and IFN-α response in tumors from TGCA-LUAD and TGCA-LUSC cohorts. Bubble plots reported the correlation coefficients for miR-455-5p expression with the indicated variables. The size of the bubbles indicates statistical significance calculated by the Spearman correlation analysis. Right: Bar plot reporting the Thorsson immune subtype of TGCA-LUAD and - LUSC tumors according to miR-455-5p expression. P-value was calculated by using the t test for equality of proportions (High vs Low). ****P
    Nci H1975, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nci h1975 - by Bioz Stars, 2022-12
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    90
    ATCC human lung adenocarcinoma nci h1975 cells
    KW6002 limits cisplatin accumulation in the kidney but not in tumors. RPTEC/hTERT1 and <t>H1975</t> <t>cells</t> were exposed to cisplatin and/or KW6002. n = 3 independent experiments. ( A and E ) Platinum quantification (ng/μg proteins) in RPTEC/hTERT1 ( A ) and H1975 ( E ) cells. *** P
    Human Lung Adenocarcinoma Nci H1975 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human lung adenocarcinoma nci h1975 cells/product/ATCC
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    Price from $9.99 to $1999.99
    human lung adenocarcinoma nci h1975 cells - by Bioz Stars, 2022-12
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    Image Search Results


    miR-455-5p overexpression decreases T cell apoptosis. ( A ) Representative flow cytometry histogram plot (upper panel) and quantification (lower panel) of PD-1 MFI in Jurkat cells stimulated either with ± CD3/CD28/CD2 soluble antibody complexes for 72 hours. Results are shown as fold change of MFI relative to not active cells. Data are mean ± SEM (N=4). P-value was calculated by one sample t-test. ( B and C ) NCI-H1975 cells transfected with the indicated oligos were exposed to IFN-γ for 8 hours and then co-cultured for 72 hours with Jurkat cells in the presence of T cell activator. (B) Representative flow cytometry histogram plots (left) and quantification (right) of PD-L1 MFI at the indicated experimental conditions. Results are shown as fold change of MFI relative to control conditions. Data are mean ± SEM (N=4). P-values were calculated by one sample t-test. (C) Analysis of Jurkat apoptosis rate co-cultured with the indicated cell lines by AnnexinV/7-AAD staining. Right panels: Representative flow cytometric plots (left) and quantification (right) of apoptotic dead Jurkat cells (Annexin V+, 7-AAD+; highlighted in red). Results are shown as fold change of apoptotic dead cells relative to matched control conditions. Data are mean ± SEM (N=4). P-values were calculated by one sample t-test. ( D-E-F ) Distribution of the percentage of CD8+ cells and miR-455-5p expression, expressed as z-score, in NSCLC primary tumors from CD8-CIMA-CUN (D), CD8-CSS Cohort (E) and after pooling together the two cohorts (F). Tumors were stratified in high and low CD8-tumors based on the median value of CD8+ z-score. ( G ) Left: correlation analysis of miR-455-5p levels with PD-L1 mRNA, gene signature for exhausted CD8+ T cell (GET), IFN-γ and IFN-α response in tumors from TGCA-LUAD and TGCA-LUSC cohorts. Bubble plots reported the correlation coefficients for miR-455-5p expression with the indicated variables. The size of the bubbles indicates statistical significance calculated by the Spearman correlation analysis. Right: Bar plot reporting the Thorsson immune subtype of TGCA-LUAD and - LUSC tumors according to miR-455-5p expression. P-value was calculated by using the t test for equality of proportions (High vs Low). ****P

    Journal: bioRxiv

    Article Title: miRNome profiling of lung cancer metastases revealed a key role for miRNA-PD-L1 axis in the modulation of chemotherapy response

    doi: 10.1101/2022.11.23.517634

    Figure Lengend Snippet: miR-455-5p overexpression decreases T cell apoptosis. ( A ) Representative flow cytometry histogram plot (upper panel) and quantification (lower panel) of PD-1 MFI in Jurkat cells stimulated either with ± CD3/CD28/CD2 soluble antibody complexes for 72 hours. Results are shown as fold change of MFI relative to not active cells. Data are mean ± SEM (N=4). P-value was calculated by one sample t-test. ( B and C ) NCI-H1975 cells transfected with the indicated oligos were exposed to IFN-γ for 8 hours and then co-cultured for 72 hours with Jurkat cells in the presence of T cell activator. (B) Representative flow cytometry histogram plots (left) and quantification (right) of PD-L1 MFI at the indicated experimental conditions. Results are shown as fold change of MFI relative to control conditions. Data are mean ± SEM (N=4). P-values were calculated by one sample t-test. (C) Analysis of Jurkat apoptosis rate co-cultured with the indicated cell lines by AnnexinV/7-AAD staining. Right panels: Representative flow cytometric plots (left) and quantification (right) of apoptotic dead Jurkat cells (Annexin V+, 7-AAD+; highlighted in red). Results are shown as fold change of apoptotic dead cells relative to matched control conditions. Data are mean ± SEM (N=4). P-values were calculated by one sample t-test. ( D-E-F ) Distribution of the percentage of CD8+ cells and miR-455-5p expression, expressed as z-score, in NSCLC primary tumors from CD8-CIMA-CUN (D), CD8-CSS Cohort (E) and after pooling together the two cohorts (F). Tumors were stratified in high and low CD8-tumors based on the median value of CD8+ z-score. ( G ) Left: correlation analysis of miR-455-5p levels with PD-L1 mRNA, gene signature for exhausted CD8+ T cell (GET), IFN-γ and IFN-α response in tumors from TGCA-LUAD and TGCA-LUSC cohorts. Bubble plots reported the correlation coefficients for miR-455-5p expression with the indicated variables. The size of the bubbles indicates statistical significance calculated by the Spearman correlation analysis. Right: Bar plot reporting the Thorsson immune subtype of TGCA-LUAD and - LUSC tumors according to miR-455-5p expression. P-value was calculated by using the t test for equality of proportions (High vs Low). ****P

    Article Snippet: Cell lines NCI-H2023, NCI-H1993, NCI-H1975, NCI-H838, NCI-H1944, NCI-H1437, NCI-H1573, NCI-H2126, NCI-H322M, BEAS-2B and Jurkat were obtained from ATCC and cultured in RPMI (Gibco) with 5% FBS, 1% penicillin/streptomycin except for Jurkat medium, which was supplemented with 10% FBS.

    Techniques: Over Expression, Flow Cytometry, Transfection, Cell Culture, Staining, Expressing

    miR-455-5p modulates cisplatin resistance in vitro and in vivo . ( A ) qRT-PCR of miR-455-5p in NCI-H1993 transfected with a miR-455-5p mimic (NCI-H1993 OE) or a negative control mimic (NCI-H1993 CTRL). Data, expressed as normalized Cq (Cqn), are mean ± SEM (N=5). P-value was calculated by the Mann-Whitney U test. ( B ) Dose-response curves of NCI-H1993 CTRL and NCI-H1993 OE cells treated with cisplatin for 72 hours. Error bars indicate SEM (N=4). ( C ) Bar plot of cisplatin potency and efficacy of NCI-H1993 CTRL and NCI-H1993 OE cells. Data are mean ± SEM (N=4). Fold change is relative to NCI-H1993 CTRL. P-value was calculated by one sample t-test. *P

    Journal: bioRxiv

    Article Title: miRNome profiling of lung cancer metastases revealed a key role for miRNA-PD-L1 axis in the modulation of chemotherapy response

    doi: 10.1101/2022.11.23.517634

    Figure Lengend Snippet: miR-455-5p modulates cisplatin resistance in vitro and in vivo . ( A ) qRT-PCR of miR-455-5p in NCI-H1993 transfected with a miR-455-5p mimic (NCI-H1993 OE) or a negative control mimic (NCI-H1993 CTRL). Data, expressed as normalized Cq (Cqn), are mean ± SEM (N=5). P-value was calculated by the Mann-Whitney U test. ( B ) Dose-response curves of NCI-H1993 CTRL and NCI-H1993 OE cells treated with cisplatin for 72 hours. Error bars indicate SEM (N=4). ( C ) Bar plot of cisplatin potency and efficacy of NCI-H1993 CTRL and NCI-H1993 OE cells. Data are mean ± SEM (N=4). Fold change is relative to NCI-H1993 CTRL. P-value was calculated by one sample t-test. *P

    Article Snippet: Cell lines NCI-H2023, NCI-H1993, NCI-H1975, NCI-H838, NCI-H1944, NCI-H1437, NCI-H1573, NCI-H2126, NCI-H322M, BEAS-2B and Jurkat were obtained from ATCC and cultured in RPMI (Gibco) with 5% FBS, 1% penicillin/streptomycin except for Jurkat medium, which was supplemented with 10% FBS.

    Techniques: In Vitro, In Vivo, Quantitative RT-PCR, Transfection, Negative Control, MANN-WHITNEY

    miR-455-5p modulates the expression of genes involved in interferon response. ( A ) Volcano plot showing differentially expressed genes found by microarray analysis. Left panel: pN2 vs. pN0 (MED-samples). Central panel: NCI-H1993 CTRL vs NCI-H1993 OE cells (N=2). Right panel: CDDP-R CTRL (N=2) vs CDDP-R OE cells (N=2). Grey dot, unchanged genes; Blue dot, downregulated genes (p-value

    Journal: bioRxiv

    Article Title: miRNome profiling of lung cancer metastases revealed a key role for miRNA-PD-L1 axis in the modulation of chemotherapy response

    doi: 10.1101/2022.11.23.517634

    Figure Lengend Snippet: miR-455-5p modulates the expression of genes involved in interferon response. ( A ) Volcano plot showing differentially expressed genes found by microarray analysis. Left panel: pN2 vs. pN0 (MED-samples). Central panel: NCI-H1993 CTRL vs NCI-H1993 OE cells (N=2). Right panel: CDDP-R CTRL (N=2) vs CDDP-R OE cells (N=2). Grey dot, unchanged genes; Blue dot, downregulated genes (p-value

    Article Snippet: Cell lines NCI-H2023, NCI-H1993, NCI-H1975, NCI-H838, NCI-H1944, NCI-H1437, NCI-H1573, NCI-H2126, NCI-H322M, BEAS-2B and Jurkat were obtained from ATCC and cultured in RPMI (Gibco) with 5% FBS, 1% penicillin/streptomycin except for Jurkat medium, which was supplemented with 10% FBS.

    Techniques: Expressing, Microarray

    LN-signature predicts chemotherapy response of chemo-naïve lung metastatic tumor tissue collected by mediastinoscopy. ( A ) Hierarchical clustering analysis of the LN-signature in MED-samples. Data are log 2 -ratio. LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; NSCLC, other non-small cell lung subtypes; NA, no available data. ( B ) Heat map showing gene expression of the indicated marker analyzed by qRT-PCR in LNmets (EBUS-samples, N=5; and MED-samples, N=5). NCI-H2023 and NCI-H1993 lung cancer cells (LC, yellow) were used as positive controls for the expression of epithelial marker while HUVEC (EN, orange), WI38 (FI, red) and HL-60 cells (LK, magenta) were used as positive control for endothelial, fibroblast and immune-like markers expression, respectively. Data are log 2 -ratio. ( C ) Pie chart showing the number of miRNAs of LN-signature (N=16) that were found differentially expressed between pN0 and pN2 samples in MED-cohort. ( D ) Schematic representation of strategy adopted to derive miRNA-based NACT predictive models. ( E to G ) Upper panels: receiver operating characteristic (ROC) curves of the 16-miRNA model (E), 14-miRNA model (F) and 4-miRNA model (G) in the validation set (MED-samples, red). Lower panels : box plot of the predicted probability of being a responder according to the 16-miRNA model (E), 14-miRNA model (F) and 4-miRNA model (G).

    Journal: bioRxiv

    Article Title: miRNome profiling of lung cancer metastases revealed a key role for miRNA-PD-L1 axis in the modulation of chemotherapy response

    doi: 10.1101/2022.11.23.517634

    Figure Lengend Snippet: LN-signature predicts chemotherapy response of chemo-naïve lung metastatic tumor tissue collected by mediastinoscopy. ( A ) Hierarchical clustering analysis of the LN-signature in MED-samples. Data are log 2 -ratio. LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; NSCLC, other non-small cell lung subtypes; NA, no available data. ( B ) Heat map showing gene expression of the indicated marker analyzed by qRT-PCR in LNmets (EBUS-samples, N=5; and MED-samples, N=5). NCI-H2023 and NCI-H1993 lung cancer cells (LC, yellow) were used as positive controls for the expression of epithelial marker while HUVEC (EN, orange), WI38 (FI, red) and HL-60 cells (LK, magenta) were used as positive control for endothelial, fibroblast and immune-like markers expression, respectively. Data are log 2 -ratio. ( C ) Pie chart showing the number of miRNAs of LN-signature (N=16) that were found differentially expressed between pN0 and pN2 samples in MED-cohort. ( D ) Schematic representation of strategy adopted to derive miRNA-based NACT predictive models. ( E to G ) Upper panels: receiver operating characteristic (ROC) curves of the 16-miRNA model (E), 14-miRNA model (F) and 4-miRNA model (G) in the validation set (MED-samples, red). Lower panels : box plot of the predicted probability of being a responder according to the 16-miRNA model (E), 14-miRNA model (F) and 4-miRNA model (G).

    Article Snippet: Cell lines NCI-H2023, NCI-H1993, NCI-H1975, NCI-H838, NCI-H1944, NCI-H1437, NCI-H1573, NCI-H2126, NCI-H322M, BEAS-2B and Jurkat were obtained from ATCC and cultured in RPMI (Gibco) with 5% FBS, 1% penicillin/streptomycin except for Jurkat medium, which was supplemented with 10% FBS.

    Techniques: Expressing, Marker, Quantitative RT-PCR, Positive Control

    miRNA-expression profiling of LNmets collected by EBUS-TBNA. ( A ) Strategy used for miRNA-expression profiling of LNmets NSCLC cells (EBUS-samples). ( B ) Upper panels: brightfield images of three representative primary LNmets cell lines obtained as described in (A). Scale bar, 100 µm. Lower panels: representative confocal analysis of Pan-Citokeratins (PanCK) in LNmets cell lines. Pan-Citokeratins (red) identifies epithelial cells; DAPI (light blue) visualizes nuclei. Scale bar: 50µm. (C) Heat map showing qRT-PCR results of airway cell markers in five individual LN-metastatic cell lines. Two commercial lung cancer cells (LC; yellow) established from LNmets of stage IIIA NSCLC patients (NCI-H2023 and NCI-H1993) were used as positive controls for airway markers expression, while the breast cancer cells (BC; orange) MDA-MB-231 and leukemic cells (LK; magenta) HL-60 were used as negative controls. Data are log 2 -ratio. ( D ) Bar plot showing the number and percentage of miRNAs detected (yellow) or not detected (blue) in EBUS-samples. ( E ) Violin plot showing expression levels (Cqn) of all miRNAs detected in EBUS-samples. ( F ) Volcano plot showing differentially expressed miRNAs in chemoresistant (pN2; N=7) vs. chemosensitive (pN0; N=5) LNmets. Grey dot, unchanged; Blue dot, downregulated (p

    Journal: bioRxiv

    Article Title: miRNome profiling of lung cancer metastases revealed a key role for miRNA-PD-L1 axis in the modulation of chemotherapy response

    doi: 10.1101/2022.11.23.517634

    Figure Lengend Snippet: miRNA-expression profiling of LNmets collected by EBUS-TBNA. ( A ) Strategy used for miRNA-expression profiling of LNmets NSCLC cells (EBUS-samples). ( B ) Upper panels: brightfield images of three representative primary LNmets cell lines obtained as described in (A). Scale bar, 100 µm. Lower panels: representative confocal analysis of Pan-Citokeratins (PanCK) in LNmets cell lines. Pan-Citokeratins (red) identifies epithelial cells; DAPI (light blue) visualizes nuclei. Scale bar: 50µm. (C) Heat map showing qRT-PCR results of airway cell markers in five individual LN-metastatic cell lines. Two commercial lung cancer cells (LC; yellow) established from LNmets of stage IIIA NSCLC patients (NCI-H2023 and NCI-H1993) were used as positive controls for airway markers expression, while the breast cancer cells (BC; orange) MDA-MB-231 and leukemic cells (LK; magenta) HL-60 were used as negative controls. Data are log 2 -ratio. ( D ) Bar plot showing the number and percentage of miRNAs detected (yellow) or not detected (blue) in EBUS-samples. ( E ) Violin plot showing expression levels (Cqn) of all miRNAs detected in EBUS-samples. ( F ) Volcano plot showing differentially expressed miRNAs in chemoresistant (pN2; N=7) vs. chemosensitive (pN0; N=5) LNmets. Grey dot, unchanged; Blue dot, downregulated (p

    Article Snippet: Cell lines NCI-H2023, NCI-H1993, NCI-H1975, NCI-H838, NCI-H1944, NCI-H1437, NCI-H1573, NCI-H2126, NCI-H322M, BEAS-2B and Jurkat were obtained from ATCC and cultured in RPMI (Gibco) with 5% FBS, 1% penicillin/streptomycin except for Jurkat medium, which was supplemented with 10% FBS.

    Techniques: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification

    Pictures of NCI-H1975 cells cultured on monolayer and PCL scaffolds for 3 and 6 days displayed by confocal laser scanning microscope (CLSM) at a magnification of ×200 (scale bars 100 µm). Actin cytoskeleton was stained with rhodamine-phalloidin (red) and nucleus with DAPI (blue).

    Journal: Scientific Reports

    Article Title: The solvent chosen for the manufacturing of electrospun polycaprolactone scaffolds influences cell behavior of lung cancer cells

    doi: 10.1038/s41598-022-23655-2

    Figure Lengend Snippet: Pictures of NCI-H1975 cells cultured on monolayer and PCL scaffolds for 3 and 6 days displayed by confocal laser scanning microscope (CLSM) at a magnification of ×200 (scale bars 100 µm). Actin cytoskeleton was stained with rhodamine-phalloidin (red) and nucleus with DAPI (blue).

    Article Snippet: NCI-H1975 human NSCLC cell line was purchased from the American Type Culture Collection (ATCC; Rockvville, MD, USA).

    Techniques: Cell Culture, Laser-Scanning Microscopy, Confocal Laser Scanning Microscopy, Staining

    Scanning electronic microscopy (SEM) images from NCI-H1975 cells attached to ( A ) AA-PCL scaffolds, ( B ) AA-FA-PCL scaffolds, ( C ) Ace-PCL scaffolds, ( D ) Chl-EtOH-PCL scaffolds, and ( E ) Chl-DCM-PCL scaffolds (scale bar: 30 μm). ( F ) SEM picture from a NCI-H1975 cell attached to a Chl-DCM-PCL nanofiber due to roughness (scale bar 6 μm). ( G) SEM picture from NCI-H1975 cells attached to a monolayer (scale bar 300 μm). ( H) SEM picture from NCI-H1975 cells attached to a monolayer (scale bar 30 μm). Representative cells are indicated by arrows.

    Journal: Scientific Reports

    Article Title: The solvent chosen for the manufacturing of electrospun polycaprolactone scaffolds influences cell behavior of lung cancer cells

    doi: 10.1038/s41598-022-23655-2

    Figure Lengend Snippet: Scanning electronic microscopy (SEM) images from NCI-H1975 cells attached to ( A ) AA-PCL scaffolds, ( B ) AA-FA-PCL scaffolds, ( C ) Ace-PCL scaffolds, ( D ) Chl-EtOH-PCL scaffolds, and ( E ) Chl-DCM-PCL scaffolds (scale bar: 30 μm). ( F ) SEM picture from a NCI-H1975 cell attached to a Chl-DCM-PCL nanofiber due to roughness (scale bar 6 μm). ( G) SEM picture from NCI-H1975 cells attached to a monolayer (scale bar 300 μm). ( H) SEM picture from NCI-H1975 cells attached to a monolayer (scale bar 30 μm). Representative cells are indicated by arrows.

    Article Snippet: NCI-H1975 human NSCLC cell line was purchased from the American Type Culture Collection (ATCC; Rockvville, MD, USA).

    Techniques: Microscopy

    Cell viability and protein adsorption of NCI-H1975 cells cultured on PCL scaffolds for ( A ) 3 and ( B ) 6 days. Cell viability is represented in boxplot form on the left axis. Protein adsorption was normalized by the scaffold weight and is shown as mean with circles on the right axis. Cell viability per fiber of NCI-H1975 cells cultured on PCL scaffolds for ( C ) 3 and ( D ) 6 days. ( E ) Cell viability per fiber (V′). The cell viability was normalized by the porosity and layers of the scaffold to obtain a parameter which represents the cell viability on fibers of each layer.

    Journal: Scientific Reports

    Article Title: The solvent chosen for the manufacturing of electrospun polycaprolactone scaffolds influences cell behavior of lung cancer cells

    doi: 10.1038/s41598-022-23655-2

    Figure Lengend Snippet: Cell viability and protein adsorption of NCI-H1975 cells cultured on PCL scaffolds for ( A ) 3 and ( B ) 6 days. Cell viability is represented in boxplot form on the left axis. Protein adsorption was normalized by the scaffold weight and is shown as mean with circles on the right axis. Cell viability per fiber of NCI-H1975 cells cultured on PCL scaffolds for ( C ) 3 and ( D ) 6 days. ( E ) Cell viability per fiber (V′). The cell viability was normalized by the porosity and layers of the scaffold to obtain a parameter which represents the cell viability on fibers of each layer.

    Article Snippet: NCI-H1975 human NSCLC cell line was purchased from the American Type Culture Collection (ATCC; Rockvville, MD, USA).

    Techniques: Adsorption, Cell Culture

    SFRE diminishes mitochondrial oxidative phosphorylation. Mitochondrial respiration analysis by flux analysis of the oxygen consumption rate (OCR) of H1299 cells (A) and H1975 (B) previously pre-treated with SFRE (1/2xIC 50 , 1xIC 50 and 2xIC 50 ) for 48 h The basal respiration rate, spare respiratory capacity, ATP production, and proton leak of 10,000 cells per condition are compared. Data represent mean ± SEM of three independent experiments, each performed with four to six replicates. Asterisks *, **, *** indicate p values

    Journal: Frontiers in Oncology

    Article Title: Phenolic diterpenes from Rosemary supercritical extract inhibit non-small cell lung cancer lipid metabolism and synergise with therapeutic drugs in the clinic

    doi: 10.3389/fonc.2022.1046369

    Figure Lengend Snippet: SFRE diminishes mitochondrial oxidative phosphorylation. Mitochondrial respiration analysis by flux analysis of the oxygen consumption rate (OCR) of H1299 cells (A) and H1975 (B) previously pre-treated with SFRE (1/2xIC 50 , 1xIC 50 and 2xIC 50 ) for 48 h The basal respiration rate, spare respiratory capacity, ATP production, and proton leak of 10,000 cells per condition are compared. Data represent mean ± SEM of three independent experiments, each performed with four to six replicates. Asterisks *, **, *** indicate p values

    Article Snippet: Human lung adenocarcinoma cell lines, NCI-H1299 (H1299) and NCI-H1975 (H1975) were obtained from the American Type Culture Collection (ATCC).

    Techniques:

    (A) SFRE synergised with pemetrexed in the inhibition of NSCLC cell proliferation. NSCLC cells were pre-treated with the extract for 3h at a fixed dose of 1/2 IC 50 for H1299 and H1975. Combinatory index (CI) according to Chou–Talalay method. (B) SFRE diminished the expression of DHFR and GARFT , in a dose dependent manner. Results are shown in relation to non-treated cells and normalized to the endogenous control B2M . Results are expressed as the mean ± SEM of three independent experiments, each performed in triplicates. Asterisks *, **, *** indicate p-values

    Journal: Frontiers in Oncology

    Article Title: Phenolic diterpenes from Rosemary supercritical extract inhibit non-small cell lung cancer lipid metabolism and synergise with therapeutic drugs in the clinic

    doi: 10.3389/fonc.2022.1046369

    Figure Lengend Snippet: (A) SFRE synergised with pemetrexed in the inhibition of NSCLC cell proliferation. NSCLC cells were pre-treated with the extract for 3h at a fixed dose of 1/2 IC 50 for H1299 and H1975. Combinatory index (CI) according to Chou–Talalay method. (B) SFRE diminished the expression of DHFR and GARFT , in a dose dependent manner. Results are shown in relation to non-treated cells and normalized to the endogenous control B2M . Results are expressed as the mean ± SEM of three independent experiments, each performed in triplicates. Asterisks *, **, *** indicate p-values

    Article Snippet: Human lung adenocarcinoma cell lines, NCI-H1299 (H1299) and NCI-H1975 (H1975) were obtained from the American Type Culture Collection (ATCC).

    Techniques: Inhibition, Expressing

    SFRE synergised with pembrolizumab in the inhibition of NSCLC cell proliferation. H1299 and H1975 NSCLC cells were treated concomitantly with SFRE at a fixed dose of 1/2xIC 50 and with pembrolizumab at the indicated doses. (A) The combinatory index (CI) according to Chou–Talay method indicated a positive synergism between SFRE and pembrolizumab in the inhibition of NSCLC proliferation. (B) FACs analysis of the expression of PD-L1 after 3h and 24h SFRE treatment of NSCLC cells at two different doses (1/2xIC 50 , and 1xIC 50 . (C) FACS analysis of the double expression of PD-L1 and CXCR1 after 3h and 24h SFRE treatment of NSCLC cells at two different doses (1/2 IC50, and 1xIC50. Data are expressed as fold induction respect to the expression levels of non-treated cells. Results are expressed as the mean ± SEM of three independent experiments, each performed in triplicates. Asterisks * indicates p-values

    Journal: Frontiers in Oncology

    Article Title: Phenolic diterpenes from Rosemary supercritical extract inhibit non-small cell lung cancer lipid metabolism and synergise with therapeutic drugs in the clinic

    doi: 10.3389/fonc.2022.1046369

    Figure Lengend Snippet: SFRE synergised with pembrolizumab in the inhibition of NSCLC cell proliferation. H1299 and H1975 NSCLC cells were treated concomitantly with SFRE at a fixed dose of 1/2xIC 50 and with pembrolizumab at the indicated doses. (A) The combinatory index (CI) according to Chou–Talay method indicated a positive synergism between SFRE and pembrolizumab in the inhibition of NSCLC proliferation. (B) FACs analysis of the expression of PD-L1 after 3h and 24h SFRE treatment of NSCLC cells at two different doses (1/2xIC 50 , and 1xIC 50 . (C) FACS analysis of the double expression of PD-L1 and CXCR1 after 3h and 24h SFRE treatment of NSCLC cells at two different doses (1/2 IC50, and 1xIC50. Data are expressed as fold induction respect to the expression levels of non-treated cells. Results are expressed as the mean ± SEM of three independent experiments, each performed in triplicates. Asterisks * indicates p-values

    Article Snippet: Human lung adenocarcinoma cell lines, NCI-H1299 (H1299) and NCI-H1975 (H1975) were obtained from the American Type Culture Collection (ATCC).

    Techniques: Inhibition, FACS, Expressing

    SFRE diminishes aerobic glycolysis. Aerobic glycolysis analysis by the flux analysis of the extracellular acidification rate (ECAR) of H1299 cells (A) and H1975 (B) , previously pre-treated with SFRE at three doses (1/2xIC 50 , 1xIC 50 and 2xIC 50 ) for 48 h The basal ECAR, glycolysis and maximal ECAR of 10,000 cells per condition are compared. Data represent mean ± SEM of three independent experiments, each performed four to six replicates. Asterisks *, *** indicate p-values

    Journal: Frontiers in Oncology

    Article Title: Phenolic diterpenes from Rosemary supercritical extract inhibit non-small cell lung cancer lipid metabolism and synergise with therapeutic drugs in the clinic

    doi: 10.3389/fonc.2022.1046369

    Figure Lengend Snippet: SFRE diminishes aerobic glycolysis. Aerobic glycolysis analysis by the flux analysis of the extracellular acidification rate (ECAR) of H1299 cells (A) and H1975 (B) , previously pre-treated with SFRE at three doses (1/2xIC 50 , 1xIC 50 and 2xIC 50 ) for 48 h The basal ECAR, glycolysis and maximal ECAR of 10,000 cells per condition are compared. Data represent mean ± SEM of three independent experiments, each performed four to six replicates. Asterisks *, *** indicate p-values

    Article Snippet: Human lung adenocarcinoma cell lines, NCI-H1299 (H1299) and NCI-H1975 (H1975) were obtained from the American Type Culture Collection (ATCC).

    Techniques:

    Effects of SFRE on lipid metabolism related genes after 48 h of treatment H1975 NSCLC cells. SFRE diminishes the expression of SREBF1, FASN, SCD1, HMGCR ( de novo lipogenesis and cholesterogenesis); ACSL1, ACSL3 and ACSL4 (fatty acid activation); ABCA1 (efflux of cholesterol and tumour microenvironment remodelling); CHKA and AGPAT1 (biosynthesis of phospholipids and plasmatic membrane phospholipid remodelling) and oncogenic EGFR . On the contrary, SFRE upregulated the expression of APOA1 . Results are shown in relation to non-treated cells and normalized to the endogenous control B2M . Results are expressed as the mean ± SEM of three independent experiments, each performed in triplicates. Asterisks *, **, *** indicate p values

    Journal: Frontiers in Oncology

    Article Title: Phenolic diterpenes from Rosemary supercritical extract inhibit non-small cell lung cancer lipid metabolism and synergise with therapeutic drugs in the clinic

    doi: 10.3389/fonc.2022.1046369

    Figure Lengend Snippet: Effects of SFRE on lipid metabolism related genes after 48 h of treatment H1975 NSCLC cells. SFRE diminishes the expression of SREBF1, FASN, SCD1, HMGCR ( de novo lipogenesis and cholesterogenesis); ACSL1, ACSL3 and ACSL4 (fatty acid activation); ABCA1 (efflux of cholesterol and tumour microenvironment remodelling); CHKA and AGPAT1 (biosynthesis of phospholipids and plasmatic membrane phospholipid remodelling) and oncogenic EGFR . On the contrary, SFRE upregulated the expression of APOA1 . Results are shown in relation to non-treated cells and normalized to the endogenous control B2M . Results are expressed as the mean ± SEM of three independent experiments, each performed in triplicates. Asterisks *, **, *** indicate p values

    Article Snippet: Human lung adenocarcinoma cell lines, NCI-H1299 (H1299) and NCI-H1975 (H1975) were obtained from the American Type Culture Collection (ATCC).

    Techniques: Expressing, Activation Assay

    (A) SFRE reduced the intracellular neutral lipids content of H1299 and H1975 cells after 48 h of treatment at the indicated doses. Relative content of neutral lipids in cells pre-treated with SFRE after 48 h at different doses, compared with untreated control cells, normalized by the number of viable cells. Results are shown mean ± SEM of three independent experiments (three replicates). (B) SFRE reduced the total content of main phospholipids. Relative content of phospholipids in cells pre-treated with SFRE after 48 h at the fixed dose of IC 50 for each cell line, compared with untreated control cells, normalized by protein content. Results are shown mean ± SEM of three independent experiments (three replicates). Asterisks *, **, *** indicate p values

    Journal: Frontiers in Oncology

    Article Title: Phenolic diterpenes from Rosemary supercritical extract inhibit non-small cell lung cancer lipid metabolism and synergise with therapeutic drugs in the clinic

    doi: 10.3389/fonc.2022.1046369

    Figure Lengend Snippet: (A) SFRE reduced the intracellular neutral lipids content of H1299 and H1975 cells after 48 h of treatment at the indicated doses. Relative content of neutral lipids in cells pre-treated with SFRE after 48 h at different doses, compared with untreated control cells, normalized by the number of viable cells. Results are shown mean ± SEM of three independent experiments (three replicates). (B) SFRE reduced the total content of main phospholipids. Relative content of phospholipids in cells pre-treated with SFRE after 48 h at the fixed dose of IC 50 for each cell line, compared with untreated control cells, normalized by protein content. Results are shown mean ± SEM of three independent experiments (three replicates). Asterisks *, **, *** indicate p values

    Article Snippet: Human lung adenocarcinoma cell lines, NCI-H1299 (H1299) and NCI-H1975 (H1975) were obtained from the American Type Culture Collection (ATCC).

    Techniques:

    SFRE inhibits cell proliferation of NSCLC cell lines -H1299 and H1975. Dose–response curves of the cell proliferation assay after 48 h of treatment with increasing concentrations of SFRE. Data represent mean ± SEM of three independent experiments, each performed in triplicates. Values corresponding to IC 50 , GI 50 , TGI and LC 50 after 48 h of SFRE treatment are indicated. Results are shown as the mean ± SEM of three independent experiments, with 4 replicates/experiment. Asterisks * and *** indicate p values

    Journal: Frontiers in Oncology

    Article Title: Phenolic diterpenes from Rosemary supercritical extract inhibit non-small cell lung cancer lipid metabolism and synergise with therapeutic drugs in the clinic

    doi: 10.3389/fonc.2022.1046369

    Figure Lengend Snippet: SFRE inhibits cell proliferation of NSCLC cell lines -H1299 and H1975. Dose–response curves of the cell proliferation assay after 48 h of treatment with increasing concentrations of SFRE. Data represent mean ± SEM of three independent experiments, each performed in triplicates. Values corresponding to IC 50 , GI 50 , TGI and LC 50 after 48 h of SFRE treatment are indicated. Results are shown as the mean ± SEM of three independent experiments, with 4 replicates/experiment. Asterisks * and *** indicate p values

    Article Snippet: Human lung adenocarcinoma cell lines, NCI-H1299 (H1299) and NCI-H1975 (H1975) were obtained from the American Type Culture Collection (ATCC).

    Techniques: Proliferation Assay

    (A) Dose–response curves of the cell proliferation after 48 h of treatment with cisplatin in NSCLC H1299 and H1975. IC 50 values after cisplatin treatment in the two different NSCLC cell lines (H1299 and H1975) after 48h of treatment are indicated (upper panel). SFRE synergised with cisplatin in the inhibition of NSCLC cell proliferation. NSCLC cells were pre-treated with the extract for 3h at a fixed dose of 1/2 IC 50 for H1299 and H1975. Combinatory index (CI) according to Chou–Talalay method (lower panel). (B) SFRE diminished the expression of TK1 and TYMS , in a dose dependent manner. Results are shown in relation to non-treated cells and normalized to the endogenous control B2M . Results are expressed as the mean ± SEM of three independent experiments, each performed in triplicates. Asterisks *, **, *** indicate p-values

    Journal: Frontiers in Oncology

    Article Title: Phenolic diterpenes from Rosemary supercritical extract inhibit non-small cell lung cancer lipid metabolism and synergise with therapeutic drugs in the clinic

    doi: 10.3389/fonc.2022.1046369

    Figure Lengend Snippet: (A) Dose–response curves of the cell proliferation after 48 h of treatment with cisplatin in NSCLC H1299 and H1975. IC 50 values after cisplatin treatment in the two different NSCLC cell lines (H1299 and H1975) after 48h of treatment are indicated (upper panel). SFRE synergised with cisplatin in the inhibition of NSCLC cell proliferation. NSCLC cells were pre-treated with the extract for 3h at a fixed dose of 1/2 IC 50 for H1299 and H1975. Combinatory index (CI) according to Chou–Talalay method (lower panel). (B) SFRE diminished the expression of TK1 and TYMS , in a dose dependent manner. Results are shown in relation to non-treated cells and normalized to the endogenous control B2M . Results are expressed as the mean ± SEM of three independent experiments, each performed in triplicates. Asterisks *, **, *** indicate p-values

    Article Snippet: Human lung adenocarcinoma cell lines, NCI-H1299 (H1299) and NCI-H1975 (H1975) were obtained from the American Type Culture Collection (ATCC).

    Techniques: Inhibition, Expressing

    KW6002 limits cisplatin accumulation in the kidney but not in tumors. RPTEC/hTERT1 and H1975 cells were exposed to cisplatin and/or KW6002. n = 3 independent experiments. ( A and E ) Platinum quantification (ng/μg proteins) in RPTEC/hTERT1 ( A ) and H1975 ( E ) cells. *** P

    Journal: The Journal of Clinical Investigation

    Article Title: Istradefylline protects from cisplatin-induced nephrotoxicity and peripheral neuropathy while preserving cisplatin antitumor effects

    doi: 10.1172/JCI152924

    Figure Lengend Snippet: KW6002 limits cisplatin accumulation in the kidney but not in tumors. RPTEC/hTERT1 and H1975 cells were exposed to cisplatin and/or KW6002. n = 3 independent experiments. ( A and E ) Platinum quantification (ng/μg proteins) in RPTEC/hTERT1 ( A ) and H1975 ( E ) cells. *** P

    Article Snippet: Human lung adenocarcinoma NCI-H1975 cells (ATCC) were cultured in RPMI GlutaMAX (Thermo Fisher Scientific) containing 10% FCS and 1% penicillin-streptomycin.

    Techniques: