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Effects of Ncs on apoptosis and cell cycle. ( A ) Cells were treated with 50 nM Ncs for 24 h and stained with calcein-AM/PI, followed by image capture using fluorescence microscopy (green: live cells, red; dead cells), scale bars = 50 μm. ( B ) cells were treated with 50 nM Ncs for 24 h and stained with annexin V and PI and followed by flow cytometric analysis of apoptosis. Apoptotic cells were quantified with early and late apoptotic cell populations. ( C ) For cell cycle arrays, 50 nM Ncs-treated cells were fixed with 70% ethanol and stained with PI, followed by flow cytometry analysis. ( D and E ) Cells were treated with indicated concentrations (0–100 nM) of Ncs for 24 h and cell lysates were subjected to immunoblotting analysis using the <t>indicated</t> <t>antibodies.</t> Expression levels of <t>p-EGFR</t> and p-ERK1/2 were quantified from immunoblot bands and normalized to total EGFR and total ERK1/2 expression levels, respectively ( D ). Data were presented as mean values from three independent experiments and error bars represent standard deviations. * p < 0.05, ** p < 0.01
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Effects of Ncs on apoptosis and cell cycle. ( A ) Cells were treated with 50 nM Ncs for 24 h and stained with calcein-AM/PI, followed by image capture using fluorescence microscopy (green: live cells, red; dead cells), scale bars = 50 μm. ( B ) cells were treated with 50 nM Ncs for 24 h and stained with annexin V and PI and followed by flow cytometric analysis of apoptosis. Apoptotic cells were quantified with early and late apoptotic cell populations. ( C ) For cell cycle arrays, 50 nM Ncs-treated cells were fixed with 70% ethanol and stained with PI, followed by flow cytometry analysis. ( D and E ) Cells were treated with indicated concentrations (0–100 nM) of Ncs for 24 h and cell lysates were subjected to immunoblotting analysis using the <t>indicated</t> <t>antibodies.</t> Expression levels of <t>p-EGFR</t> and p-ERK1/2 were quantified from immunoblot bands and normalized to total EGFR and total ERK1/2 expression levels, respectively ( D ). Data were presented as mean values from three independent experiments and error bars represent standard deviations. * p < 0.05, ** p < 0.01
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Effects of Ncs on apoptosis and cell cycle. ( A ) Cells were treated with 50 nM Ncs for 24 h and stained with calcein-AM/PI, followed by image capture using fluorescence microscopy (green: live cells, red; dead cells), scale bars = 50 μm. ( B ) cells were treated with 50 nM Ncs for 24 h and stained with annexin V and PI and followed by flow cytometric analysis of apoptosis. Apoptotic cells were quantified with early and late apoptotic cell populations. ( C ) For cell cycle arrays, 50 nM Ncs-treated cells were fixed with 70% ethanol and stained with PI, followed by flow cytometry analysis. ( D and E ) Cells were treated with indicated concentrations (0–100 nM) of Ncs for 24 h and cell lysates were subjected to immunoblotting analysis using the indicated antibodies. Expression levels of p-EGFR and p-ERK1/2 were quantified from immunoblot bands and normalized to total EGFR and total ERK1/2 expression levels, respectively ( D ). Data were presented as mean values from three independent experiments and error bars represent standard deviations. * p < 0.05, ** p < 0.01

Journal: Journal of Translational Medicine

Article Title: Narciclasine as a potential therapeutic agent to overcome EGFR-TKI resistance in non-small cell lung cancer

doi: 10.1186/s12967-025-07368-4

Figure Lengend Snippet: Effects of Ncs on apoptosis and cell cycle. ( A ) Cells were treated with 50 nM Ncs for 24 h and stained with calcein-AM/PI, followed by image capture using fluorescence microscopy (green: live cells, red; dead cells), scale bars = 50 μm. ( B ) cells were treated with 50 nM Ncs for 24 h and stained with annexin V and PI and followed by flow cytometric analysis of apoptosis. Apoptotic cells were quantified with early and late apoptotic cell populations. ( C ) For cell cycle arrays, 50 nM Ncs-treated cells were fixed with 70% ethanol and stained with PI, followed by flow cytometry analysis. ( D and E ) Cells were treated with indicated concentrations (0–100 nM) of Ncs for 24 h and cell lysates were subjected to immunoblotting analysis using the indicated antibodies. Expression levels of p-EGFR and p-ERK1/2 were quantified from immunoblot bands and normalized to total EGFR and total ERK1/2 expression levels, respectively ( D ). Data were presented as mean values from three independent experiments and error bars represent standard deviations. * p < 0.05, ** p < 0.01

Article Snippet: Primary antibodies against total EGFR (#4267, Cell Signaling Technology(CST)), CDK1 (#9116, CST), phospho-EGFR Y1068 (#44-788 G, Invitrogen), total STAT3 (#4904, CST), phospho-STAT3 Y705 (#9145, CST), phospho-Src (#6943,CST), total-Src (#2109, CST), phospho-CDK1 Y15 (#4539, CST), phospho-Wee1 (#4910, CST), Myt1 (#4282, CST), p21 (#2947, CST), Cdc25B (#9525, CST), GAPDH (#5174, CST), β-actin (#A700-057, Bethyl Laboratory), phospho-ERK1/2 (#9101, CST), Bcl-2 (#sc-7382, Santa Cruz), and c-Myc (#sc-789, Santa Cruz) were used for immunoblotting.

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

Effects of Ncs on cell growth in PC-9 and PC-9-GR cells. ( A ) PC-9 and PC-9-GR cells (3 × 10 3 cells/well) were plated in 96-well plates and treated with gefitinib in a dose-dependent manner for 48 h. ( B ) Cells were treated with Ncs at the indicated concentrations for 48 hours, and cell growth rates were measured using the WST-1 assay. ( C ) Cells were treated with Ncs for 48 hours, and representative images are shown. ( D ) Cells (1 × 10 4 cells/well) were plated in 24-well plates and treated with Ncs at the indicated concentrations for 48 h. Live and dead cells were counted using the trypan blue staining assay. ( E and F ) Cells were treated with Ncs for 48 h, fixed with 70% ethanol, stained with PI, and analyzed by flow cytometry to assess cell cycle distribution. ( G ) PC-9 and PC-9-GR cells were treated with the indicated concentrations (0-50 nM) of Ncs for 24 h. Cell lysates were then subjected to immunoblotting analysis using the indicated antibodies. Expression levels of p-EGFR, p-Src, and p-STAT3 were quantified and normalized to total EGFR, total Src, and total STAT3 expression levels, respectively. Data were presented as mean values from three independent experiments and error bars represent standard deviations of the mean of three measurements. Magnification: x 100, n.s. >0.05, *P < 0.05, **P < 0.01

Journal: Journal of Translational Medicine

Article Title: Narciclasine as a potential therapeutic agent to overcome EGFR-TKI resistance in non-small cell lung cancer

doi: 10.1186/s12967-025-07368-4

Figure Lengend Snippet: Effects of Ncs on cell growth in PC-9 and PC-9-GR cells. ( A ) PC-9 and PC-9-GR cells (3 × 10 3 cells/well) were plated in 96-well plates and treated with gefitinib in a dose-dependent manner for 48 h. ( B ) Cells were treated with Ncs at the indicated concentrations for 48 hours, and cell growth rates were measured using the WST-1 assay. ( C ) Cells were treated with Ncs for 48 hours, and representative images are shown. ( D ) Cells (1 × 10 4 cells/well) were plated in 24-well plates and treated with Ncs at the indicated concentrations for 48 h. Live and dead cells were counted using the trypan blue staining assay. ( E and F ) Cells were treated with Ncs for 48 h, fixed with 70% ethanol, stained with PI, and analyzed by flow cytometry to assess cell cycle distribution. ( G ) PC-9 and PC-9-GR cells were treated with the indicated concentrations (0-50 nM) of Ncs for 24 h. Cell lysates were then subjected to immunoblotting analysis using the indicated antibodies. Expression levels of p-EGFR, p-Src, and p-STAT3 were quantified and normalized to total EGFR, total Src, and total STAT3 expression levels, respectively. Data were presented as mean values from three independent experiments and error bars represent standard deviations of the mean of three measurements. Magnification: x 100, n.s. >0.05, *P < 0.05, **P < 0.01

Article Snippet: Primary antibodies against total EGFR (#4267, Cell Signaling Technology(CST)), CDK1 (#9116, CST), phospho-EGFR Y1068 (#44-788 G, Invitrogen), total STAT3 (#4904, CST), phospho-STAT3 Y705 (#9145, CST), phospho-Src (#6943,CST), total-Src (#2109, CST), phospho-CDK1 Y15 (#4539, CST), phospho-Wee1 (#4910, CST), Myt1 (#4282, CST), p21 (#2947, CST), Cdc25B (#9525, CST), GAPDH (#5174, CST), β-actin (#A700-057, Bethyl Laboratory), phospho-ERK1/2 (#9101, CST), Bcl-2 (#sc-7382, Santa Cruz), and c-Myc (#sc-789, Santa Cruz) were used for immunoblotting.

Techniques: WST-1 Assay, Staining, Flow Cytometry, Western Blot, Expressing

Effects of Ncs on EGFR activity and EGFR signaling. ( A ) H1975 cells were treated with 50 nM of Ncs for indicated times and cell lysates were subjected to immunoblotting analysis using the indicated antibodies. ( B ) Cells were treated with 50 nM of Ncs for the indicated durations, and total RNA was isolated. EGFR mRNA levels were determined by real-time PCR analysis and normalized to GAPDH expression. ( C ) Cells were treated with 20 μM of cycloheximide (CHX) for the indicated time points in the presence of either DMSO or 50 nM of Ncs for 24 h. Cell lysates were subjected to immunoblotting analysis using the indicated antibodies (upper panel). EGFR expression levels were quantified from immunoblot bands and normalized to GAPDH, comparing CHX treatment in the presence of DMSO or Ncs (lower panel). ( D ) Cells were treated with 50 nM of Ncs for 24 h in the presence of BafA1 (20 nM, 12 h) or MG132 (20 μM, 2 h). Cell lysates were subjected to immunoblotting analysis using indicated antibodies. ( E ) Cells were plated on coverslips and treated with DMSO (control) or 50 nM of Ncs for 24 h. Cells were then fixed with 4% paraformaldehyde (PFA) and subjected to immunofluorescence staining using DAPI, EGFR, and LAMP1 antibody. ( F ) Cells were treated with 50 nM of Ncs for 24 h, followed by EGF (10 nM, 15 min) stimulation. Cell lysates were subjected to immunoblotting analysis using the specified antibodies. Similar results were observed in three independent experiments. Error bars represent standard deviations of the mean of three measurements, *P < 0.05, **P < 0.01, ***P < 0.001

Journal: Journal of Translational Medicine

Article Title: Narciclasine as a potential therapeutic agent to overcome EGFR-TKI resistance in non-small cell lung cancer

doi: 10.1186/s12967-025-07368-4

Figure Lengend Snippet: Effects of Ncs on EGFR activity and EGFR signaling. ( A ) H1975 cells were treated with 50 nM of Ncs for indicated times and cell lysates were subjected to immunoblotting analysis using the indicated antibodies. ( B ) Cells were treated with 50 nM of Ncs for the indicated durations, and total RNA was isolated. EGFR mRNA levels were determined by real-time PCR analysis and normalized to GAPDH expression. ( C ) Cells were treated with 20 μM of cycloheximide (CHX) for the indicated time points in the presence of either DMSO or 50 nM of Ncs for 24 h. Cell lysates were subjected to immunoblotting analysis using the indicated antibodies (upper panel). EGFR expression levels were quantified from immunoblot bands and normalized to GAPDH, comparing CHX treatment in the presence of DMSO or Ncs (lower panel). ( D ) Cells were treated with 50 nM of Ncs for 24 h in the presence of BafA1 (20 nM, 12 h) or MG132 (20 μM, 2 h). Cell lysates were subjected to immunoblotting analysis using indicated antibodies. ( E ) Cells were plated on coverslips and treated with DMSO (control) or 50 nM of Ncs for 24 h. Cells were then fixed with 4% paraformaldehyde (PFA) and subjected to immunofluorescence staining using DAPI, EGFR, and LAMP1 antibody. ( F ) Cells were treated with 50 nM of Ncs for 24 h, followed by EGF (10 nM, 15 min) stimulation. Cell lysates were subjected to immunoblotting analysis using the specified antibodies. Similar results were observed in three independent experiments. Error bars represent standard deviations of the mean of three measurements, *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Primary antibodies against total EGFR (#4267, Cell Signaling Technology(CST)), CDK1 (#9116, CST), phospho-EGFR Y1068 (#44-788 G, Invitrogen), total STAT3 (#4904, CST), phospho-STAT3 Y705 (#9145, CST), phospho-Src (#6943,CST), total-Src (#2109, CST), phospho-CDK1 Y15 (#4539, CST), phospho-Wee1 (#4910, CST), Myt1 (#4282, CST), p21 (#2947, CST), Cdc25B (#9525, CST), GAPDH (#5174, CST), β-actin (#A700-057, Bethyl Laboratory), phospho-ERK1/2 (#9101, CST), Bcl-2 (#sc-7382, Santa Cruz), and c-Myc (#sc-789, Santa Cruz) were used for immunoblotting.

Techniques: Activity Assay, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Expressing, Control, Immunofluorescence, Staining

Anti-cancer effects of ncs in vivo. ( A ) Adult-stage C. elegans models: wild type (WT) and jgIs25 strains. The jgIs25 strain exhibits polyps on the ventral side (arrows in the lower panel). Scale bar = 20 μm. ( B ) Effects of gefitinib and ncs treatment on jgIs25 . the arrows in the upper panel indicate polyps, and the rectangular area is magnified in the lower panel. Scale bar = 200 μm (upper), 20 μm (lower). ( C ) Quantification of polyp formation in jgIs25 following treatment with the indicated drugs. ( D and G ) In vivo tumor model using subcutaneous transplantation of A549 and H1975 cells into nude mice, followed by ncs treatment. Tumor volumes were measured at the indicated time points in control and ncs-treated mice. Tumor weights were measured after sacrifice. ( E and H ) Representative image of xenograft tumors after sacrifice. ( F and I ) Immunohistochemical (IHC) staining of xenograft tumors using anti-EGFR, anti-phospho-EGFR (Y1068), and anti-phospho-STAT3 (Y705) antibodies. Scale bar = 500 μm. Similar results were observed in three independent experiments. Error bars represent standard deviations of the mean of three measurements, *p < 0.05, **p < 0.01, *p < 0.001

Journal: Journal of Translational Medicine

Article Title: Narciclasine as a potential therapeutic agent to overcome EGFR-TKI resistance in non-small cell lung cancer

doi: 10.1186/s12967-025-07368-4

Figure Lengend Snippet: Anti-cancer effects of ncs in vivo. ( A ) Adult-stage C. elegans models: wild type (WT) and jgIs25 strains. The jgIs25 strain exhibits polyps on the ventral side (arrows in the lower panel). Scale bar = 20 μm. ( B ) Effects of gefitinib and ncs treatment on jgIs25 . the arrows in the upper panel indicate polyps, and the rectangular area is magnified in the lower panel. Scale bar = 200 μm (upper), 20 μm (lower). ( C ) Quantification of polyp formation in jgIs25 following treatment with the indicated drugs. ( D and G ) In vivo tumor model using subcutaneous transplantation of A549 and H1975 cells into nude mice, followed by ncs treatment. Tumor volumes were measured at the indicated time points in control and ncs-treated mice. Tumor weights were measured after sacrifice. ( E and H ) Representative image of xenograft tumors after sacrifice. ( F and I ) Immunohistochemical (IHC) staining of xenograft tumors using anti-EGFR, anti-phospho-EGFR (Y1068), and anti-phospho-STAT3 (Y705) antibodies. Scale bar = 500 μm. Similar results were observed in three independent experiments. Error bars represent standard deviations of the mean of three measurements, *p < 0.05, **p < 0.01, *p < 0.001

Article Snippet: Primary antibodies against total EGFR (#4267, Cell Signaling Technology(CST)), CDK1 (#9116, CST), phospho-EGFR Y1068 (#44-788 G, Invitrogen), total STAT3 (#4904, CST), phospho-STAT3 Y705 (#9145, CST), phospho-Src (#6943,CST), total-Src (#2109, CST), phospho-CDK1 Y15 (#4539, CST), phospho-Wee1 (#4910, CST), Myt1 (#4282, CST), p21 (#2947, CST), Cdc25B (#9525, CST), GAPDH (#5174, CST), β-actin (#A700-057, Bethyl Laboratory), phospho-ERK1/2 (#9101, CST), Bcl-2 (#sc-7382, Santa Cruz), and c-Myc (#sc-789, Santa Cruz) were used for immunoblotting.

Techniques: In Vivo, Transplantation Assay, Control, Immunohistochemical staining, Immunohistochemistry