Journal: Nutrition Research and Practice

Article Title: Vaccinium oldhamii fruits improve insulin resistance by inhibiting inflammation in macrophages and adipocytes

doi: 10.4162/nrp.2025.19.6.839

Figure Lengend Snippet: VOW, Vaccinium oldhamii water extract; LPS, lipopolysaccharide; p-ERK, phosphorylated extracellular signal-regulated kinase; ERK, extracellular signal-regulated kinase; p-JNK, phosphorylated Jun N-terminal kinase; JNK, Jun N-terminal kinase; p-p38, phosphorylated p38; p-IKKα/β, phosphorylated IKKα/β; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; NO, nitric oxide.

Article Snippet: Primary antibodies (phosphorylated extracellular signal-regulated kinase [p-ERK, #4370; Cell Signaling Technology; CST, Beverly, MA, USA], extracellular signal-regulated kinase [ERK, #4695; CST], phosphorylated JNK [p-JNK, #9255; CST], JNK [#9252; CST], phosphorylated p38 [p-p38, #4511; CST], p38 [#8690; CST], phosphorylated IKKα/β [p-IKKα/β, #2697; CST], IKKα [#61294; CST], NF-κB [#8242; CST], inducible nitric oxide synthase [iNOS, #13120; CST], cyclooxygenase-2 [COX-2, #4842; CST], glucose transporter type 4 [GLUT4, #2213; CST], phosphorylated AK strain transforming [p-AKT, #9271; CST], Ak strain transforming [AKT, #9272; CST], phosphorylated phosphatidylinositol 3-kinase [p-PI3K, #4228; CST], phosphatidylinositol 3-kinase [PI3K, #4292; CST], phosphorylated insulin receptor substrate [p-IRS (Ser 307), #2381; CST], p-IRS (Tyr 632) [sc-17196; Santa Cruz, Dallas, TX, USA], and insulin receptor substrate [#2382; CST]) overnight with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit or goat anti-mouse [1:10,000]).

Techniques:

Journal: Nutrition Research and Practice

Article Title: Vaccinium oldhamii fruits improve insulin resistance by inhibiting inflammation in macrophages and adipocytes

doi: 10.4162/nrp.2025.19.6.839

Figure Lengend Snippet: VOW, Vaccinium oldhamii water extract; GLUT4, glucose transporter type 4; p-AKT, phosphorylated AK strain transforming; AKT, AK strain transforming; p-PI3K, phosphorylated phosphatidylinositol 3-kinase; PI3K, phosphatidylinositol 3-kinase; p-IRS, phosphorylated insulin receptor substrate; IRS, insulin receptor substrate.

Article Snippet: Primary antibodies (phosphorylated extracellular signal-regulated kinase [p-ERK, #4370; Cell Signaling Technology; CST, Beverly, MA, USA], extracellular signal-regulated kinase [ERK, #4695; CST], phosphorylated JNK [p-JNK, #9255; CST], JNK [#9252; CST], phosphorylated p38 [p-p38, #4511; CST], p38 [#8690; CST], phosphorylated IKKα/β [p-IKKα/β, #2697; CST], IKKα [#61294; CST], NF-κB [#8242; CST], inducible nitric oxide synthase [iNOS, #13120; CST], cyclooxygenase-2 [COX-2, #4842; CST], glucose transporter type 4 [GLUT4, #2213; CST], phosphorylated AK strain transforming [p-AKT, #9271; CST], Ak strain transforming [AKT, #9272; CST], phosphorylated phosphatidylinositol 3-kinase [p-PI3K, #4228; CST], phosphatidylinositol 3-kinase [PI3K, #4292; CST], phosphorylated insulin receptor substrate [p-IRS (Ser 307), #2381; CST], p-IRS (Tyr 632) [sc-17196; Santa Cruz, Dallas, TX, USA], and insulin receptor substrate [#2382; CST]) overnight with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit or goat anti-mouse [1:10,000]).

Techniques:

Journal: Nutrition Research and Practice

Article Title: Vaccinium oldhamii fruits improve insulin resistance by inhibiting inflammation in macrophages and adipocytes

doi: 10.4162/nrp.2025.19.6.839

Figure Lengend Snippet: MCM, macrophage-conditioned media; VOW, Vaccinium oldhamii water extract; GLUT4, glucose transporter type 4; p-AKT, phosphorylated AK strain transforming; AKT, AK strain transforming; p-PI3K, phosphorylated phosphatidylinositol 3-kinase; PI3K, phosphatidylinositol 3-kinase; p-IRS, phosphorylated insulin receptor substrate; IRS, insulin receptor substrate.

Article Snippet: Primary antibodies (phosphorylated extracellular signal-regulated kinase [p-ERK, #4370; Cell Signaling Technology; CST, Beverly, MA, USA], extracellular signal-regulated kinase [ERK, #4695; CST], phosphorylated JNK [p-JNK, #9255; CST], JNK [#9252; CST], phosphorylated p38 [p-p38, #4511; CST], p38 [#8690; CST], phosphorylated IKKα/β [p-IKKα/β, #2697; CST], IKKα [#61294; CST], NF-κB [#8242; CST], inducible nitric oxide synthase [iNOS, #13120; CST], cyclooxygenase-2 [COX-2, #4842; CST], glucose transporter type 4 [GLUT4, #2213; CST], phosphorylated AK strain transforming [p-AKT, #9271; CST], Ak strain transforming [AKT, #9272; CST], phosphorylated phosphatidylinositol 3-kinase [p-PI3K, #4228; CST], phosphatidylinositol 3-kinase [PI3K, #4292; CST], phosphorylated insulin receptor substrate [p-IRS (Ser 307), #2381; CST], p-IRS (Tyr 632) [sc-17196; Santa Cruz, Dallas, TX, USA], and insulin receptor substrate [#2382; CST]) overnight with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit or goat anti-mouse [1:10,000]).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Cepharanthine may inhibit the proliferation of prostate cells by blocking the EGFR/PI3K/AKT signaling pathway: comprehensive network analysis, molecular docking, and experimental evaluation

doi: 10.3389/fphar.2025.1654757

Figure Lengend Snippet: Western blot confirmed that CEP can inhibit the proliferation of WPMY-1 by inhibiting the EGFR/PI3K/AKT signaling pathway. (A) Western blot analysis showed that CEP could significantly inhibit the expression of FN1 and also had a certain inhibitory effect on the total protein expression of AKT and EGFR, but there was no statistical difference; (B) Compared with the DMSO group, 2.5 μM CEP treatment for 48 h significantly reduced the expression of p-AKT and p-EGFR, and 5 μM CEP further aggravated the inhibitory effect; (C) Quantitative analysis of AKT, p-AKT, EGFR, p-EGFR and FN1 by Western blot; (D) phosphorylation level analysis simultaneously showed that the ratios of p-AKT/AKT and p-EGFR/EGFR in the CEP group were significantly decreased. Data are presented as mean ± SD, and were analyzed with One-way ANOVA with Tukey’s post-hoc test. ****p < 0.0001. NS: Non-significant; Cep: Cepharanthine.

Article Snippet: Antibodies against AKT (No. 75692), phosphorylated AKT (Ser473) (No. 4060), EGFR (No. 4267), phosphorylated EGFR (Tyr1068) (No. 3777), FN1 (No. A0056-3), and GAPDH (No. 2118) were purchased from Cell Signaling Technology, Inc. (Boston, USA).

Techniques: Western Blot, Expressing, Phospho-proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Activating NEDD4L suppresses EGFR-driven lung adenocarcinoma growth via facilitating EGFR proteasomal degradation

doi: 10.1186/s13046-025-03528-y

Figure Lengend Snippet: NEDD4L promotes EGFR protein ubiquitination and proteasome-mediated degradation. A A total of 153 EGFR-mutant LUAD patients treated with the third-generation EGFR-TKIs as the first-line treatment. The Progress-free survival curves of these patients in with and without EGFR amplification group. B Top 20 E3 ubiquitin ligases of EGFR predicted by UbiBrowser database. C Western blot to detect the protein level of EGFR in LUAD cells (H1299, HCC827, H1975, H1975OR) stably expressing NEDD4L. D HEK293T cells were co-transfected with Flag-EGFR (bearing C797S only, or 19del/T790M/C797S, or L858R/T790M/C797S mutations) and His-NEDD4L expressing plasmids for 36 h, followed by western blot analyses. E H1299 or H1975 cells stably expressing His-NEDD4L were incubated in the absence or presence of serum (10%) for 12 h, and then treated with cyclohexamide (CHX) (50 µg/mL) for indicated time periods, followed by western blot analyses. F HEK293T cells were co-transfected with Flag-EGFR (bearing L858R/T790M/C797S mutations) and His-NEDD4L expressing plasmids for 36 h, and then treated with cyclohexamide (CHX) (50 µg/mL) for indicated time periods, followed by western blot analyses. G - L Quantitative analysis of ( E , F ) results. M HEK293T cells were co-transfected with HA-ubiquitin, Flag-EGFR, and either His-NEDD4L expressing plasmids for 36 h, and then treated with MG132 (20 μM) for 4 h, followed by IP-Western analyses. N H1299, H1975 or H1975OR cells stably expressing His-NEDD4L were treated with MG132 (20 μM) for 6 h, followed by western blot analyses.

Article Snippet: The antibodies used were listed as follows: anti-EGFR (Cat. #4267, Cell Signaling Technology; 1:50), anti-phosphorylated EGFR (Cat. #3777, Cell Signaling Technology;1:200), anti-NEDD4L (Cat. #ab46521, Abcam; 1:200), anti-FOXM1 (Cat. #ab207298, Abcam; 1:200), and anti-Ki67 (Cat. #ab16667, Abcam; 1:200).

Techniques: Ubiquitin Proteomics, Mutagenesis, Amplification, Western Blot, Stable Transfection, Expressing, Transfection, Incubation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Activating NEDD4L suppresses EGFR-driven lung adenocarcinoma growth via facilitating EGFR proteasomal degradation

doi: 10.1186/s13046-025-03528-y

Figure Lengend Snippet: NEDD4L interacts with EGFR. A H1299, HCC827 or H1975 cells were transfected with His-NEDD4L expressing plasmids for 36 h, and then treated with MG132 (20 μM) for 6 h, followed by Immunofluorescence assays of NEDD4L (red) and EGFR (green) and DAPI (blue). Scale bar = 10 µm. B HEK293T cells were co-transfected with His-NEDD4L and indicated Flag-EGFR-mutant expressing plasmids for 36 h, and then treated with MG132 (20 μM) for 6 h, followed by IP-Western analyses. C Schematic representation of EGFR truncated mutants. FL, full length; ICD, intracellular domain; ECD, extracellular domain. D Schematic representation of NEDD4L truncated mutants. Del, deletion. E HEK293T cells were co-transfected with His-NEDD4L and indicated truncated Flag-EGFR-mutant expressing plasmids for 36 h, and then treated with MG132 (20 μM) for 6 h, followed by IP-Western analyses. F HEK293T cells were co-transfected with Flag-EGFR and indicated truncated His-NEDD4L-mutant expressing plasmids for 36 h, and then treated with MG132 (20 μM) for 6 h, followed by IP-Western analyses.

Article Snippet: The antibodies used were listed as follows: anti-EGFR (Cat. #4267, Cell Signaling Technology; 1:50), anti-phosphorylated EGFR (Cat. #3777, Cell Signaling Technology;1:200), anti-NEDD4L (Cat. #ab46521, Abcam; 1:200), anti-FOXM1 (Cat. #ab207298, Abcam; 1:200), and anti-Ki67 (Cat. #ab16667, Abcam; 1:200).

Techniques: Transfection, Expressing, Immunofluorescence, Mutagenesis, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Activating NEDD4L suppresses EGFR-driven lung adenocarcinoma growth via facilitating EGFR proteasomal degradation

doi: 10.1186/s13046-025-03528-y

Figure Lengend Snippet: NEDD4L inhibits LUAD cell proliferation through decreasing EGFR protein expression. A - B H1299, H1975 or H1975OR cells stably expressing either vector or His-NEDD4L were incubated for indicated time periods, followed by ( A ) CCK8 assays, or ( B ) EdU assays. Scale bar = 100 µm. C Quantitative analysis of ( B ) results. D H1299, H1975 or H1975OR cells stably expressing shNEDD4L, or shEGFR, or both were incubated for 10 days, followed by colony formation assays. E Quantitative analysis of ( D ) results. F Experimental design of CDX model. H1975OR cells (bearing EGFR L858R/T790M/C797S mutations) stably expressing either Flag-EGFR L858R/T790M/C797S , or His-NEDD4L, or both were injected subcutaneously in female BALB/c nude mice ( n = 5/group) for inoculation of 21 days, then tumors were harvested for analysis. G Western blot analyses of NEDD4L, EGFR and phosphorylated EGFR protein expression in final shaped tumors. H Photos of final shaped tumors. I Tumor growth curve. J Final tumor weight. K Experimental design of transgenic mice model. Female EGFR 19del/T790M transgenic mice ( n = 3/group) were intranasally administrated with vector or NEDD4L lentivirus once Daily for 3 consecutive days. One week after the first lentiviral administration, DOX was added to feed the mice for 6 weeks, then the lung samples were harvested for analysis. L Photos of final lung samples. M H&E staining of final lung samples. Scale bar = 1mm (up) or 200 µm (down). N Numbers of observable nodules in the final lung surface. O Western blot analyses of NEDD4L, EGFR, phosphorylated EGFR and Ki67 protein expression in final lung nodule tissues. ** P < 0.01, *** P < 0.001.

Article Snippet: The antibodies used were listed as follows: anti-EGFR (Cat. #4267, Cell Signaling Technology; 1:50), anti-phosphorylated EGFR (Cat. #3777, Cell Signaling Technology;1:200), anti-NEDD4L (Cat. #ab46521, Abcam; 1:200), anti-FOXM1 (Cat. #ab207298, Abcam; 1:200), and anti-Ki67 (Cat. #ab16667, Abcam; 1:200).

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Incubation, Injection, Western Blot, Transgenic Assay, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Activating NEDD4L suppresses EGFR-driven lung adenocarcinoma growth via facilitating EGFR proteasomal degradation

doi: 10.1186/s13046-025-03528-y

Figure Lengend Snippet: FOXM1 directly binds to NEDD4L promoter and represses its expression to increase EGFR protein level. A Prediction of potential upstream transcription factors of NEDD4L in LUAD. Four approaches including hTFtarget and KnockTF 2.0 database to identify potential upstream transcription factors of NEDD4L and TCGA-LUAD cohort and GEO dataset ( GSE31210 ) to search for NEDD4L-related DEGs. B H1299, H1975, or H1975OR cells stably expressing either shControl or shFOXM1 were incubated for 24 h, followed by qPCR analyses. C Map of the position weight matrix (PWM) for FOXM1 binding motif sequence from MotifMap database. D Schematic illustration of the predicted FOXM1 binding site in the NEDD4L promoter using hTFtarget. TSS,transcriptional start site. E ChIP analysis of FOXM1 binding to the NEDD4L promoter in H1299 cells. F Schema of the putative FOXM1 binding site in the promoter of NEDD4L, the consensus and mutant sequences for FOXM1 binding were boxed. G H1299 cells stably expressing either shControl or shFOXM1 were co-transfected with NEDD4L promoter-wt or -mutant plasmids for 36 h, followed by dual luciferase reporter assay. H H1299, H1975 or H1975OR cells stably expressing shFOXM1, shNEDD4L, or both were incubated for 36 h, followed by western blot analyses. ** P < 0.01, *** P < 0.001.

Article Snippet: The antibodies used were listed as follows: anti-EGFR (Cat. #4267, Cell Signaling Technology; 1:50), anti-phosphorylated EGFR (Cat. #3777, Cell Signaling Technology;1:200), anti-NEDD4L (Cat. #ab46521, Abcam; 1:200), anti-FOXM1 (Cat. #ab207298, Abcam; 1:200), and anti-Ki67 (Cat. #ab16667, Abcam; 1:200).

Techniques: Expressing, Stable Transfection, Incubation, Binding Assay, Sequencing, Mutagenesis, Transfection, Luciferase, Reporter Assay, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Activating NEDD4L suppresses EGFR-driven lung adenocarcinoma growth via facilitating EGFR proteasomal degradation

doi: 10.1186/s13046-025-03528-y

Figure Lengend Snippet: FOXM1 promotes EGFR-driven LUAD growth through inhibiting NEDD4L expression. A H1299, H1975 or H1975OR cells stably expressing shFOXM1, shNEDD4L, or both were incubated for 10 days, followed by colony formation assays. B Quantitative analysis of ( A ) results. C Experimental design of CDX model. H1975OR cells (bearing EGFR L858R/T790M/C797S mutations) stably expressing either shFOXM1, or shNEDD4L, or both were injected subcutaneously in female BALB/c nude mice ( n = 5/group) for inoculation of 21 days, then tumors were harvested for analysis. D Photos of final shaped tumors. E Tumor growth curve. F Final tumor weight. G Western blot analyses of FOXM1, NEDD4L, EGFR, phosphorylated EGFR and Ki67 protein expression in final shaped tumors. H Experimental design of transgenic mice model. DOX-induced female EGFR 19del/T790M transgenic mice ( n = 3/group) were intraperitoneally treated with FOXM1 inhibitor FDI-6 (50 mg/kg) Daily for a period of 21 days, then the lung samples were harvested for analysis. I Photos of final lung samples. J H&E staining of final lung samples. Scale bar = 1mm (up) or 200 µm (down). K Numbers of observable nodules in the final lung surface. L Western blot analyses of FOXM1, NEDD4L, EGFR, phosphorylated EGFR and Ki67 protein expression in final lung nodule tissues. ** P < 0.01, *** P < 0.001.

Article Snippet: The antibodies used were listed as follows: anti-EGFR (Cat. #4267, Cell Signaling Technology; 1:50), anti-phosphorylated EGFR (Cat. #3777, Cell Signaling Technology;1:200), anti-NEDD4L (Cat. #ab46521, Abcam; 1:200), anti-FOXM1 (Cat. #ab207298, Abcam; 1:200), and anti-Ki67 (Cat. #ab16667, Abcam; 1:200).

Techniques: Expressing, Stable Transfection, Incubation, Injection, Western Blot, Transgenic Assay, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Activating NEDD4L suppresses EGFR-driven lung adenocarcinoma growth via facilitating EGFR proteasomal degradation

doi: 10.1186/s13046-025-03528-y

Figure Lengend Snippet: Both high-FOXM1 and low-NEDD4L expression predict poor clinical outcomes of LUAD patients. A - B The comparative expression of NEDD4L( A ) and FOXM1( B ) mRNA between LUAD and normal tissues in TCGA database. C - D The comparative expression of NEDD4L( C ) and FOXM1( D ) mRNA between LUAD and normal tissues in GSE31210 dataset. E – F The comparison expression of NEDD4L ( E ) and FOXM1 ( F ) protein between LUAD and paired normal tissues from tissue microarray slides for IHC analyses. Staining was quantified by H-score. G Representative images of IHC staining. Scale bar = 50 µm. H Western blot analyses of FOXM1, NEDD4L and EGFR protein in twelve pairs of LUAD and adjacent samples collected from our cancer center. I Western blot analyses of FOXM1, NEDD4L and EGFR protein expression level in LUAD cell lines (A549, H1299, PC9, HCC827, H1975) and the normal lung epithelial cell line (BEAS-2B). J The comparison of FOXM1, NEDD4L and EGFR protein level in low- and high-NEDD4L expression groups from LUAD tissue microarray slides for IHC analyses. K Representative images of IHC staining. Scale bar = 50 µm. L - M The comparative expression of FOXM1, NEDD4L and EGFR mRNA level in low- and high-NEDD4L expression groups in TCGA database ( L ) and GSE31210 dataset ( M ). ns, not significant. N – O The expression correlation of FOXM1 and NEDD4L in LUAD from TCGA ( N ) database and GSE31210 ( O ) dataset. P Kaplan–Meier plots of overall survival (OS) of LUAD patients with high or low expression of NEDD4L. Q Kaplan–Meier plots of overall survival (OS) of LUAD patients with high or low expression of FOXM1.

Article Snippet: The antibodies used were listed as follows: anti-EGFR (Cat. #4267, Cell Signaling Technology; 1:50), anti-phosphorylated EGFR (Cat. #3777, Cell Signaling Technology;1:200), anti-NEDD4L (Cat. #ab46521, Abcam; 1:200), anti-FOXM1 (Cat. #ab207298, Abcam; 1:200), and anti-Ki67 (Cat. #ab16667, Abcam; 1:200).

Techniques: Expressing, Comparison, Microarray, Staining, Immunohistochemistry, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Activating NEDD4L suppresses EGFR-driven lung adenocarcinoma growth via facilitating EGFR proteasomal degradation

doi: 10.1186/s13046-025-03528-y

Figure Lengend Snippet: Verteporfin is a novel inhibitor of FOXM1 in facilitating NEDD4L-mediated EGFR proteasomal degradation and inhibiting LUAD growth. A Flow chart showing the strategy used to screen FOXM1 inhibitors. B Molecular docking diagram of the interaction between FOXM1 protein and verteporfin. Purple represents FOXM1, and green represents verteporfin. C H1975 cells stably expressing empty vector or HA-FOXM1 were treated with verteporfin for 36 h, followed by Immunofluorescence staining. Scale bar = 10 µm. D H1299, H1975 or H1975OR cells stably expressing empty vector or HA-FOXM1 were treated with verteporfin for 36 h, followed by western blot analyses. E H1299, H1975 or H1975OR cells were firstly treated with or without verteporfin for 24 h, then added cyclohexamide (CHX) (50 µg/mL) to co-incubate for indicated time periods, followed by western blot analyses. F H1299, H1975 or H1975OR cells were treated with indicated doses of verteporfin for 48 h, followed by CCK8 assays. G Experimental design of CDX model. H1975OR cells (bearing EGFR L858R/T790M/C797S mutations) stably expressing either FOXM1 or shNEDD4L were injected subcutaneously in female BALB/c nude mice ( n = 5/group) for inoculation. When the tumor size reach about 100 mm 3 , mice were intraperitoneally treated with verteporfin (30 mg/kg) Daily for a period of 21 days, then the tumors were harvested for analysis. H Photos of final shaped tumors. I Tumor growth curve. J Final tumor weight. K Western blot analyses of FOXM1, NEDD4L, EGFR and phosphorylated EGFR protein expression in final shaped tumors. VP, verteporfin. *** P < 0.001.

Article Snippet: The antibodies used were listed as follows: anti-EGFR (Cat. #4267, Cell Signaling Technology; 1:50), anti-phosphorylated EGFR (Cat. #3777, Cell Signaling Technology;1:200), anti-NEDD4L (Cat. #ab46521, Abcam; 1:200), anti-FOXM1 (Cat. #ab207298, Abcam; 1:200), and anti-Ki67 (Cat. #ab16667, Abcam; 1:200).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Immunofluorescence, Staining, Western Blot, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Activating NEDD4L suppresses EGFR-driven lung adenocarcinoma growth via facilitating EGFR proteasomal degradation

doi: 10.1186/s13046-025-03528-y

Figure Lengend Snippet: Verteporfin plus osimertinib exert an additively inhibitory effect on EGFR-mutated LUAD growth compared to monotherapy. A H1975 or H1975OR cells infected with vector or His-NEDD4L lentivirus were treated with indicated dose of osimertinib for 48 h, followed by CCK8 assays. B H1975 or H1975OR cells stably expressing shFOXM1 alone or together with shNEDD4L were treated with an indicated dose of osimertinib for 48 h, followed by CCK8 assays. C - D H1975 or H1975OR cells incubated with verteporfin (6 µM), or together with FOXM1( C ) or shNEDD4L( D ) lentivirus were treated with indicated dose of osimertinib for 48 h, followed by CCK8 assays. E Experimental design of CDX model. H1975OR cells were injected subcutaneously in female BALB/c nude mice ( n = 5/group) for inoculation. When the tumor size reach about 100 mm 3 , mice were treated with osimertinib (5 mg/kg, p.o., daily), or verteporfin (30 mg/kg, i.p., daily), or their combination for a period of 21 days, then the tumors were harvested for analysis. F Photos of final shaped tumors. G Tumor growth curve. H Final tumor weight. I IHC analyses of FOXM1, NEDD4L, EGFR, phosphorylated EGFR and Ki67 protein level in final tumor tissues. Scale bar = 10 µm. J Experimental design of transgenic mice model. DOX-induced female EGFR 19del/T790M transgenic mice ( n = 3/group) were intraperitoneally treated with osimertinib (2 mg/kg, p.o., daily), or verteporfin (20 mg/kg, i.p., daily), or their combination for a period of 21 days, then the lung samples were harvested for analysis. K H&E staining of final lung nodule samples. L Numbers of observable nodules in the final lung surface. M Western blot analyses of FOXM1, NEDD4L, EGFR, phosphorylated EGFR and Ki67 protein expression in final lung nodule tissues. N The proposed working model. VP, verteporfin. ** P < 0.01, *** P < 0.001, ns, not significant.

Article Snippet: The antibodies used were listed as follows: anti-EGFR (Cat. #4267, Cell Signaling Technology; 1:50), anti-phosphorylated EGFR (Cat. #3777, Cell Signaling Technology;1:200), anti-NEDD4L (Cat. #ab46521, Abcam; 1:200), anti-FOXM1 (Cat. #ab207298, Abcam; 1:200), and anti-Ki67 (Cat. #ab16667, Abcam; 1:200).

Techniques: Infection, Plasmid Preparation, Stable Transfection, Expressing, Incubation, Injection, Transgenic Assay, Staining, Western Blot