Journal: Future science OA

Article Title: Adverse predictive value of ASPM on lung adenocarcinoma overall survival depended on chemotherapy status.

doi: 10.1080/20565623.2025.2489328

Figure Lengend Snippet: Figure 1. (A) TCGA TARGET GTEx pan-cancer dataset analysis showed the relationship between ASPM gene expression and patient prognosis in each tumor. (B) In TCGA dataset of LUAD, high ASPM expression was related to worse overall survival (Log-rank test p = 2.4e-6). (C) In proteomics dataset of LUAD, the difference of prognosis was not significant between ASPM high or low expression groups (Log-rank test p = 0.26).

Article Snippet: Immunohistochemistry and special staining the Luad tissues (n = 160) were obtained from surgically resected specimens and made into tissue microarray. the sections were de-paraffinized and hydrated, and the endogenous peroxidase was blocked. antigen retrieval was performed using the dako target Retrieval Solution, High pH (dako ominis, agilent technologies, Santa Clara, Ca, uSa), in a ptLink set at 98 °C for 25 min. the tissue sections were incubated with an anti-aSpm rabbit polyclonal antibody (1:200, a02584-1, Boster, China) 1h at 37 °C. detection of immunostaining was achieved by an enzyme-conjugated polymer complex (K8002, dako, denmark) adapted for autostainers from daKo (dako autostainer, agilent technologies). the staining intensity in selected areas was scored as follows: 0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining. the stained area was scored as follows: 0, no staining; 1, 1–10% positive cells; 2, 10–50% positive cells; 3, > 50% positive cells. the final score was evaluated by multiplying the staining intensity and stained area percentage. the negative and weak staining cases were considered as low expression group while the moderate and strong staining cases were defined as high expression group in later analysis.

Techniques: Gene Expression, Expressing

Journal: Future science OA

Article Title: Adverse predictive value of ASPM on lung adenocarcinoma overall survival depended on chemotherapy status.

doi: 10.1080/20565623.2025.2489328

Figure Lengend Snippet: Figure 2. (A) The representative immunohistochemical staining of ASPM negative, weak, moderate, and strong cases. (B) In our cohort, high ASPM expression predicted better LUAD prognosis (Log-rank test p = 5.4e-4). (C) In patients who received chemotherapy, high ASPM expression was related to better overall survival (Log-rank test p = 5.0e-4). (D) In patients who did not received chemotherapy, ASPM expression was not related to patient overall survival (Log-rank test p = 0.48). (E) Multivariate regression analysis showed ASPM was not an independent prognostic factor while other protective factor included chemother apy and risk factor included clinical stages. (F) time dependent ROC analysis was conducted at time points 365, 1095, and 1825 days. The AUC and confidence intervals were evaluated using the ci function of pROC.

Article Snippet: Immunohistochemistry and special staining the Luad tissues (n = 160) were obtained from surgically resected specimens and made into tissue microarray. the sections were de-paraffinized and hydrated, and the endogenous peroxidase was blocked. antigen retrieval was performed using the dako target Retrieval Solution, High pH (dako ominis, agilent technologies, Santa Clara, Ca, uSa), in a ptLink set at 98 °C for 25 min. the tissue sections were incubated with an anti-aSpm rabbit polyclonal antibody (1:200, a02584-1, Boster, China) 1h at 37 °C. detection of immunostaining was achieved by an enzyme-conjugated polymer complex (K8002, dako, denmark) adapted for autostainers from daKo (dako autostainer, agilent technologies). the staining intensity in selected areas was scored as follows: 0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining. the stained area was scored as follows: 0, no staining; 1, 1–10% positive cells; 2, 10–50% positive cells; 3, > 50% positive cells. the final score was evaluated by multiplying the staining intensity and stained area percentage. the negative and weak staining cases were considered as low expression group while the moderate and strong staining cases were defined as high expression group in later analysis.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Future science OA

Article Title: Adverse predictive value of ASPM on lung adenocarcinoma overall survival depended on chemotherapy status.

doi: 10.1080/20565623.2025.2489328

Figure Lengend Snippet: Figure 3. (A) heatmap of signature proteins in ASPM low or high groups from proteomics dataset. (B) High ASPM1 related signature proteins were enriched mainly in cell cycle and mitosis processes by Metascape analysis. (C) ASPM positive group in proteomics dataset showed higher expression of proliferation related proteins mainly including AURKB, PLK1, KIFC3, EZH2,CDKN2A, BRD3, CASP2, MKI67, CDK1, and MDC1.

Article Snippet: Immunohistochemistry and special staining the Luad tissues (n = 160) were obtained from surgically resected specimens and made into tissue microarray. the sections were de-paraffinized and hydrated, and the endogenous peroxidase was blocked. antigen retrieval was performed using the dako target Retrieval Solution, High pH (dako ominis, agilent technologies, Santa Clara, Ca, uSa), in a ptLink set at 98 °C for 25 min. the tissue sections were incubated with an anti-aSpm rabbit polyclonal antibody (1:200, a02584-1, Boster, China) 1h at 37 °C. detection of immunostaining was achieved by an enzyme-conjugated polymer complex (K8002, dako, denmark) adapted for autostainers from daKo (dako autostainer, agilent technologies). the staining intensity in selected areas was scored as follows: 0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining. the stained area was scored as follows: 0, no staining; 1, 1–10% positive cells; 2, 10–50% positive cells; 3, > 50% positive cells. the final score was evaluated by multiplying the staining intensity and stained area percentage. the negative and weak staining cases were considered as low expression group while the moderate and strong staining cases were defined as high expression group in later analysis.

Techniques: Expressing

Journal: Future science OA

Article Title: Adverse predictive value of ASPM on lung adenocarcinoma overall survival depended on chemotherapy status.

doi: 10.1080/20565623.2025.2489328

Figure Lengend Snippet: Figure 4. (A) Proteomics data analysis showed ASPM positive group had higher MKI67 expression. (B) Western blot analysis of A549 cells and H1299 cells confirmed effectiveness of ASPM transfection and in vitro overexpression. (C-D) Statistical anal ysis of western blot images. (E-F) CCK8 assay proved overexpression of ASPM promoted tumor cell proliferation in A549 and H1299 cells. (G-H) When treated with cisplatin, overexpression of ASPM enhanced the lethality on A549 and H1299 tumor cells. (P value *<0.05, **<0.01, ***<0.001).

Article Snippet: Immunohistochemistry and special staining the Luad tissues (n = 160) were obtained from surgically resected specimens and made into tissue microarray. the sections were de-paraffinized and hydrated, and the endogenous peroxidase was blocked. antigen retrieval was performed using the dako target Retrieval Solution, High pH (dako ominis, agilent technologies, Santa Clara, Ca, uSa), in a ptLink set at 98 °C for 25 min. the tissue sections were incubated with an anti-aSpm rabbit polyclonal antibody (1:200, a02584-1, Boster, China) 1h at 37 °C. detection of immunostaining was achieved by an enzyme-conjugated polymer complex (K8002, dako, denmark) adapted for autostainers from daKo (dako autostainer, agilent technologies). the staining intensity in selected areas was scored as follows: 0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining. the stained area was scored as follows: 0, no staining; 1, 1–10% positive cells; 2, 10–50% positive cells; 3, > 50% positive cells. the final score was evaluated by multiplying the staining intensity and stained area percentage. the negative and weak staining cases were considered as low expression group while the moderate and strong staining cases were defined as high expression group in later analysis.

Techniques: Expressing, Western Blot, Transfection, In Vitro, Over Expression, CCK-8 Assay

Journal: Frontiers in Genetics

Article Title: Assembly factor for spindle microtubules ( ASPM ) promotes osimertinib resistance in lung cancer by increasing EGFR stability

doi: 10.3389/fgene.2025.1593314

Figure Lengend Snippet: ASPM enhances EGFR resistance in lung cancer cells. (A) Dose‒effect relationship curves of osimertinib for PC-9 cells; (B) Dose‒effect relationship curves of osimertinib for PC-9 OR cells. (C) mRNA expression levels of ASPM and EGFR in PC-9 and PC-9 OR cells. (D) Protein expression of ASPM and EGFR in PC-9 and PC-9 OR cells. The histogram represents the relative gray values of the intracellular EGFR and ASPM proteins, and the data are presented as the means ± standard deviations (means ± SDs, n = 3 fields). (E) Silencing of the ASPM gene and mRNA expression levels of ASPM in PC-9 and PC-9 OR cells. (F) Dose‒effect relationship curves of osimertinib in PC-9 si ASPM cells. (G) Dose‒effect relationship curves of osimertinib in PC-9 OR si ASPM cells. (H) mRNA expression levels of ASPM were detected in PC-9 cells after overexpressing the ASPM gene. (I) Dose‒effect curves of osimertinib after PC-9 overexpression of ASPM . (J) The viability of the cells in each group was assessed via a CCK-8 assay. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The membrane was blocked with 5% skim milk for 60 min and incubated with primary antibodies, including ASPM (Proteintech, 26223-1-AP) (1:600) and EGFR (Cusabio, CSB-PA005571LA01HU) (1:1000), overnight at 4 °C, followed by 2 h of incubation with HRP-conjugated goat anti-rabbit (Proteintech, SA00001-2) (1:2000) at room temperature.

Techniques: Expressing, Over Expression, CCK-8 Assay

Journal: Frontiers in Genetics

Article Title: Assembly factor for spindle microtubules ( ASPM ) promotes osimertinib resistance in lung cancer by increasing EGFR stability

doi: 10.3389/fgene.2025.1593314

Figure Lengend Snippet: ASPM plays a key role in the stabilization of drug-resistant cell lines. (A) Scatter plot of the correlation between ASPM and EGFR expression; p < 0.05. (B) Silencing of ASPM followed by ASPM and EGFR mRNA expression. (C) Protein expression of EGFR after silencing ASPM. (D) CHX administration at various time points. The right panel represents the relative gray values of the intracellular EGFR proteins, and the data are presented as the means ± standard deviations (means ± SDs, n = 3 fields). (E) IF images of PC-9 and PC-9 OR cells incubated with primary antibodies against EGFR (red) and ASPM (green). Detection was performed using secondary antibodies coupled to Alexa Fluor 488 and Alexa Fluor 555. The cell nuclei were stained with DAPI (blue), Scale = 7.5 μm. (F) mRNA expression levels of ASPM in PC-9 OR cells after silencing of ASPM -2. (G) PC-9 OR cells in the NC group/si ASPM -2 group were stained with propidium iodide (PI), and the DNA content was detected via flow cytometry. The red color represents the theoretical curve that we fit via ModFit software on the basis of the DNA content distribution data of the cells. (H) Statistics of the flow cytometric results in H plots. n = 3, a p value <0.05 was considered statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The membrane was blocked with 5% skim milk for 60 min and incubated with primary antibodies, including ASPM (Proteintech, 26223-1-AP) (1:600) and EGFR (Cusabio, CSB-PA005571LA01HU) (1:1000), overnight at 4 °C, followed by 2 h of incubation with HRP-conjugated goat anti-rabbit (Proteintech, SA00001-2) (1:2000) at room temperature.

Techniques: Expressing, Incubation, Staining, Flow Cytometry, Software

Journal: Frontiers in Genetics

Article Title: Assembly factor for spindle microtubules ( ASPM ) promotes osimertinib resistance in lung cancer by increasing EGFR stability

doi: 10.3389/fgene.2025.1593314

Figure Lengend Snippet: ASPM is significantly upregulated in NSCLC tumor tissues and strongly associated with reduced patient survival. ASPM silencing attenuates PC-9 and PC-9 OR malignant phenotypes, including proliferation and invasion, and sensitizes resistant cells to osimertinib. In addition, inhibiting the expression of ASPM effectively reduces damage to the cell cycle and protein stability of drug-resistant cells, thereby restoring the expression and function of EGFR.

Article Snippet: The membrane was blocked with 5% skim milk for 60 min and incubated with primary antibodies, including ASPM (Proteintech, 26223-1-AP) (1:600) and EGFR (Cusabio, CSB-PA005571LA01HU) (1:1000), overnight at 4 °C, followed by 2 h of incubation with HRP-conjugated goat anti-rabbit (Proteintech, SA00001-2) (1:2000) at room temperature.

Techniques: Expressing

Journal: Exploration

Article Title: ASPM Induces Radiotherapy Resistance by Disrupting Microtubule Stability Leading to Chromosome Malsegregation in Non‐Small Cell Lung Cancer

doi: 10.1002/EXP.20230024

Figure Lengend Snippet: High levels of ASPM perturb the stability of microtubules in A549‐R cells. (A) Immunofluorescence staining using anti‐ASPM, anti‐α‐tubulin, anti‐γ‐tubulin, and DAPI in A549‐R cells to detect ASPM co‐localization with microtubules during mitosis. Scale bar: 12 µm. (B) Immunofluorescence images of A549 cells and A549‐R cells with anti‐α‐tubulin (green) and anti‐acetylated α‐tubulin (red) antibodies and DAPI (blue). Scale bar: 10 µm. (C) Immunoblots showing levels of acetylated α‐tubulin in A549 cells and A549‐R cells. (D) Immunofluorescence images of A549 cells and A549‐R cells incubated on ice for the indicated time, followed by staining with anti‐α‐tubulin antibodies (green) and DAPI (blue). Scale bar: 10 µm, with quantification of microtubule intensity in cells treated as described above at various times afterward. n ≥ 12 in each group. (E) Immunostaining with anti‐α‐tubulin antibodies (green) and DAPI (blue) in A549 cells and A549‐R cells incubated on ice for 45 min to induce complete microtubule disassembly and incubating at 37°C for the indicated times. Scale bar: 10 µm. The panel below shows quantification of microtubule intensity in cells treated as described above at various times afterward. n ≥ 6 in each group. (F) Time‐lapse images showing variations in microtubule length in HeLa cells stably expressing GFP‐tagged α‐tubulin over time after irradiation with 2 Gy. Quantification of changes in microtubule length (G) and accumulated changes in microtubule length (H) from panel F. (I) Immunostaining with anti‐acetylated α‐tubulin antibodies and DAPI in A549‐R cells transfected with control or ASPM siRNAs. Scale bar: 10 µm. (J) Immunoblots showing levels of ASPM, acetylated α‐tubulin, α‐tubulin, in A549‐R cells transfected with control or ASPM siRNAs. ** p < 0.01. *** p < 0.001.

Article Snippet: Antibodies targeting ASPM (26223‐1‐AP), β‐actin (20536‐1‐AP), and acetylated α‐tubulin (66200‐1‐Ig) were purchased from ProteinTech (Rosemont, IL).

Techniques: Immunofluorescence, Staining, Western Blot, Incubation, Immunostaining, Stable Transfection, Expressing, Irradiation, Transfection, Control

Journal: Exploration

Article Title: ASPM Induces Radiotherapy Resistance by Disrupting Microtubule Stability Leading to Chromosome Malsegregation in Non‐Small Cell Lung Cancer

doi: 10.1002/EXP.20230024

Figure Lengend Snippet: Loss of ASPM prevents spindle misorientation in A549‐R cells. (A) Schematic depicting the process for measuring spindle angles (α). Representative immunofluorescence images (B), spindle angles (C), and spindle angle distribution (D) of metaphase A549 cells and A549‐R cells stained with anti‐α‐tubulin (red) and anti‐γ‐tubulin (green) antibodies and DAPI (blue). Scale bar: 6 µm. n = 62 in A549 group. n = 59 in A549‐R group. (E) Immunoblots showing the effectiveness of ASPM siRNAs in A549‐R cells. Representative immunofluorescence images (F), spindle angles (G), and spindle angle distribution (H) of metaphase A549‐R cells transfected with control or ASPM siRNAs and stained with anti‐α‐tubulin (red) and anti‐γ‐tubulin (green) antibodies and DAPI (blue). Scale bar: 6 µm. *** p < 0.001. n = 35 in siControl group. n = 38 in siASPM#1 group. n = 42 in siASPM#1 group.

Article Snippet: Antibodies targeting ASPM (26223‐1‐AP), β‐actin (20536‐1‐AP), and acetylated α‐tubulin (66200‐1‐Ig) were purchased from ProteinTech (Rosemont, IL).

Techniques: Immunofluorescence, Staining, Western Blot, Transfection, Control

Journal: Exploration

Article Title: ASPM Induces Radiotherapy Resistance by Disrupting Microtubule Stability Leading to Chromosome Malsegregation in Non‐Small Cell Lung Cancer

doi: 10.1002/EXP.20230024

Figure Lengend Snippet: ASPM promotes incorrect chromosome segregation and formation of micronuclei. (A) Schematic diagram showing the effects of microtubule disruption during cytokinesis on chromosome mis‐segregation. (B) Immunofluorescence images of A549 cells and A549‐R cells during cytokinesis with anti‐α‐tubulin (green) and anti‐acetylated α‐tubulin (red) antibodies and DAPI (blue). Scale bar: 10 µm. n = 10 in A549 group. n = 12 in A549‐R group. (C) Fluorescence images of lung biopsy samples from patients with RT‐sensitive or RT‐resistant disease showing chromosomal segregation after metaphase of cancer cells with anti‐pan CK (green) and DAPI (blue), with quantification of chromosomal mis‐segregation. Scale bar: 20 µm (left) and 10 µm (right). n = 6 in Sensitive group and n = 8 in resistant group. (D) Immunofluorescence images of A549 cells and A549‐R cells with anti‐α‐tubulin (green) and anti‐γ‐tubulin (red) antibodies and DAPI (blue) showing generation of micronuclei in the cells, with quantification of micronuclei. Scale bar: 10 µm. n = 12 in each group. (E) Ploidy score showing the correlation between ASPM expression and generation of ploidy, analyzed with The Cancer Genome Atlas database. (F) Immunoblots showing the effectiveness of ASPM siRNAs in A549‐R cells. (G) Immunofluorescence images of A549‐R cells with control or ASPM siRNAs treated and stained with anti‐α‐tubulin (green) and anti‐acetylated α‐tubulin (red) antibodies and DAPI (blue) during cytokinesis, with quantification of chromosomal mis‐segregation. Scale bar: 10 µm. n = 12 in each group. (H) Immunofluorescence images of A549‐R cells transfected with control or ASPM siRNAs, followed by staining with anti‐α‐tubulin (green) and anti‐γ‐tubulin (red) antibodies and DAPI (blue) showing generation of micronuclei in the cell, with quantification of micronuclei. Scale bar: 10 µm. ** p < 0.01. *** p < 0.001. The experiment was repeated twelve times, at least 36 cells in each group.

Article Snippet: Antibodies targeting ASPM (26223‐1‐AP), β‐actin (20536‐1‐AP), and acetylated α‐tubulin (66200‐1‐Ig) were purchased from ProteinTech (Rosemont, IL).

Techniques: Disruption, Immunofluorescence, Fluorescence, Expressing, Western Blot, Control, Staining, Transfection

Journal: Exploration

Article Title: ASPM Induces Radiotherapy Resistance by Disrupting Microtubule Stability Leading to Chromosome Malsegregation in Non‐Small Cell Lung Cancer

doi: 10.1002/EXP.20230024

Figure Lengend Snippet: ASPM depletion re‐sensitizes RT‐resistant cells to RT‐induced apoptosis. (A) Immunofluorescence images of γH2AX foci in A549‐R cells treated with control or ASPM siRNAs followed by irradiation to 2 Gy, with quantification of foci per cell. Scale bar: 8 µm. The experiment was repeated three times, 30 cells in each group. (B) Immunoblots showing levels of ASPM, γH2AX, cleaved caspase‐3, and β‐actin in A549‐R cells transfected with control or ASPM siRNAs followed by treatment with 2 Gy. (C) Representative analyses of numbers of A549‐R clones treated with control or ASPM siRNAs and then treatment with 2 Gy. (D) Flow cytometry analysis of apoptotic A549‐R cells treated with control or ASPM siRNAs followed by treatment with 2 Gy. The experiment was repeated five times in each group, n = 5. (E) Immunoblots showing levels of ASPM, cleaved caspase‐3, acetylated α‐tubulin, α‐tubulin, and β‐actin in subcutaneous tumors from nude mice implanted with cells incorporating control‐ or ASPM siRNAs after treatment with 2 Gy. (F) Subcutaneous tumor volumes formed by A549‐R cells treated with ASPM siRNAs were reduced after treatment with 2 Gy. Five mice in each group, n = 5. NS, not significant; ** p < 0.01. *** p < 0.001. The experiment was repeated three times, 6 mice in each group. (G) Schematic diagram showing the consequences of irradiating RT‐resistant cells with high or low expression of ASPM.

Article Snippet: Antibodies targeting ASPM (26223‐1‐AP), β‐actin (20536‐1‐AP), and acetylated α‐tubulin (66200‐1‐Ig) were purchased from ProteinTech (Rosemont, IL).

Techniques: Immunofluorescence, Control, Irradiation, Western Blot, Transfection, Clone Assay, Flow Cytometry, Expressing