Journal: iScience

Article Title: A Non-genetic Mechanism Involving the Integrin β4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer

doi: 10.1016/j.isci.2020.101496

Figure Lengend Snippet: Knocking Down ITGB4 Attenuates Proliferation and Migration and Increases Sensitivity of Cisplatin-Resistant Cells H2009 and H1993 stable cell lines expressing mKate2 were transfected with control (Si Scramble) or ITGB4-specific (Si ITGB4) siRNA. (A and B) (A) ITGB4 knockdown cells (red) had a significantly reduced proliferation rate than control cells (black). (∗∗∗∗p < 0.0001 two-way ANOVA). (B) Immunoblotting and qPCR data confirming the knockdown. (C and D) (C) A scratch wound assay demonstrating the effect of knocking down ITGB4. ITGB4 knockdown (red) significantly halted the migration and did not close the wound completely after 96 h in both resistant cell lines. (∗∗∗∗p < 0.0001 two-way ANOVA). (D) Rate at which the wound closed was also significantly decreased in ITGB4 knockdown cells (red). (∗∗∗∗p < 0.0001 and ∗p < 0.0156 two-way ANOVA). (E) Cisplatin (10 μM) treatment for 72 h reduced expression of phosphorylated PXN, PXN, and total FAK, but not ITGB4. (LE = low exposure, HE = high exposure). (F) ITGB4 knockdown in H1993 cells inhibited proliferation and addition of cisplatin had a cytotoxic effect (∗∗∗∗p < 0.0001 two-way ANOVA). (G) ITGB4 knockdown in H1993 cells increased caspase-3/7 activity. Treating ITGB4 knockdown cells with cisplatin had an added effect of inducing caspase activity, but drug treatment alone did not have a cytotoxic effect (∗∗∗∗p < 0.0001 two-way ANOVA). (H) ITGB4 knockdown in H2009 cells inhibited proliferation by 72 h and addition of cisplatin had a cytostatic effect (∗∗∗∗p < 0.0001 two-way ANOVA). (I) ITGB4 knockdown in H2009 cells did not induce caspase activity. Cisplatin treatment to ITGB4 knockdown cells for 72 h had an additive effect to induce caspase activity (∗∗∗∗p < 0.0001 two-way ANOVA). (J) Immunoblot showing that MET protein expression in H1993 cells was reduced 4 days after knocking down ITGB4. Data are represented as mean ± SD.

Article Snippet: We tested three small interfering RNA (siRNA) constructs from OriGene in the commonly employed NSCLC cell lines H358 and A549. siRNA C was found to be most effective in knocking down ITGB4 and inhibiting proliferation ( B–S3D).

Techniques: Migration, Stable Transfection, Expressing, Transfection, Control, Knockdown, Western Blot, Scratch Wound Assay Assay, Activity Assay

Journal: iScience

Article Title: A Non-genetic Mechanism Involving the Integrin β4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer

doi: 10.1016/j.isci.2020.101496

Figure Lengend Snippet: Knocking Down Both PXN and ITGB4 has a Synergistic Effect on Attenuating Cisplatin Resistance in 2D and 3D Cultures (A) Double knockdown of both PXN and ITGB4 (red) in H1993 cells had a synergistic effect on inhibiting proliferation compared with single knockdown of either gene (green/blue) and control (black). (∗∗∗∗p < 0.0001 two-way ANOVA). (B) Adding 10 μM cisplatin to the double knockdown cells (red) had an even greater effect on inhibiting proliferation compared with single knockdown of either gene (green/blue) and control (black) treated with cisplatin. (∗∗∗∗p < 0.0001 two-way ANOVA). (C) Double knockdown of both PXN and ITGB4 (red) in H2009 cells also had a synergistic effect on inhibition of proliferation compared with single knockdown of either gene (green/blue) and control (black). (∗∗∗∗p < 0.0001 two-way ANOVA). (D) With double knockdown of PXN and ITGB4 in combination with cisplatin (red), proliferation was greatly inhibited compared with single knockdown of either gene (green/blue) and control (black) treated with cisplatin. (∗∗∗∗p < 0.0001 two-way ANOVA). (E) Immunoblotting confirmed siRNA-mediated knockdown of PXN and ITGB4. PXN knockdown alone increased expression of p27 and decreased levels of phospho-Rb (S807/811), indicating cell-cycle arrest. (F) Cell cycle analysis revealed that knocking down PXN induced G1-S arrest, whereas knocking down ITGB4 arrested cells in G2-M. Double knockdown of PXN and ITGB4 arrested cells in G1-S and G2-M. (G) H2009 cells expressing mKate2 were transfected with siRNA, seeded in a 96-well ultra-low attachment plate (5,000 cells/well), and allowed to form a compact spheroid overnight. Images acquired by the IncuCyte Live Cell Imaging System showed that spheroids with a single knockdown start disintegrating by day 3 and even earlier for double knockdown spheroids (Day 1). (H) Immunoblotting confirmed that the PXN and ITGB4 siRNA-mediated knockdown was still effective after 72 h in 3D culture. (I and J) To quantitate spheroid viability, (I) red fluorescence area and (J) mean intensity were measured. Both parameters showed that double knockdown had a synergistic effect on attenuating spheroid viability (∗∗∗∗p < 0.0001 two-way ANOVA). Data are represented as mean ± SD.

Article Snippet: We tested three small interfering RNA (siRNA) constructs from OriGene in the commonly employed NSCLC cell lines H358 and A549. siRNA C was found to be most effective in knocking down ITGB4 and inhibiting proliferation ( B–S3D).

Techniques: Knockdown, Control, Inhibition, Western Blot, Expressing, Cell Cycle Assay, Transfection, Live Cell Imaging, Fluorescence

Journal: iScience

Article Title: A Non-genetic Mechanism Involving the Integrin β4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer

doi: 10.1016/j.isci.2020.101496

Figure Lengend Snippet: Signaling Pathways of PXN and ITGB4 Converge in Mediating Cisplatin Resistance (A) Total RNA was extracted from single and double knockdown cells 48 h after siRNA transfection. Total RNA-seq revealed the number of genes downregulated with single knockdown of PXN and ITGB4 and when both genes are knocked down simultaneously. (B) Total RNA-seq after single and double knockdown of PXN and ITGB4 revealed that five common genes were downregulated compared with control cells (SCR). (C) Major pathways affected by double knockdown of PXN and ITGB4 were MYC targets, G2-M checkpoint, and E2-F targets. (D) Bar diagram showing the major Hallmark pathways affected by the double knockdown. The top 10 pathways were arranged in descending order of their enrichment scor23e. (E) Heatmap representation of top 10 genes that belong to hallmark MYC target V1 pathways were analyzed after single or double knockdown. The heatmap is representative of two experimental repeats. (F) Immunoblotting confirmed decreased expression of top three genes (G3BP1, USP1, and VDAC1) that are downregulated in MYC pathway after knockdown of PXN and/or ITGB4. (G) qPCR results with single and double knockdown of PXN and ITGB4 confirmed downregulation of VDAC1 and USP1 at the mRNA level (∗∗∗∗p < 0.0001 two-way ANOVA). Data are represented as mean ± SD.

Article Snippet: We tested three small interfering RNA (siRNA) constructs from OriGene in the commonly employed NSCLC cell lines H358 and A549. siRNA C was found to be most effective in knocking down ITGB4 and inhibiting proliferation ( B–S3D).

Techniques: Protein-Protein interactions, Knockdown, Transfection, RNA Sequencing, Control, Western Blot, Expressing

Journal: iScience

Article Title: A Non-genetic Mechanism Involving the Integrin β4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer

doi: 10.1016/j.isci.2020.101496

Figure Lengend Snippet: Knocking Down MYC Pathway Genes USP1 and VDAC1 Attenuates Cisplatin Resistance and USP1 is Involved in Genomic Stability (A) Gene expression profiles of patients with LUAD were extracted from TCGA database, and expressions of USP1 and VDAC1 were higher compared with normal tissue. (B) siRNA constructs A, B, or C against top three downregulated genes (VDAC1, USP1, and G3BP1) were tested to determine the construct with maximum effect on inhibiting proliferation. (∗∗∗∗p < 0.0001 two-way ANOVA). (C) Double knockdown of PXN and ITGB4 (Si ITGB4+Si PXN) decreased expression of USP1 and VDAC1. However, knocking down USP1 (Si USP1) or VDAC1 (Si VDAC1) did not affect expression of ITGB4 or PXN. (D) In H2009 cells, knocking down USP1 (blue) attenuated proliferation (∗∗∗∗p < 0.0001 two-way ANOVA). (E) In H2009 cells, knocking down USP1 (blue) induced apoptosis and sensitized cells to a lower dose of cisplatin (2 μM) (red) (∗∗∗∗p < 0.0001 two-way ANOVA). (F) After knocking down PXN/ITGB4 and USP1, γH2AX foci were detected via immunofluorescence, imaged with confocal microscopy, and counted with QuPath image analysis software (∗∗p = 0.004, ∗∗∗∗p < 0.0001). (G) Knocking down VDAC1 (blue) inhibited cell proliferation by 50% within 72 h, and cisplatin (red) addition has an additive effect to VDAC1 knockdown in inhibiting proliferation (∗∗∗∗p < 0.0001 two-way ANOVA). (H) Knocking down VDAC1 (blue) could induce apoptosis only at a later time point (∗∗∗p = 0.0001 two-way ANOVA). Data are represented as mean ± SD.

Article Snippet: We tested three small interfering RNA (siRNA) constructs from OriGene in the commonly employed NSCLC cell lines H358 and A549. siRNA C was found to be most effective in knocking down ITGB4 and inhibiting proliferation ( B–S3D).

Techniques: Gene Expression, Construct, Knockdown, Expressing, Immunofluorescence, Confocal Microscopy, Software

Journal: iScience

Article Title: A Non-genetic Mechanism Involving the Integrin β4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer

doi: 10.1016/j.isci.2020.101496

Figure Lengend Snippet: Mathematical Modeling Suggests that Cisplatin Resistance Can Be Stochastic and Reversible (A) Chromatin immunoprecipitation was performed with an acetylated H3K27 antibody 72 h after siRNA-mediated knockdown of PXN/ITGB4. With the knockdown, H3K27 acetylation at the promoter region of USP1 was greatly reduced compared with that of an upstream region, indicating the roles of ITGB4 and PXN in USP1 transcriptional activation. (B) A double-negative feedback loop between ITGB4 and miR-1-3p leads to bistability. (C) To test this mathematical model, RACIPE algorithm generated an ensemble (n = 100,000) with varying parameter sets then plotted to represent robust dynamical patterns. Results showed that ITGB4 and miR-1-3p exhibit bimodality: two distinct subpopulations of cells that are negatively correlated, reinforcing the previously described negative feedback loop. (D) A mathematical model stimulating the dynamics of ITGB4 and miR-1-3p showed cisplatin resistance to be a reversible state. High ITGB4 and low miR-1-3p render cells to be resistant, whereas low ITGB4 and high miR-1-3p represents a more sensitive state. (E) H2009 cells were stained with ITGB4 antibody conjugated to Alexa Fluor 488 and sorted based on gates set to high and low ~10% of ITGB4-expressing population using the FACSAria Fusion instrument. Sorted cells were subsequently cultured for 48 h and then treated with 1 μM cisplatin for 48 h. Then, using the Attune NxT Flow Cytometer, equal numbers of cells were stained again and analyzed to determine shifts in population between untreated and treated cells. Low sorted cells treated with cisplatin had a greater cell population that shifted toward higher ITGB4 expression. High sorted cells treated with cisplatin did not undergo significant changes in population compared with untreated. (F) Schematic depicting the interaction between ITGB4 and PXN regulating downstream proteins USP1 and VDAC1 at the transcriptional level to coordinate cisplatin resistance. Data are represented as mean ± SD.

Article Snippet: We tested three small interfering RNA (siRNA) constructs from OriGene in the commonly employed NSCLC cell lines H358 and A549. siRNA C was found to be most effective in knocking down ITGB4 and inhibiting proliferation ( B–S3D).

Techniques: Chromatin Immunoprecipitation, Knockdown, Activation Assay, Generated, Staining, Expressing, Cell Culture, Flow Cytometry

Journal: BMC Cancer

Article Title: Identification and functional characterization of genes associated with lipopolysaccharide in lung adenocarcinoma

doi: 10.1186/s12885-025-15387-z

Figure Lengend Snippet: Expression of ITGB4 in Lung Adenocarcinoma Cells and Tissues. A Comparison of ITGB4 expression between 483 LUAD samples and 347 normal samples. B K–M survival curves for high and low ITGB4 expression groups. C – D ITGB4 expression levels in normal lung epithelial cell lines and LUAD cell lines analyzed by qRT-PCR and WB. E WB analysis of ITGB4 expression in 12 paired LUAD tissues. F IHC detection of ITGB4 expression in LUAD tissues. * p < 0.05, ** p < 0.01

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with the following primary antibodies: ITGB4 (Proteintech, 21738-1-AP), GAPDH (Proteintech, 10494-1-AP), MMP2 (Proteintech, 10373-2-AP), MMP9 (Proteintech, 10375-2-AP), p53 (Proteintech, 10442-1-AP), Caspase-3 (Cell Signaling Technology, 9664 T), and Bcl-2 (Cell Signaling Technology, 4223 T).

Techniques: Expressing, Comparison, Quantitative RT-PCR

Journal: BMC Cancer

Article Title: Identification and functional characterization of genes associated with lipopolysaccharide in lung adenocarcinoma

doi: 10.1186/s12885-025-15387-z

Figure Lengend Snippet: Knockdown of ITGB4 Inhibits Proliferation, Migration, and Invasion of Lung Adenocarcinoma Cells A.Efficiency of ITGB4 knockdown in A549 and PC9 cells validated by qRT-PCR and WB. B.Cell proliferation assessed using the CCK-8 assay. C.Cell migration evaluated by wound healing assay. D.Expression levels of migration-related proteins (MMP2, MMP9) detected by WB. E. Transwell assay used to assess cell migration and invasion abilities. *** p < 0.001, **** p < 0.0001

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with the following primary antibodies: ITGB4 (Proteintech, 21738-1-AP), GAPDH (Proteintech, 10494-1-AP), MMP2 (Proteintech, 10373-2-AP), MMP9 (Proteintech, 10375-2-AP), p53 (Proteintech, 10442-1-AP), Caspase-3 (Cell Signaling Technology, 9664 T), and Bcl-2 (Cell Signaling Technology, 4223 T).

Techniques: Knockdown, Migration, Quantitative RT-PCR, CCK-8 Assay, Wound Healing Assay, Expressing, Transwell Assay

Journal: BMC Cancer

Article Title: Identification and functional characterization of genes associated with lipopolysaccharide in lung adenocarcinoma

doi: 10.1186/s12885-025-15387-z

Figure Lengend Snippet: ITGB4 Knockdown Induces G1 Phase Arrest and Promotes Apoptosis. A Effects of ITGB4 knockdown on the cell cycle of PC9 cells. B Effects of ITGB4 knockdown on apoptosis in PC9 cells. C Changes in the expression of apoptosis-related proteins (Caspase-3, P53, Bcl-2) in PC9 cells. *** p < 0.001

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with the following primary antibodies: ITGB4 (Proteintech, 21738-1-AP), GAPDH (Proteintech, 10494-1-AP), MMP2 (Proteintech, 10373-2-AP), MMP9 (Proteintech, 10375-2-AP), p53 (Proteintech, 10442-1-AP), Caspase-3 (Cell Signaling Technology, 9664 T), and Bcl-2 (Cell Signaling Technology, 4223 T).

Techniques: Knockdown, Expressing

Journal: BMC Cancer

Article Title: Identification and functional characterization of genes associated with lipopolysaccharide in lung adenocarcinoma

doi: 10.1186/s12885-025-15387-z

Figure Lengend Snippet: Functional and Pathway Analysis Regulated by ITGB4. A Volcano plot of differentially expressed genes. B GO enrichment analysis. C – D KEGG enrichment analysis

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with the following primary antibodies: ITGB4 (Proteintech, 21738-1-AP), GAPDH (Proteintech, 10494-1-AP), MMP2 (Proteintech, 10373-2-AP), MMP9 (Proteintech, 10375-2-AP), p53 (Proteintech, 10442-1-AP), Caspase-3 (Cell Signaling Technology, 9664 T), and Bcl-2 (Cell Signaling Technology, 4223 T).

Techniques: Functional Assay

Journal: BMC Cancer

Article Title: Identification and functional characterization of genes associated with lipopolysaccharide in lung adenocarcinoma

doi: 10.1186/s12885-025-15387-z

Figure Lengend Snippet: ITGB4 Promotes Tumor Growth In Vivo. A Xenograft model in nude mice. B Tumor volume measurement. C Tumor weight evaluation. D ITGB4 expression levels detected in the orthotopic lung cancer model by IHC. ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with the following primary antibodies: ITGB4 (Proteintech, 21738-1-AP), GAPDH (Proteintech, 10494-1-AP), MMP2 (Proteintech, 10373-2-AP), MMP9 (Proteintech, 10375-2-AP), p53 (Proteintech, 10442-1-AP), Caspase-3 (Cell Signaling Technology, 9664 T), and Bcl-2 (Cell Signaling Technology, 4223 T).

Techniques: In Vivo, Expressing, Paraffin-embedded Immunohistochemistry

Journal: Cancers

Article Title: Fibroblasts Promote Resistance to KRAS Silencing in Colorectal Cancer Cells

doi: 10.3390/cancers16142595

Figure Lengend Snippet: List of anti-human antibodies used for flow cytometry.

Article Snippet: CD104 , FITC , REA236 , 130-124-266 , Miltenyi Biotec.

Techniques: Cytometry

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: Increased SOX2 gene amplification and protein overexpression in LUSC (A) Analysis conducted on the C-Bio portal Pan-Cancer Analysis revealed SOX2 gene amplification in 39.4% of LUSC. (B) The GEPIA interactive software was used to determine the expression of stem cell markers SOX2, EPCAM, CD133, CD44, and ITGB4 in the LUSC TCGA dataset. The significant changes in expression between normal and tumor tissue were determined (∗p < 0.01). (C and D) The gene expression analysis of SOX2 and ITGB4 was performed on different sub-histologies of LUSC within the TCGA dataset, utilizing the GEPIA interactive software. The analysis demonstrated a statistically significant association (∗p < 0.01). (E) The overall survival of LUSC subtypes expressing median high or low levels of ITGB4 normalized to SOX2 expression was investigated. Except for the basal subtype, the overall survival was poor for all other subtypes. (F) Immunofluorescence analysis was performed on a LUSC tumor microarray, demonstrating variations in the expression and spatial distribution of ITGB4 and SOX2. The SOX2 protein was represented by red fluorescence, ITGB4 by yellow fluorescence, and DAPI staining was used for blue visualization.

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Amplification, Over Expression, Software, Expressing, Gene Expression, Immunofluorescence, Microarray, Fluorescence, Staining

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: Patient-derived cells exhibited the stem cell-like phenotype (A) Schematic representation illustrating the process of tumor tissue processing, isolation, and characterization of patient-derived primary cell lines. (B) Flow cytometry analysis demonstrating the expression of cancer stem cell surface markers EPCAM, CD133, and CD44. (C) Quantitative PCR analysis revealing the expression levels of stem cell markers, including SOX2, CD44, ALDH1A1, EPCAM, ITGB4, and PXN, in two primary patient-derived cell lines. Statistical significance was determined using ordinary one-way ANOVA (∗p < 0.05, ∗∗p < 0.001, ∗∗∗p = 0.0001, ∗∗∗∗p < 0.0001). (D) Immunoblotting analysis showcasing variations in the expression of stem cell markers among the BEAS2B, COH1, and COH2 cell lines. (E) Spheroid formation assay depicting the ability of COH2 cells to form spheroids in specialized media, while BEAS2B cells failed to form spheroids, indicating a stemness phenotype in COH2 cells. (F and G) Cell viability assay was conducted on COH2 primary cells in both attached and spheroid conditions after a 3-day treatment with cisplatin. The results are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA (∗∗p < 0.01). (H) Western blot analysis was performed on COH2 cells following treatment with increasing concentrations of cisplatin for 3 days, revealing no significant changes in the expression of SOX2 or ITGB4.

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Derivative Assay, Isolation, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Tube Formation Assay, Viability Assay

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: Subclones retain cisplatin-resistant phenotype, and ITGB4 knockdown sensitizes the primary cells to cisplatin (A) Schematic representation illustrating the isolation of single clones from the primary patient-derived cell line. (B and C) Representative images demonstrating the differential growth patterns of subclones derived from single cells. (D and E) Immunoblotting analysis depicting the differences in the expression of SOX2 and ITGB4 in the subclones compared to the mixed parental cells. (F and G) Cell viability assay (CCK8 assay) was conducted on subclones in both attached and spheroid conditions after a 3-day treatment with cisplatin. The subclones exhibited a resistant phenotype similar to parental cells. Results are presented as mean ± SD. Statistical significance was determined using one-way ANOVA (ns, not significant). (H) Western blot analysis was performed after 3 days of cisplatin treatment on subclone 1, revealing no significant changes in the expression of SOX2 or ITGB4. (I) Immunoblotting analysis conducted after 72 h of transfection with siRNA targeting SOX2, siRNA targeting ITGB4, or both, in COH2 cells. ITGB4 knockdown resulted in suppressed SOX2 expression. The numbers below each immunoblot represent the percentage difference in intensity compared to the control. The band intensity was normalized to actin and then compared to the control. (J) The knockdown of ITGB4 or SOX2 in COH2 cells sensitized the cells to cisplatin. Statistical significance was determined using ordinary one-way ANOVA. (K) Stable cell lines expressing ITGB4 shRNA were generated using COH2 cells. Immunoblotting confirmed ITGB4 knockdown and a reduction in SOX2 expression. The numbers below each immunoblot represent the percentage difference in intensity compared to the control. (L) These stable cell lines expressing ITGB4 shRNA exhibited high sensitivity to cisplatin. The bar graph represents the mean ± SD. Statistical significance was determined using one-way ANOVA (∗∗∗p = 0.0001, ∗∗∗∗p < 0.0001).

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Knockdown, Isolation, Clone Assay, Derivative Assay, Western Blot, Expressing, Viability Assay, CCK-8 Assay, Transfection, Control, Stable Transfection, shRNA, Generated

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: Inhibition of SOX2 expression by ITGB4 knockdown (A) Western blot analysis revealing the expression levels of SOX2 and ITGB4 in different cell lines of LUSC and SCLC. H520 and SBC5 cell lines exhibit higher expression of SOX2 and lower expression of ITGB4. (B) Cell viability assays were performed on H520 and SBC5 cells after 3 days of cisplatin treatment. The IC50 value indicates that SBC5 is resistant to cisplatin. (C) Immunoblotting data showed no significant reduction in the expression patterns of SOX2 and ITGB4 after 3 days of cisplatin treatment. (D and E) The knockdown of SOX2 sensitizes H520 and SBC5 cells to lower concentrations (2μM) of cisplatin. Results are presented as mean ± SD. Statistical significance was determined using one-way ANOVA (ns, not significant, ∗∗p < 0.001, ∗∗∗∗p < 0.0001). (F) Immunoblots confirmed reduced expression of SOX2 in the H520 and SBC5 cells with no significant change in ITGB4 expression. (G and H) H520 and SBC5 cells with ITGB4 knockdown exhibited sensitivity to cisplatin treatment at a lower concentration of 2μM. Results are presented as mean ± SD. Statistical significance was determined using one-way ANOVA (ns, not significant, ∗∗p < 0.001, ∗∗∗∗p < 0.0001). (I) Immunoblots confirmed ITGB4 knockdown in both H520 and SBC5 cell lines in addition to reduced expression of SOX2 and YAP1. The percentage reduction compared to the control is quantified and mentioned below each immunoblot. (J) Chromatin immunoprecipitation was performed using H3K27Ac and H3Kme4 antibodies on H520 control and ITGB4 knockdown cell lines. A significant reduction in histone acetylation was observed at the SOX2 promoter site. Results are presented as mean ± SD. Statistical significance was determined using one-way ANOVA (ns-not significant, ∗∗∗p = 0.0001).

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Inhibition, Expressing, Knockdown, Western Blot, Concentration Assay, Control, Chromatin Immunoprecipitation

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: Sensitivity of primary cell lines to proteasome inhibitors (A) CCK8 assay performed on COH2 cells after 3 days of treatment with increasing concentrations of carfilzomib (CFZ) or ixazomib (IXA) in 2D culture. (B) CCK8 assay conducted on COH2 cells treated with increasing concentrations of CFZ or IXA in 3D culture. Data are presented as mean ± SD (n = 3), and IC50 values were calculated using GraphPad Prism 9.0. (C) Immunoblotting analysis of SOX2, ITGB4, ALDH1A1, EPCAM, and cytokeratin in COH2 cells after 3 days of CFZ and IXA treatment at their respective IC50 doses. The normalized changes in protein expression are indicated below each immunoblot. Treatment with the drugs resulted in reduced expression of SOX2 and ITGB4. (D) CCK8 assay showing the significant inhibition of Clone 1 cell viability after 3 days of CFZ treatment. Mean ± SD shown, with statistical significance determined by one-way ANOVA (ns, not significant, ∗∗∗∗p < 0.0001). (E) Immunoblot and densitometry analysis revealed reduced SOX2 expression following CFZ treatment at IC50 dose for 3 days. (F) Evaluation of the inhibitory effect of the cisplatin and CFZ combination using synergy experiments. The average synergy score was calculated using Bliss analysis. A synergy score ≥10 indicates synergism. (G) COH2 cells treated with the cisplatin and CFZ combination exhibited inhibition of CD44, EPCAM, SOX2, and ITGB4 expression, as shown by immunoblotting. (H and I) CCK8 assay was performed on H520 and SBC5 cells after 3 days of CFZ or IXA treatment in 2D culture. Data are presented as mean ± SD (n = 3), and IC50 values were calculated for both inhibitors. CFZ demonstrated efficacy at lower doses. Analysis was performed using GraphPad Prism 9.0.

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: CCK-8 Assay, Western Blot, Expressing, Inhibition

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet: CFZ reduced SOX2 expression by inhibiting promoter activity (A) Immunofluorescent images of COH2 cells treated with 40 nM and 80 nM CFZ for 3 days, showing differential expression of SOX2 (Green) among cells. High SOX2-expressing cells are indicated by white arrows, and low SOX2-expressing cells are indicated by red arrows. Blue represents DAPI staining. Scale bar: 100 μm. Quantification of high SOX2 expression cell numbers using QuPath. Data presented as mean ± SD (n = 10), ∗∗p < 0.01 one-way ANOVA. (B–D) Time course measurement of SOX2 mRNA expression in COH2, H520, and SBC5 cells after 6 h of CFZ treatment up to 24 h using qPCR. (E) qPCR assay measuring changes in SOX2 mRNA expression in COH2, H520, and SBC5 cells after 6 h of CFZ or Act D treatment. Both drugs significantly reduced SOX2 expression. (F and G) qPCR measurement of ALDH1A1, CD44, and EPCAM mRNA expression in COH2 and H520 cells after 6 h of CFZ or Act D treatment. No significant reduction was observed in these markers. (H–J) COH2, H520, and SBC5 were transfected with p-GL3-SOX2 promoter expressing plasmid. A significant reduction in promoter activity was observed after treatment with ActD and CFZ (IC50 dose, 12 h, and 24 h), but not with cisplatin. Data represented as mean ± SD (n = 3). (K) CFZ or IXA treatment significantly inhibits the binding of acetylated H3K27 at the SOX2 promoter site in COH2 cells, correlating with the reduction in its transcription. (L) SOX2 chromatin immunoprecipitation (ChIP) reveals the binding of SOX2 at the promoter region of PXN and ITGB4. Statistical significance was determined using one-way or two-way ANOVA, with p values as follows: ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and NS (not significant).

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Expressing, Activity Assay, Quantitative Proteomics, Staining, Transfection, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation

Journal: iScience

Article Title: Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors

doi: 10.1016/j.isci.2023.107302

Figure Lengend Snippet:

Article Snippet: Likewise, short hairpin RNAs (s) against human ITGB4, and SOX2 were purchased from Origene (Cat# TL309173 and Cat# TL312080).

Techniques: Control, Virus, Recombinant, Live Cell Imaging, Chromatin Immunoprecipitation, Luciferase, CCK-8 Assay, Small Interfering RNA, shRNA, Software

Journal: Breast Cancer : Basic and Clinical Research

Article Title: Male Breast Cancer—Immunohistochemical Patterns and Clinical Relevance of FASN, ATF3, and Collagen IV

doi: 10.1177/11782234211002496

Figure Lengend Snippet: Antibody reagents and conditions.

Article Snippet: β4 integrin , Atlas antibodies , ITGB4 , 1:200—28 min , ULTRA CC1-56 min.

Techniques:

Journal: Marine Drugs

Article Title: Characterisation of Elevenin-Vc1 from the Venom of Conus victoriae : A Structural Analogue of α-Conotoxins

doi: 10.3390/md21020081

Figure Lengend Snippet: Two-dimensional [ 15 N– 1 H]-HSQC spectrum of elevenin-Vc1 showing backbone amide resonance assignments. The spectrum was acquired at 303 K and pH 4 on a Bruker Avance III 600 MHz spectrometer.

Article Snippet: The human muscle nAChR clones (α1, β1, δ, and ε) were purchased from Integrated DNA Technologies (Coralville, IA, USA), the human (h) α3, α9, α10, β2, and β4 clones were purchased from OriGene (Rockville, MD, USA), and all were subsequently inserted into the pT7TS vector.

Techniques:

Journal: Marine Drugs

Article Title: Characterisation of Elevenin-Vc1 from the Venom of Conus victoriae : A Structural Analogue of α-Conotoxins

doi: 10.3390/md21020081

Figure Lengend Snippet: ( A ) Region of 600 MHz NOESY spectrum of elevenin-Vc1 showing NOEs diagnostic of the Ala11-Pro12 peptide bond conformation. Strong NOEs were observed between Ala11 Hα ( i ) and Pro12 Hδ protons ( i + 1), which are ~2.2 Å apart, indicating the trans conformation of the Ala11-Pro12 bond ( B ). In addition, a weak NOE (⁎) is observed between Ala11 Hα and Pro12 Hα protons; this may be because these protons are within 5 Å (4.4 Å). ( C ) 2D [ 13 C– 1 H]-HSQC spectrum showing Pro12 Cα and Cβ NMR assignments. The small Cβ and Cγ chemical shift difference of 4.3 ppm (<5 ppm for trans and ~10 ppm for cis ) further confirms the trans geometry of the Ala11-Pro12 peptide bond.

Article Snippet: The human muscle nAChR clones (α1, β1, δ, and ε) were purchased from Integrated DNA Technologies (Coralville, IA, USA), the human (h) α3, α9, α10, β2, and β4 clones were purchased from OriGene (Rockville, MD, USA), and all were subsequently inserted into the pT7TS vector.

Techniques: Diagnostic Assay

Journal: Advanced healthcare materials

Article Title: Fibrosis-Encapsulated Tumoroid, A Solid Cancer Assembloid Model for Cancer Research and Drug Screening.

doi: 10.1002/adhm.202402391

Figure Lengend Snippet: Figure 7. Application of FETs in cancer research. A) Evaluation of exosome-carrying miRNA delivery to FETs. Exosomes were isolated from HEK293T cells transfected with control or miR-9 vectors. a) Western blot analysis of exosome markers in isolated exosomes. b) Real-time PCR analysis of miR-9 levels in control and miR-9 exosomes. c) Schematic process of exosome treatment to FETs. d) Images of exosome-treated FETs on days 1 and 4. e) Total area (TA) and f) signal integrated density (SID) of FETumoroids treated with control or miR-9 exosomes on days 1 and 4. g) Fold changes (FCs) in TA and SID of FETumoroids in exosome-treated FETs. FCs were calculated by dividing the values of FETumoroids on day 4 by those on day 1. h) Cancer cell cluster number in FETs treated with control or miR-9 exosomes on day 4. B) Evaluation of ITGB4 expression effects on FETumoroids. a) Western blot analysis of ITGB4 expression in control and ITGB4 knockdown (KD) MDA cells. b) Images of FETs formed with control and ITGB4 KD MDA cells on days 1 and 3. c) TA and d) SID of FETumoroids formed with control or ITGB4 KD MDA cells on days 1 and 3. e) Fold changes in TA and SID of FETumoroids formed with control or ITGB4 KD MDA cells. FCs were calculated by dividing the values of FETumoroids on day 3 by those on day 1. f) Cancer cell cluster number in FETs constructed with control and ITGB4 KD MDA-MB-231 cells, analyzed on day 3.

Article Snippet: The plasmids utilized included pBABE-puro SV40 LT (Addgene plasmid #13 970), pLenti CMV GFP Puro (658-5) (Addgene #17 448), a modified pLenti CMV plasmid with RFP instead of GFP (based on Addgene plasmid #17 448), an ITGB4 Human shRNA plasmid (OriGene Technologies, Rockville, MD), and lentiviral packaging plasmids (Addgene plasmids #12 259, #12 253, and #12 251).

Techniques: Isolation, Transfection, Control, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Construct