human itgb4  (Santa Cruz Biotechnology)

Journal: Translational Lung Cancer Research

Article Title: TFAP2A -activated ITGB4 promotes lung adenocarcinoma progression and inhibits CD4 + /CD8 + T-cell infiltrations by targeting NF-κB signaling pathway

doi: 10.21037/tlcr-24-50

Figure Lengend Snippet: ITGB4 is up-regulated in LUAD tumor tissues, and patients with higher ITGB4 expression have an inferior prognosis. (A) Flowchart of the present study. (B-D) ITGB4 was significantly up-regulated in LUAD compared with adjacent normal tissues in TCGA (B), GSE31210 (C) and JSPH (D) databases. (E,F) Patients with LUAD exhibiting a higher ITGB4 expression had a worse prognosis in TCGA (E) and GSE31210 (F) datasets. (G,H) IHC analysis of ITGB4 expression in 20 paired LUAD samples; (G) representative staining images of ITGB4 expression were scored as follows: −, no staining; +, weak staining; ++, moderate staining; and +++, strong staining. Scale bar, 200 µm; (H) evaluation of the IHC score of ITGB4 expression. Values are expressed as the means ± SDs. ***, P<0.001. LUAD, lung adenocarcinoma; TCGA, The Cancer Genome Atlas; HR, hazard ratio; CI, confidence interval; IHC, immunohistochemistry; JSPH, Jiangsu Province Hospital; SDs, standard deviations.

Article Snippet: To construct ITGB4 -overexpressing plasmids (named ITGB4 OE), human ITGB4 complementary DNA (cDNA) was synthesized and cloned into the pcDNA3.1(+) vector (Invitrogen), while empty plasmids were used as a control (named vector).

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Translational Lung Cancer Research

Article Title: TFAP2A -activated ITGB4 promotes lung adenocarcinoma progression and inhibits CD4 + /CD8 + T-cell infiltrations by targeting NF-κB signaling pathway

doi: 10.21037/tlcr-24-50

Figure Lengend Snippet: ITGB4 promotes LUAD cell proliferation in vitro . (A) Relative ITGB4 expression in cell lines was determined by qRT-PCR and western blot assays. (B) The efficiency of ITGB4 knockdown and overexpression in A549 and PC9 cells was verified by qRT-PCR and western blot assays. (C,D) Cell proliferation was evaluated by CCK-8 assay after knocking down (C) and overexpressing (D) ITGB4 in A549 and PC9 cells. (E,F) Effects of ITGB4 silencing (E) and overexpression (F) on colony formation in LUAD cells. The staining method was crystal violet staining. (G,H) EdU assay was conducted to evaluate LUAD cell proliferation. Cells were stained with Apollo, and DAPI was used to label cell nuclei. Scale bar, 50 µm. Values are expressed as the means ± SDs. *, P<0.05; **, P<0.01; ***, P<0.001. NC, negative control; OE, overexpression; qRT-PCR, quantitative reverse transcription polymerase chain reaction; LUAD, lung adenocarcinoma; CCK-8, Cell Counting Kit-8; SDs, standard deviations.

Article Snippet: To construct ITGB4 -overexpressing plasmids (named ITGB4 OE), human ITGB4 complementary DNA (cDNA) was synthesized and cloned into the pcDNA3.1(+) vector (Invitrogen), while empty plasmids were used as a control (named vector).

Techniques: In Vitro, Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Over Expression, CCK-8 Assay, Staining, EdU Assay, Negative Control, Reverse Transcription, Polymerase Chain Reaction, Cell Counting

Journal: Translational Lung Cancer Research

Article Title: TFAP2A -activated ITGB4 promotes lung adenocarcinoma progression and inhibits CD4 + /CD8 + T-cell infiltrations by targeting NF-κB signaling pathway

doi: 10.21037/tlcr-24-50

Figure Lengend Snippet: ITGB4 facilitates LUAD cell migration and invasion in vitro . (A) The migration and invasion abilities of cells with ITGB4 knockdown were evaluated using Transwell assays. The staining method was crystal violet staining. Scale bar, 100 µm. (B) The migration and invasion abilities of cells with ITGB4 overexpression were assessed. The staining method was crystal violet staining. Scale bar, 100 µm. (C,D) Wound healing assays were conducted to investigate the migratory ability of ITGB4-overexpressing and ITGB4-knockdown LUAD cells. Scale bar, 100 µm. Values are expressed as the means ± SDs. **, P<0.01; ***, P<0.001. NC, negative control; OE, overexpression; LUAD, lung adenocarcinoma; SDs, standard deviations.

Article Snippet: To construct ITGB4 -overexpressing plasmids (named ITGB4 OE), human ITGB4 complementary DNA (cDNA) was synthesized and cloned into the pcDNA3.1(+) vector (Invitrogen), while empty plasmids were used as a control (named vector).

Techniques: Migration, In Vitro, Knockdown, Staining, Over Expression, Negative Control

Journal: Translational Lung Cancer Research

Article Title: TFAP2A -activated ITGB4 promotes lung adenocarcinoma progression and inhibits CD4 + /CD8 + T-cell infiltrations by targeting NF-κB signaling pathway

doi: 10.21037/tlcr-24-50

Figure Lengend Snippet: ITGB4 promotes LUAD cell tumorigenesis and metastasis in vivo . (A) Gross appearance of subcutaneous xenograft tumors from the sh-NC and sh-ITGB4 groups (n=10 for each group); (B) tumor volumes were measured every 3 days and the growth curves were calculated; (C) tumor weights were evaluated; (D) HE and Ki-67 IHC staining of xenograft tumors. Scale bar, 100 µm; (E) representative images and histogram analysis of the fluorescence intensity in the lungs after injection of LUAD cells into the tail veins of mice; (F) representative images and histogram analysis of the lung metastatic nodules (arrowheads). (G) HE staining of the lung metastatic nodules (arrowheads). Scale bar, 200 µm. Values are expressed as the means ± SDs. **, P<0.01; ***, P<0.001. NC, negative control; HE, hematoxylin and eosin; LUAD, lung adenocarcinoma; IHC, immunohistochemistry; W, weeks; SDs, standard deviations.

Article Snippet: To construct ITGB4 -overexpressing plasmids (named ITGB4 OE), human ITGB4 complementary DNA (cDNA) was synthesized and cloned into the pcDNA3.1(+) vector (Invitrogen), while empty plasmids were used as a control (named vector).

Techniques: In Vivo, Immunohistochemistry, Fluorescence, Injection, Staining, Negative Control

Journal: Translational Lung Cancer Research

Article Title: TFAP2A -activated ITGB4 promotes lung adenocarcinoma progression and inhibits CD4 + /CD8 + T-cell infiltrations by targeting NF-κB signaling pathway

doi: 10.21037/tlcr-24-50

Figure Lengend Snippet: ITGB4 activates the NF-κB signaling pathway by interacting with IκBα . (A,B) KEGG pathway enrichment analysis was conducted in TCGA dataset (A) and RNA transcriptome sequencing data (B). (C) The luciferase activity of NF-κB was measured after knocking down or overexpressing ITGB4 in A549 and PC9 cells. (D) Western blot analysis of p-p65, total p65, p-IκBα, total IκBα, p-IKKα/β, and total IKKα/β protein expression in ITGB4-knockdown or ITGB4-overexpressing LUAD cells. (E) Western blot analysis of nuclear and cytoplasmic p65 protein expression in ITGB4-knockdown or ITGB4-overexpressing LUAD cells. Lamin B1 was used as a nuclear loading control, while GAPDH was used as a cytoplasmic loading control for normalization. (F) Western blot assays were performed on the IP protein ( IκBα ) identified through mass spectrometry in the IP assay of ITGB4 in A549 cells. (G) The binding of IκBα to ITGB4 in A549 and PC9 cells was detected by co-IP assays. (H) Representative images and histogram analysis of ITGB4 and IκBα IHC analysis in xenograft tumors. The staining method was immunohistochemical staining. Scale bar, 100 µm. (I,J) The cell migration of ITGB4-overexpressing LUAD cells treating with either DMSO or the NF-κB inhibitor PDTC was evaluated by Transwell (I) and wound healing (J) assays. The staining method for Transwell assay was crystal violet staining. Scale bar, 100 µm. Values are expressed as the means ± SDs. **, P<0.01; ***, P<0.001. ECM, extracellular matrix; AGE, advanced glycation end product; RAGE, receptor for AGE; HTLV-1, human T-lymphotropic virus type 1; TNF, tumor necrosis factor; NC, negative control; OE, overexpression; IgG, immunoglobulin G; IP, immunoprecipitation; DMSO, dimethyl sulfoxide; PDTC, pyrrolidine dithiocarbamate; LUAD, lung adenocarcinoma; KEGG, Kyoto Encyclopedia of Genes and Genomes; TCGA, The Cancer Genome Atlas; IHC, immunohistochemistry; SDs, standard deviations.

Article Snippet: To construct ITGB4 -overexpressing plasmids (named ITGB4 OE), human ITGB4 complementary DNA (cDNA) was synthesized and cloned into the pcDNA3.1(+) vector (Invitrogen), while empty plasmids were used as a control (named vector).

Techniques: Sequencing, Luciferase, Activity Assay, Western Blot, Expressing, Knockdown, Control, Mass Spectrometry, Binding Assay, Co-Immunoprecipitation Assay, Staining, Immunohistochemical staining, Migration, Transwell Assay, Virus, Negative Control, Over Expression, Immunoprecipitation, Immunohistochemistry

Journal: Translational Lung Cancer Research

Article Title: TFAP2A -activated ITGB4 promotes lung adenocarcinoma progression and inhibits CD4 + /CD8 + T-cell infiltrations by targeting NF-κB signaling pathway

doi: 10.21037/tlcr-24-50

Figure Lengend Snippet: Laminin-5 promotes LUAD cell proliferation, migration and invasion through ITGB4 signaling activation. (A) Western blot analysis of p-IκBα, total IκBα , p-p65, total p65 , p-IKKα/β, and total IKKα/β protein expression in sh-NC or sh-ITGB4 LUAD cells, both in the presence or absence of 10 ng/mL recombinant laminin-5 protein. (B) CCK-8 assay was performed with sh-NC or sh-ITGB4 LUAD cells in the presence or absence of recombinant laminin-5 . (C) Colony formation assay was conducted to evaluate cell proliferation in sh-NC or sh-ITGB4 LUAD cells treated with or without laminin-5 . The staining method was crystal violet staining. (D,E) Transwell assays were performed to assess the migration (D) and invasion (E) capabilities of sh-NC or sh-ITGB4 LUAD cells treated with or without laminin-5 . The staining method was crystal violet staining. Scale bar, 100 µm. (F) Wound healing assay was carried out to evaluate cell migration in sh-NC or sh-ITGB4 LUAD cells treated with or without laminin-5 . Scale bar, 100 µm. Values are expressed as the means ± SDs. **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; NC, negative control; LUAD, lung adenocarcinoma; SDs, standard deviations.

Article Snippet: To construct ITGB4 -overexpressing plasmids (named ITGB4 OE), human ITGB4 complementary DNA (cDNA) was synthesized and cloned into the pcDNA3.1(+) vector (Invitrogen), while empty plasmids were used as a control (named vector).

Techniques: Migration, Activation Assay, Western Blot, Expressing, Recombinant, CCK-8 Assay, Colony Assay, Staining, Wound Healing Assay, Cell Counting, Negative Control

Journal: Translational Lung Cancer Research

Article Title: TFAP2A -activated ITGB4 promotes lung adenocarcinoma progression and inhibits CD4 + /CD8 + T-cell infiltrations by targeting NF-κB signaling pathway

doi: 10.21037/tlcr-24-50

Figure Lengend Snippet: ITGB4 suppresses CD4 + and CD8 + T-cell infiltrations in the tumor immune microenvironment of LUAD. (A) Representative images of ITGB4 , CD4 and CD8 IHC analysis in LUAD tumor tissues from 20 patients. Scale bars, 200 and 50 µm. (B,C) Correlation between ITGB4 and CD4 (B), CD8 (C) IHC scores of 20 LUAD samples. (D-G) An immunocompetent mouse model was established by injecting LLC-sh-NC or LLC-sh-ITGB4 cells into the right flanks of C57BL/6J mice: (D,E) gross appearance of subcutaneous tumors from the LLC-sh-NC or LLC-sh-ITGB4 groups (n=10 for each group); (F) tumor volumes were measured every 3 days and growth curves were calculated; (G) tumor weights were evaluated. (H-K) HE and IHC staining of tumors from the immunocompetent mouse model: (H) representative images of IHC staining for ITGB4 , CD4 and CD8 in tumors from the LLC-sh-NC or LLC-sh-ITGB4 groups. Red arrowheads indicated the positive area. Scale bar, 100 µm; (I-K) IHC scores of ITGB4 (I), CD4 (J) and CD8 (K) protein expression were analyzed. (L,M) The populations of CD4 + (L) and CD8 + (M) T cells in CD45 + TILs in tumors from LLC-sh-NC or LLC-sh-ITGB4 group were analyzed by flow cytometry. Values are expressed as the means ± SDs. *, P<0.05; **, P<0.01; ***, P<0.001. NC, negative control; LLC, Lewis lung carcinoma; HE, hematoxylin and eosin; IHC, immunohistochemistry; FSC-H, forward scatter-height; LUAD, lung adenocarcinoma; SDs, standard deviations.

Article Snippet: To construct ITGB4 -overexpressing plasmids (named ITGB4 OE), human ITGB4 complementary DNA (cDNA) was synthesized and cloned into the pcDNA3.1(+) vector (Invitrogen), while empty plasmids were used as a control (named vector).

Techniques: Immunohistochemistry, Expressing, Flow Cytometry, Negative Control

Journal: Translational Lung Cancer Research

Article Title: TFAP2A -activated ITGB4 promotes lung adenocarcinoma progression and inhibits CD4 + /CD8 + T-cell infiltrations by targeting NF-κB signaling pathway

doi: 10.21037/tlcr-24-50

Figure Lengend Snippet: TFAP2A activates the transcription of ITGB4 in LUAD cells. (A) The JASPAR and AnimalTFDB3 databases were used to predict the transcription factor that activated ITGB4 transcription in LUAD cells. (B) Correlation between TFAP2A and ITGB4 expression was evaluated by co-expression analysis based on TCGA database. (C) TFAP2A was significantly up-regulated in LUAD compared with adjacent normal tissues in TCGA database. (D) LUAD patients with a higher TFAP2A expression had a worse prognosis in TCGA dataset. (E,F) Relative mRNA (E) and protein (F) levels of ITGB4 after TFAP2A knockdown or overexpression in A549 and PC9 cells. (G) The binding site of TFAP2A in the ITGB4 promoter was at the region −235 to −222 bp. The ITGB4 promoter was cloned into the pGL3-basic luciferase vector (pGL3-ITGB4 wild-type, named ITGB4-WT), while the TFAP2A -binding motif was removed from the ITGB4 promoter vector (pGL3-ITGB4 mutant, named ITGB4-MUT). (H) Dual-luciferase reporter assay was performed to evaluate the effect of TFAP2A on the activity of ITGB4-WT and ITGB4-MUT. (I) ChIP qPCR analyses were performed to evaluate the enrichment of the DNA fragment containing the putative TFAP2A -binding site in the chromatin that was precipitated by the anti-TFAP2A antibody. Anti-IgG antibody was used as a negative control. (J) Schematic diagram illustrating the molecular mechanism by which ITGB4 promoted LUAD progression and suppressed CD4 + /CD8 + T-cell infiltrations. The red arrows represent enhanced cell proliferation, migration, and invasion, while the blue arrows indicate reduced infiltration of CD4 + and CD8 + T cells. Values are expressed as the means ± SDs. **, P<0.01; ***, P<0.001. ns, not significant; TCGA, The Cancer Genome Atlas; HR, hazard ratio; NC, negative control; IgG, immunoglobulin G; LUAD, lung adenocarcinoma; ChIP, Chromatin immunoprecipitation; SDs, standard deviations; qPCR, quantitative polymerase chain reaction.

Article Snippet: To construct ITGB4 -overexpressing plasmids (named ITGB4 OE), human ITGB4 complementary DNA (cDNA) was synthesized and cloned into the pcDNA3.1(+) vector (Invitrogen), while empty plasmids were used as a control (named vector).

Techniques: Expressing, Knockdown, Over Expression, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Mutagenesis, Reporter Assay, Activity Assay, Negative Control, Migration, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: The American journal of pathology

Article Title: Periostin in Cancer-Associated Fibroblasts Promotes Esophageal Squamous Cell Carcinoma Progression by Enhancing Cancer and Stromal Cell Migration.

doi: 10.1016/j.ajpath.2023.12.010

Figure Lengend Snippet: Figure 5 The enhancement of esophageal squamous cell carcinoma cell migration by periostin is mediated via integrin b4. A and B: Integrin subunit beta 4 (ITGB4) and integrin b4 expression levels in TE cells after direct co-culture were compared with those after mono-culture using real-time quantitative PCR (qPCR; A) and Western blot analysis (B). Beta actin (ACTB) was used as a control in the Western blot analysis. CeE: TE-9, TE-10, and TE-15 cells were transfected with siRNA targeting ITGB4 (siITGB4; 20 nmol/L) and negative control siRNA (siNC; 20 nmol/L). ITGB4 knockdown was confirmed using qPCR (C), RT-PCR (D), and Western blot analysis (E). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ACTB were used as a control in RT-PCR and Western blot analysis, respectively. F: Transwell migration assays were performed to confirm the effect of ITGB4 knockdown on the enhanced migration of TE-9, TE-10, and TE-15 cells by adding recombinant human periostin (rhPOSTN; 100 pg/mL). Migrating cells were counted in five random fields in each chamber after 48 hours of incubation. G: TE-9, TE-10, and TE-15 cells transfected with siNC and siITGB4 were treated with rhPOSTN (100 pg/mL), and time-dependent changes in Akt, phosphorylated Akt (p-Akt; Ser473), p-Akt (Thr308), extracellular signal-regulated kinase (Erk), and phosphorylated Erk (p-Erk; Thr202/Tyr204) levels in each cell were then determined using Western blot analysis. ACTB was used as a control. Data are presented as means SEM (A, C, and F). **P < 0.01, ***P < 0.001. N.S., not significant.

Article Snippet: For ITGB4 knockdown, TE-9, TE-10, and TE-15 cells were transfected with siRNA targeting human ITGB4 (20 pmol; sc-35678; Santa Cruz Biotechnology) using Lipofectamine RNAiMAX (Invitrogen) for 48 hours, according to the manufacturer’s instructions.

Techniques: Migration, Expressing, Co-Culture Assay, Real-time Polymerase Chain Reaction, Western Blot, Control, Transfection, Negative Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Recombinant, Incubation

Journal: The American journal of pathology

Article Title: Periostin in Cancer-Associated Fibroblasts Promotes Esophageal Squamous Cell Carcinoma Progression by Enhancing Cancer and Stromal Cell Migration.

doi: 10.1016/j.ajpath.2023.12.010

Figure Lengend Snippet: Figure 9 Schematic diagram of roles of periostin (POSTN) in the esophageal squamous cell carcinoma (ESCC) microenvironment. Direct contact with ESCC cells leads mesenchymal stem cells (MSCs) to become cancer-associated fibroblast (CAF)elike cells, which secrete periostin and activate the Akt and extra- cellular signal-regulated kinase (Erk) pathways via integrin subunit beta 4 (ITGB4) in ESCC cells, promoting their migration. Periostin also promotes MSC and macrophage migration and contributes to the activation of tumor-associated macrophage (TAM)elike macrophage properties. MF, macrophage.

Article Snippet: For ITGB4 knockdown, TE-9, TE-10, and TE-15 cells were transfected with siRNA targeting human ITGB4 (20 pmol; sc-35678; Santa Cruz Biotechnology) using Lipofectamine RNAiMAX (Invitrogen) for 48 hours, according to the manufacturer’s instructions.

Techniques: Migration, Activation 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: 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: 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: The small interfering RNA oligonucleotides for SOX2, and ITGB4 were purchased from Origene (Cat #:SR321861, Cat #:SR302473CL).

Techniques: Amplification, Over Expression, Software, Expressing, 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: The small interfering RNA oligonucleotides for SOX2, and ITGB4 were purchased from Origene (Cat #:SR321861, Cat #:SR302473CL).

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: The small interfering RNA oligonucleotides for SOX2, and ITGB4 were purchased from Origene (Cat #:SR321861, Cat #:SR302473CL).

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: The small interfering RNA oligonucleotides for SOX2, and ITGB4 were purchased from Origene (Cat #:SR321861, Cat #:SR302473CL).

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: The small interfering RNA oligonucleotides for SOX2, and ITGB4 were purchased from Origene (Cat #:SR321861, Cat #:SR302473CL).

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: The small interfering RNA oligonucleotides for SOX2, and ITGB4 were purchased from Origene (Cat #:SR321861, Cat #:SR302473CL).

Techniques: Expressing, Activity Assay, 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: The small interfering RNA oligonucleotides for SOX2, and ITGB4 were purchased from Origene (Cat #:SR321861, Cat #:SR302473CL).

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

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: Short hairpin RNA ITGB4 , Origene , TL312080.

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