Journal: bioRxiv

Article Title: The Specificity of IQGAP1 Toward the PI3K-Akt Pathway is Dependent on the IQ3 motif

doi: 10.1101/250936

Figure Lengend Snippet: ( a ) IQGAP1 WT and ΔIQ3 mutant associated cell surface receptors were analyzed by immunoprecipitation. IQGAP1 WT and ΔIQ3 mutant interact with EGFR, Integrin α3 and Integrin β4 at similar levels. HEK293FT cells post 24 h of transient transfection with Myc-tagged IQGAP1 WT or Myc-tagged IQGAP1 ΔIQ3 were starved for 24 h and then treated with 10 ng/ml EGF for 15 min as indicated. The whole cell lysates were collected and processed for immunoprecipitation using anti-Myc antibody conjugated Protein A/G agarose beads. ( b-d ) Quantification of immunoblots in a . n=3. Error bars denote SD.

Article Snippet: Antibodies for immunoblotting, immunofluorescent (IF) staining and immunoprecipitation were from Cell Signaling Technology, including, pAkt (Ser473, 4058), ERK (4695), pErk (Thr202/Tyr204, 4370), PDK1 (13037), p85α (4292), PIPK1α (9693), Santa Cruz Biotechnology, including IQGAP1(H-109) and EGFR (SC-03), Abcam, including Akt (ab126811), Merck Millipore, Myc-tag (05-724), Sigma-Aldrich, including Myc-tag (C3956), Novus Biologicals, including integrin α3 (NBP2-48514) and R&D Systems, including integrin β4 (MAB4060).

Techniques: Mutagenesis, Immunoprecipitation, Transfection, Western Blot

Journal: bioRxiv

Article Title: The Specificity of IQGAP1 Toward the PI3K-Akt Pathway is Dependent on the IQ3 motif

doi: 10.1101/250936

Figure Lengend Snippet: PLA demonstrated the interaction between IQGAP1 WT/ ΔIQ3 mutant and EGFR/Integrin α3β1/Integrin α6β4/PIPK1α/p85α. HEK293FT cells post 24 h of transient transfection with Myctagged IQGAP1 WT or Myc-tagged IQGAP1I ΔIQ3 were starved for 24 h and then treated with 10 ng/ml EGF for 15 min. The cells were fixed and processed for PLA to determine the direct interaction between the Myc-tagged IQGAP1 WT/ΔIQ3 mutant and EGFR/Integrin α3/Integrin β4/PIPK1α/p85α. ⋆⋆P<0.01, n=10. Error bars denote SD. Scale bar, 5 μm.

Article Snippet: Antibodies for immunoblotting, immunofluorescent (IF) staining and immunoprecipitation were from Cell Signaling Technology, including, pAkt (Ser473, 4058), ERK (4695), pErk (Thr202/Tyr204, 4370), PDK1 (13037), p85α (4292), PIPK1α (9693), Santa Cruz Biotechnology, including IQGAP1(H-109) and EGFR (SC-03), Abcam, including Akt (ab126811), Merck Millipore, Myc-tag (05-724), Sigma-Aldrich, including Myc-tag (C3956), Novus Biologicals, including integrin α3 (NBP2-48514) and R&D Systems, including integrin β4 (MAB4060).

Techniques: Mutagenesis, Transfection

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: 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

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: A) Five LUAD cell lines with wild type (WT) KRAS and 5 cell lines with mutant (MT) KRAS were treated with 10 μM cisplatin for 72 h and cell viability was determined using Cell Counting Kit-8 assay. B) Immunoblot showing that the most cisplatin-resistant cell lines (H1993 and H2009) have high expression of both PXN and ITGB4. C) and D) qPCR results demonstrating high mRNA expression of PXN and ITGB4 in cisplatin-resistant cell lines. E) Gene expression profiles of LUAD patients were extracted from TCGA database and expression of PXN and ITGB4 were higher compared to normal tissue. F) Kaplan-Meier curves indicate significant survival difference (p=0.000089) between the two groups (PXN low + ITGB4 low and PXN high + ITGB4 high). G) and H) Immunohistochemistry staining of tissue microarrays showing ubiquitous expression of PXN (yellow) and differential expression of ITGB4 (pink). (**** p<0.0001 One-way ANOVA)

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Mutagenesis, Cell Counting, Western Blot, Expressing, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: A) Immunohistochemistry staining of lung adenocarcinoma tumor tissue showed that Case 1 had high PXN and intermediate ITGB4 expression. In Case 2, PXN expression is low and ITGB4 expression is high. In Case 3, both PXN and ITGB4 expression are low.

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Immunohistochemistry, Staining, Expressing

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: A) Stable cell lines were generated to express nuclear mKate2 red fluorescent protein (RFP) using the NucLight Red Lentivirus Reagent (Essen BioScience). Upon selection with puromycin, cells were analyzed with the IncuCyte software to create a mask around each individual nucleus and obtain accurate real-time cell counts for proliferation assays. B) Doubling times for H2009 and H1993 cells transfected with scramble siRNA (siControl) were measured and compared to that of cells with ITGB4 knockdown (siITGB4). C) Scratch wound healing assays were performed by creating an initial scratch wound with the WoundMaker tool. Wound closure was quantitated by monitoring cells that migrated to fill the initial wound. Cells transfected with scramble siRNA (Si Scramble) were able to completely fill the scratch wound by day 3 whereas ITGB4 knockdown cells (Si ITGB4) were not. D) H2009 cells transfected with10 nM of siRNA constructs A, B, and C, targeting ITGA7 had minimal effect on proliferation. E) Knocking down ITGB4 increased mRNA expression of other integrin beta forms such as ITGB1, ITGB2, and ITGB3 but had no significant effect on the expression of ITGA7. F) In order to nullify the effect of ITGB3 rescue, H2009 cells transfected with siRNA ITGB4 were treated with ITGB3 inhibitors. There was no significant effect in the fold change in proliferation compared to the ITGB4 knockdown cells

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Stable Transfection, Generated, Selection, Software, Transfection, Construct, Expressing

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: H2009 and H1993 stable cell lines expressing mKate2 were transfected with control (Si Scramble) or ITGB4-specific (Si ITGB4) siRNA. 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) A scratch wound assay demonstrating the effect of knocking down ITGB4. ITGB4 knockdown (red) significantly halted 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 in phosphorylated and total FAK and PXN but not ITGB4. F) and G) ITGB4 knockdown in H1993 cells inhibited proliferation and increased caspase-3/7 activity. Treating ITGB4 knockdown cells with cisplatin had an additive effect to induce caspase activity but drug treatment alone did not have a cytotoxic effect. (****p<0.0001 Two-way ANOVA) H) and I) ITGB4 knockdown in H2009 cells inhibited proliferation by 72 h but 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 MET protein expression in H1993 cells was reduced 4 days after knocking down ITGB4.

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

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

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: A) Proliferation and apoptosis assays were executed with stable cell lines expressing nuclear RFP and the IncuCyte Caspase-3/7 Green Reagent (Essen BioScience), which emits green fluorescence when cleaved by activated caspase-3/7. Apoptosis is induced with knockdown of ITGB4 (Si ITGB4) by day 4 and enhanced with added cisplatin (Si ITGB4+Cisplatin). B) Nuclear RFP-expressing cell line H1650 was transfected with ITGB4 siRNA (Si ITGB4) and monitored in real-time with the IncuCyte. Over the course of 10 h, ITGB4 knockdown induced cells with an intact membrane to undergo anoikis-like bursting, attenuating cell proliferation in Si ITGB4 (red) cells compared to control (black). C) H1993 cells harbor MET amplification and upon ITGB4 knockdown, MET expression at only the protein level decreases but no change in the mRNA expression, indicating ITGB4 is required for MET protein stability but not for transcriptional regulation. However, ITGB4 knockdown increased apoptosis significantly compared to control cells. D) H2009 cells that do not have amplified MET did not show any significant change in the protein as well as mRNA expression. They also did not show significant increase in apoptosis with ITGB4 knockdown. E) H2009 cells expressing nuclear RFP were seeded in an ultra-low attachment 96-well plate to facilitate spheroid formation. After 4 h, double knockdown of PXN and ITGB4 impeded cells from forming a compact spheroid as observed in control and single knockdown conditions. F) In H2009 cells, single knockdown of ITGB4 (Si ITGB4) significantly induced apoptosis and double knockdown of PXN and ITGB4 (Si ITGB4+Si PXN) had an enhanced effect.

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Stable Transfection, Expressing, Fluorescence, Transfection, Amplification

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: H1993 cells were treated with an antibody (10 μg/ml) targeting the ITGB4 extracellular epitope in an ultra-low attachment 96-well plate for 6 h then transferred to a tissue culture-treated 96-well plate and allowed to attach overnight. A) and B) After 96 h, proliferation was inhibited, and apoptosis was significantly induced in cells treated with the ITGB4 antibody (red) compared to untreated (black) and cells treated with an IgG antibody (green) used as a control. (****p<0.0001 Two-way ANOVA, multiple comparison) C) Immunoblotting confirmed decreased expression of ITGB4, PXN, and MET with ITGB4 antibody treatment after 48 h compared to IgG antibody control. D) and E) ITGB4 antibody (10 μg/ml) treatment in combination with a lower dose of cisplatin (2.5 μM instead of 10 μM) exhibited a synergistic effect on inhibition of proliferation and increased caspase activity. (****p<0.0001 Two-way ANOVA) F) Double knockdown of both PXN and ITGB4 (red) in H1993 cells had a synergistic effect on inhibiting proliferation compared to single knockdown of either gene (green/blue) and control (black). G) Adding 10 μM cisplatin to the double knockdown cells (red) had an even greater effect on inhibiting proliferation compared to single knockdown of either gene (green/blue) and control (black) treated with cisplatin. (****p<0.0001 Two-way ANOVA) H) ITGB4 knockdown and double knockdown of PXN and ITGB4 induced apoptosis in H1993 and rendered cells more prone to toxic effects of cisplatin. (****p<0.0001 Two-way ANOVA) I) Double knockdown of both PXN and ITGB4 (red) in H2009 cells also had a synergistic effect on inhibition of proliferation compared to single knockdown of either gene (green/blue) and control (black). (****p<0.0001 Two-way ANOVA) J) and K) Double knockdown of PXN and ITGB4 also induced strong apoptosis and in combination with cisplatin, proliferation was greatly inhibited and caspase activity increased at an earlier time point of 24 h. (****p<0.0001 Two-way ANOVA) L) 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. M) 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.

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Inhibition, Activity Assay, Cell Cycle Assay

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: H2009 cells expressing RFP were transfected with siRNA, seeded in a 96-well ultra-low attachment plate (5000 cells/well), and allowed to form a compact spheroid overnight. A) Images acquired by the IncuCyte Live Cell Imaging System showed spheroids with single knockdown to start disintegrating by Day 3 and even earlier for double knockdown spheroids (Day 1). B) and C) To quantitate spheroid viability, red fluorescence area and intensity were measured. Both parameters showed that double knockdown had a synergistic effect on attenuating spheroid viability. (****p<0.0001 Two-way ANOVA) D) and E) Spheroids with ITGB4 single knockdown and PXN/ITGB4 double knockdown were sensitized to cisplatin (10 μM) treatment indicated by a decrease in red fluorescence area and intensity. (****p<0.0001 Two-way ANOVA) F) Immunobloting confirmed PXN and ITGB4 siRNA-mediated knockdown is still present after 72 h in 3D culture. G) Double knockdown spheroids treated with cisplatin had the greatest cytotoxic effect indicated by the green fluorescence in the confocal images acquired by a Zeiss LSM 880 microscope. (**p<0.002, ***p<0.0009 Two-way ANOVA) H) Total RNA was extracted from single and double knockdown cells 48 h after siRNA transfection. Total RNASeq revealed the number of genes downregulated with each single knockdown of PXN and ITGB4 and when both genes are knocked down simultaneously. I) Bar diagram showing the major Hallmark pathways affected by the double knockdown. The top 10 pathways were arranged in descending order of their enrichment score. J) Heat map representation of top 10 genes that belong to hallmark MYC target V1 pathways were analyzed after single or double knockdown. The heat map is representation of two experimental repeats.

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Transfection, Live Cell Imaging, Fluorescence, Western Blot, Microscopy

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: A) Total RNA sequencing after single and double knockdown of PXN and ITGB4 revealed that 5 common genes were downregulated compared to control cells (SCR). B) Major pathways affected by double knockdown of PXN and ITGB4 were MYC targets, G2-M checkpoint, and E2-F targets. C) qPCR results with single and double knockdown of PXN and ITGB4 confirmed downregulation of VDAC1, USP1, and G3BP1 at the mRNA level. D) SiRNA constructs A, B, or C against top four genes (KIF14, VDAC1, USP1, and G3BP1) down regulated were tested to determine the construct with maximum effect on inhibiting proliferation. KIF14 construct C, VDAC1 construct A, and USP1 construct B showed significant effect on cell proliferation. E) The selected constructs were tested for induction of caspase-3/7 activity by tracking green fluorescence using live cell imaging and analysis for 72 hrs. F) H2009 and H1993 cells treated with ML323, a USP1 inhibitor, did not undergo significant changes in proliferation compared to untreated cells (black). G) Spare respiratory capacity, which is the ratio of maximal respiration vs. basal respiration, did not show any significant change between control and knockdown cells. H) 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. I) H2009 and H1993 cells with PXN/ITGB4 or USP1/VDAC1 double knockdown confirmed by immunoblot to be used for ROS production assay.

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: RNA Sequencing Assay, Construct, Activity Assay, Fluorescence, Live Cell Imaging, Expressing, Western Blot

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: A) Immunobloting confirmed decreased expression of top 3 genes (G3BP1, USP1, and VDAC1) downregulated in MYC pathway after knockdown of PXN and/or ITGB4. B) Gene expression profiles of LUAD patients were extracted from TCGA database and expression of USP1 and VDAC1 were higher compared to normal tissue. C) and D) In H2009 cells, knocking down USP1 (blue) attenuated proliferation, induced apoptosis, and sensitized cells to a lower dose of cisplatin (2 μM) (red). (****p<0.0001 Two-way ANOVA) E) After knocking down PXN/ITGB4 and USP1, γH2AX foci were detected via immunofluorescence, imaged with confocal microscopy, and counted with QuPath image analysis software. Knockdown exhibited greater number of cells with higher γH2AX foci counts. Added cisplatin increased number of detected γH2AX foci. (*p=0.04, **p=0.004, ***p,<0.0001) F) and G) Similarly, knocking down VDAC1 (blue) inhibited cell proliferation by 50% within 72hrs, but could induce apoptosis only at later time point. Cisplatin (red) addition has an additive effect to VDAC1 knockdown in inhibiting proliferation. H) – J) Using the Seahorse XF Analyzer, cellular metabolic activity was measured with knockdown and in presence of cisplatin. Knocking down PXN/ITGB4 or VDAC1 increased basal (I) and maximal (J) mitochondrial oxygen consumption rate per 1000 cells. (**p=0.0029, ****p<0.0001 One-way ANOVA) K) and L) ATP-linked respiration (K) and proton leak (L) also showed an increase in the same knockdown cells. (* p=0.01 One-way ANOVA) M) Double knockdown of PXN/ITGB4 (red) and USP1/VDAC1 (blue) inhibited proliferation to similar extent whether in the absence or presence of cisplatin. (****p<0.0001 Two-way ANOVA) N) Reactive oxygen species (ROS) production was measured using the ROS-Glo™ H2O2 Assay. Double knockdown of PXN/ITGB4 and USP1/VDAC1 induced higher levels of ROS compared to control. O) ChIP 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 to that of an upstream region, indicating ITGB4 and PXN role in USP1 transcriptional activation.

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Immunofluorescence, Confocal Microscopy, Software, Activity Assay, H2O2 Assay, Activation Assay

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: A) Co-immunoprecipitation (co-IP) of H2009 whole cell lysate with an ITGB4 antibody showing FAK, PXN, and ITGA7 interact with ITGB4. Reverse co-IP with FAK and PXN antibody showing ITGB4 binds to FAK and PXN. B) Knocking down PXN did not affect the interaction between FAK and PXN. B) and D) Treating H2009 cells with 10 μM cisplatin for 48 h did not induce any changes in the interaction between ITGB4, FAK, and PXN. E) PONDR prediction algorithm determined that the N-terminal region of PXN to be intrinsically disordered whereas the C-terminal half is highly ordered. F) Circular dichroism (CD) spectra were recorded as a function of temperature 10°C to 60°C. Low ellipticity values near the 215-230 nm region indicated that the N-terminal region lacks any secondary structure. G) NMR spectroscopy using a two-dimensional 1 H- 15 N HSQC spectrum was used to analyze the LD2-LD4 region of PXN to confirm that it is indeed disordered. H) Using the binding pocket in the N-terminal region of PXN, 1440 FDA-approved drugs were virtually screened. I) Molecular structure of carfilzomib. J) H2009 cells were treated with an increasing dose of carfilzomib to determine IC50. K) Scratch wound assay using H2009 cells treated with a sublethal dose of carfilzomib (50-200 nM) showed dose-dependent inhibition of cell migration. (**p-0.002, ***p-0.0001, p-****<0.0001) L) Immunoblot of H2009 cells treated with carfilzomib showed reduced expression of ITGB4 and phospho-Rb (S807/811) and increased expression of p27, γH2AX, and cleaved PARP, indicating cell cycle inhibition and cell death.

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Spectroscopy, Binding Assay, Scratch Wound Assay Assay, Inhibition, Migration, Western Blot, Expressing

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: A) Eleven FDA-approved compounds that exhibited the greatest inhibitory effect on H358 cells were further analyzed with immunoblot. H358 cells were treated with each compound at the indicated sublethal dose for 72 h. B) and C) H2009 cells treated with the eleven FDA-approved compounds at 0.08 μM. Cell proliferation over the course of 72 h was monitored and viability percentage compared to untreated was measured using CCK-8 assay. D) Immunoblot of H2009 cells treated with sublethal dose of eleven drugs for 72 h showed reduction in both ITGB4 and PXN expression with only carfilzomib treatment. E) H1993 cells were treated with an increasing dose of carfilzomib to determine cell proliferation effect with change in dose and determine the IC50. F) Immunoblot of H1993 cells treated with carfilzomib showed reduced expression of ITGB4 and phospho-Rb (S807/811) and increased expression of p27, γH2AX, and cleaved PARP, indicating cell cycle inhibition and cell death. G) Ixazomib and CUDC-101, two compounds that induced caspase activity, were further analyzed with scratch wound healing assays in H2009 cells by taking into parameters relative wound density and wound closure rate. Only ixazomib impeded cell migration and CUDC-101 did not. H) Treating the patient-derived organoids Lcsc4 with 20 μg/ml of ITGB4 antibody decreased the spheroid area significantly, but combination with cisplatin 2.5 μM had no additive effect.

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Western Blot, CCK-8 Assay, Expressing, Inhibition, Activity Assay, Migration, Derivative Assay

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: A) Confocal images taken of H2009 spheroids treated with 300 nM and 600 nM carfilzomib for 72 h depicted disintegration and induction of caspase activity marked by green fluorescence. B) and C) Carfilzomib treatment of H2009 spheroids reduced viability of spheroids as quantitated by lower red fluorescence intensity (B) and increased caspase activity (C) in a dose-dependent manner. (****p<0.0001 Ordinary one-way ANOVA) D – I) Surgical samples from lung cancer patients were obtained and cultured to form organoids (5000 cells/well). In one patient sample (LCSC4) (D) , cisplatin (5 μM) alone and carfilzomib (2.5 μM) alone decreased the size of the organoid (E) , but there was a 5-fold greater induction of apoptosis with carfilzomib compared to cisplatin in reference to cisplatin, (****p<0.0001 Two-way ANOVA) (F) . The combination of carfilzomib and cisplatin did not have an additive effect. In another patient sample (LCSC2) (G) , cisplatin and carfilzomib decreased the spheroid area (H). Cisplatin alone did not induce any caspase activity, but carfilzomib induced a ∼50-fold increase in apoptosis. (****p<0.0001 Two-way ANOVA) (I) . Caspase activity was enhanced with the combination of both drugs. (****p<0.0001 Two-way ANOVA) J) These patient-derived organoids were treated with cisplatin and carfilzomib at indicated concentrations and collected after 48 h for immunoblot. Cisplatin treatment had a minimal effect whereas carfilzomib had a more toxic effect on cells marked by decreased expression of ITGB4, PXN, USP1, VDAC1, and CD133.

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Activity Assay, Fluorescence, Cell Culture, Derivative Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: A) A double negative feedback loop between ITGB4 and miR-1-3p leads to bistability. B) 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 state. 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) 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 to untreated. E) Human sera (500 μl) were obtained from healthy donors and LUAD patients. Exosomes were isolated, lysed, and analyzed for expression of ITGB4 and PXN by immunoblotting. Compared to that of healthy donors, exosomes of patients had increased expression of ITGB4 and PXN and also USP1, which we identified to be regulated by ITGB4. F) Schematic depicting the interaction between ITGB4 and PXN regulating downstream proteins USP1 and VDAC1 at the transcriptional level to coordinate cisplatin resistance.

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Generated, Staining, Expressing, Cell Culture, Flow Cytometry, Isolation, Western Blot

Journal: bioRxiv

Article Title: A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

doi: 10.1101/781807

Figure Lengend Snippet: A) RACIPE ensemble results (n=100,000) show ITGB4 and miR-1-3p exhibit bimodality: two distinct subpopulations of cells, as shown via z-socre distributions of ITGB4 and miR-1-3p. B) FACS analysis using an anti-ITGB4 antibody showed enrichment of high ITGB4 population upon cisplatin treatment in H2009 cells.

Article Snippet: Knockdown of ITGB4 (Cat #: SR302473C), FAK (Cat #: SR303877C), USP1 (Cat #: SR305052B), and VDAC1 (Cat #: SR305067C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: