Journal: Cancers

Article Title: Regression of Lung Cancer in Mice by Intranasal Administration of SARS-CoV-2 Spike S1

doi: 10.3390/cancers14225648

Figure Lengend Snippet: Effect of recombinant SARS-CoV-2 spike S1 on the survival of human H1299 and H358 lung cancer cells. H1299 ( A , C ) and H358 ( B , D ) cells were treated with spike S1 protein for 24 h under serum-free condition followed by monitoring cell death by LDH release ( A , B ) and MTT ( C , D ). Results are mean + SD of three different experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Same procedure was utilized for two other cell lines purchased from ATCC: H1299 (human NSCLC, p53 negative; Catalog# CRL-5803) H358 (human NSCLC, KRAS mutant; Catalog# CRL-5807) These cells were cultured in RPMI-1640 containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin.

Techniques: Recombinant

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: Definition of the earliest PanINs and their physical location in the pancreas of KC mice. A and F) The representative 3D imaging projection of the whole pancreas of 2 (A)‐ and 4 (F)‐week‐old Pdx1 ‐Cre; LSL‐ Kras G12D/+ (KC) transgenic mouse using 3D histological analysis. Red arrow: the early PanIN. Blue signal: the nucleus staining; White signal: CK19‐staining; Red signal: the blood vessel staining. Scale bar: 1 mm. B and G) Quantification of PanIN number in 2 (B)‐ and 4 (G)‐week‐old transgenic mice. Each dot represents the datum of one mouse. Values were presented as mean ± SD. C and H) The distribution of the earliest PanIN lesions from 45 and 24 of 2 (C)‐ and 4 (H) ‐week‐old KC mice, respectively. One yellow dot indicates one lesion. n = 109 (C); n = 211 (H). D and I) Stacked bar plot showing the percentage of PanIN in the pancreas's head, body, and tail of 2 (D)‐ and 4 (I)‐week‐old KC mice. E and J) Quantification of PanIN and islet association in the pancreas of 2 (E)‐ and 4 (J) ‐week‐old KC mice. An association is defined by the distance between lesion and islet within 300 µm.

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Imaging, Transgenic Assay, Staining

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: Genetic alterations in the earliest PanIN and Muc4 up‐regulation cooperate with oncogenic Kras G12D for PanIN initiation. A) The most common genetic alterations in the earliest PanINs from the pancreas of 2‐week‐old KC mice. The data analyzed in this study were obtained from 21 lesions in 13 mice, which were compared to the normal pancreatic region within the same KC mouse. In addition, two control mice were included for comparison. To ensure accuracy, all mouse‐specific single nucleotide polymorphisms (SNPs) were excluded by referencing the UCSC Genome Browser website. B) The RNA‐seq data of the Muc4 gene and Sirpb1a , comparing the PanIN sample with the control sample from a 2‐week‐old KC mouse (M1376). C) The IHC staining of Ki67 proliferation marker. Representative images (left panel) and quantification (right panel) of Ki67 staining in 4‐week‐old control and KC mice. Bar, 50 µm. Each dot represents the datum of one mouse. Values were presented as mean ± SD, n = 11 mice. *, P <0.05; **, P <0.01; ***, P < 0.001 (two‐tailed Student's t‐test). D) Plots of the survival probability of pancreatic cancer patients with MUC4 (left panel) and SIRPB1 (right panel) mRNA expression levels from the Human Protein Atlas as shown in methods. P values are calculated by log‐rank test. E–H) The Alcian Blue staining and IHC analysis with anti‐Muc4 antibody in 4‐week‐old control and KC mice. Representative images of IHC staining with Alcain Blue and Muc4 E), quantification of Alcain Blue F) and Muc4 G) in the early PanIN cells. Bar, 50 µm. The H score of Muc4 expression in 4‐week‐old control and KC mice H). Each dot represents the datum of one mouse. Values were presented as mean ± SD, n = 11 mice. ****, P < 0.0001 (two‐tailed Student's t‐test). I and J) EpCAM + /Muc4 − ‐ or double EpCAM/Muc4‐positive pancreatic cells isolated by FACS from 4‐week‐old KC mice for spheroid formation analysis I) and soft agar colony formation analysis J). N indicates independent experiments, and n indicates total measurements in all experiments. Each dot represents the datum from one measurement. Values show mean ± SD. ****, P < 0.0001 (two‐tailed Student's t‐test). K and L) Primary acinar cells were isolated from 4‐week‐old control or KC mice for spheroid formation analysis K) and soft agar colony formation analysis L). N indicates independent experiments, and n indicates total measurements. Each dot represents the datum from one measurement. Values show mean ± SD. ****, P < 0.0001 (two‐tailed Student's t‐test).

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Control, Comparison, RNA Sequencing, Immunohistochemistry, Marker, Staining, Two Tailed Test, Expressing, Isolation

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: αSMA + fibroblasts associate with the earliest PanIN cells to promote Kras G12D/+ pancreatic cell transformation and stemness properties. A) Representative image of 3D histology‐detected the earliest PanINs in the whole pancreas of 2‐week‐old KC mice. Blue signal: the nucleus staining; White signal: CK19‐staining (PanIN cells); Green signal: αSMA staining (activated fibroblasts). Bar, 100 µm. B) Quantification of the percentage of the earliest PanINs associated with αSMA + fibroblasts. C) Representative images of the IHC staining of fibroblasts with anti‐αSMA antibody in 4‐week‐old KC mice (left panel) and quantification of the percentage of early PanINs associated with αSMA + fibroblasts (right panel). Bar, 50 µm. D) Close contact with fibroblasts promotes acinar‐to‐ductal metaplasia of Kras G12D/+ pancreatic acinar cells in 3D Matrigel coculture systems. X: Kras G12D/+ pancreatic acinar cells only (1000 cells). F: coculture with Kras +/+ fibroblasts (2000 cells). I: coculture with Kras +/+ islet cells (2000 cells). Upper panel: Representative images in coculture experiment in Kras G12D/+ pancreatic cells. Bar, 50 µm. Bottom panel: Quantification of cyst number. E and F) Close contact of Kras G12D/+ pancreatic acinar cells with fibroblasts promotes sphere formation. E) 1000 Kras G12D/+ pancreatic acinar cells labeled with CellTracker Green CMFDA were cocultured with 2000 Kras +/+ fibroblasts or 2000 Kras +/+ isle cells for 14 days in a 96‐well low attached plate. (Left panel) Representative images of the cocultured Kras G12D/+ pancreatic cells. (Right panel) Quantification of sphere number. Spheres with a diameter ≥ 100 µm were counted. F) 1000 Kras G12D/+ pancreatic acinar cells labeled with CellTracker Green CMFDA were cocultured with different ratios of Kras +/+ fibroblasts for 14 days in a 96‐well low attached plate. Spheres with a diameter ≥ 100 µm were counted. In the above experiments, N indicates independent experiment, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. ****, P < 0.0001 (two‐tailed Student's t‐test). G) EpCAM + /PDGFRα + cell clusters derived from the pancreas of 4‐week‐old control or KC mice by MACS dissociation and FACS with EpCAM/PDGFRα antibodies for cyst formation analysis. Representative images of cysts without and with lumen were shown in the left panel. CK19 staining indicates pancreatic ductal cells, and αSMA staining indicates activated fibroblasts. Bar, 100 µm. The number of cysts was counted and compared in the right panel. N indicates independent experiment and n indicates cyst number. H) The same cell clusters as above, in addition two more controls including EpCAM + and non‐ EpCAM + /PDGFRα + clusters from KC mice, were used for sphere formation analysis. Representative images of spheres were shown in the upper panel and the number of spheres were counted (lower panel). Spheres with a diameter ≥ 100 µm were counted. N indicates independent experiment, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. ***, P < 0.001 (two‐tailed Student's t‐test).

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Transformation Assay, Staining, Immunohistochemistry, Labeling, Two Tailed Test, Derivative Assay, Control

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: Muc4 overexpression in Kras G12D/+ pancreatic cells promotes fibroblast activation and recruitment in the earliest PanIN. A) The mRNA levels of Muc4, Muc1, and Muc5a in the EpCAM + /PDGFRα + clusters derived from the pancreas of 4‐week‐old KC mice were measured and compared with that of the control mice after normalized with the control gene, GAPDH . Primary mPSCs in all experiments were isolated from the pancreas of 4‐week‐old control mice. For chemotaxis analysis, mPSCs were labeled with CellTracker Green CMFDA for 10 mins before the experiment. B) Expression of ACTA2 gene in mPSC treated with conditional media derived from EpCAM + /Muc4 − or EpCAM + /Muc4 + pancreatic cells isolated by FACS from 4‐week‐old control or KC mice by qPCR. C) Left panel: Representative images of µslide chemotaxis analysis of mPSC after treated with conditional media as B). Right panel: Quantitation of the percentage of chemotaxis. D–F) EpCAM + /Muc4 − ‐ or EpCAM + /Muc4 + ‐pancreatic cells were cocultured with mPSC cells for cyst formation analysis D), sphere formation analysis E), and soft colony formation analysis F). For sphere formation analysis, sphere with a diameter ≥ 100 µm were counted. G) Expression of ACTA2 gene in mPSC treated with conditional media derived from pancreatic acinar cells isolated from 4‐week‐old control or KC mice by qPCR. These primary pancreatic acinar cells were infected with lentiviral GFP or lentiviral GFP‐MUC4 (MUC4/X). H) Left panel: Representative images of µslide chemotaxis analysis of mPSC after treated with conditional media as G). Right panel: Quantitation of the percentage of chemotaxis. I and J) Pancreatic acinar cells from KC or control mice were ectopically expressed GFP‐MUC4/X or GFP only and cocultured with mPSC cells for sphere formation analysis I) and soft colony formation analysis J). For soft agar colony formation analysis, the colony with a diameter ≥ 50 µm was counted. N indicates independent experiment, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. **, P < 0.01; ****, P < 0.0001 (two‐tailed Student's t‐test).

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Over Expression, Activation Assay, Derivative Assay, Control, Isolation, Chemotaxis Assay, Labeling, Expressing, Quantitation Assay, Infection, Two Tailed Test

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: Activin A from Muc4 overexpressed and Kras G12D/+ pancreatic cells facilitate fibroblast recruitment for PanIN formation. A) Cytokines analysis of conditional media from the cell clusters isolated from 4‐week‐old control or KC mice using RayBio® Mouse Biotin‐Label Based Antibody Array (Mouse L‐308 Array, Glass Slide). B) The effect of Activin A on mPSC chemotaxis was analyzed using μ‐Slide chemotaxis analysis. Representative images (left panel) and quantification (right panel) of the chemotaxis effect. C) Quantification of Activin A in the conditional media of EpCAM + /Muc4 − ‐ or EpCAM + /Muc4 + ‐pancreatic cells using ELISA analysis. D) Anti‐Activin A antibody abolishes mPSC chemotaxis. Conditional media from EpCAM + /Muc4 + ‐pancreatic cells were pre‐treated with or without 4 µg mL −1 of anti‐Activin A antibody for 30 mins and were subjected to µslide chemotaxis analysis. E) Quantification of Activin A in the conditional media of pancreatic acinar cells infected with lentiviral GFP or lentiviral GFP‐MUC4 (MUC4/X) using ELISA. F) Anti‐Activin A antibody abolishes mPSC chemotaxis. Conditional media from lentiviral GFP‐MUC4/X‐infected pancreatic acinar cells were pre‐treated with or without 4 µg mL −1 of anti‐Activin A antibody for 30 min and were subjected to μ‐Slide chemotaxis analysis. G) Increases of Activin A secretion of media from FACS‐isolated pancreatic cells cocultured with or without fibroblasts by ELISA analysis. H) Activin A secretion from Kras G12D/+ pancreatic acinar cells infected with the indicated lentivirus after cocultured with or without fibroblasts by ELISA analysis. In the above experiments, N indicates independent experiments, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two‐tailed Student's t‐test). I) Activin A mRNA expression in PanIN and PanIN‐associated fibroblasts in 4‐week‐old KC mice was detected by an Opal Multiplex IHC Assay. Representative IHC images (left panel) and quantification of Activin A expressed in early PanIN cells (right panel). Bar, 20 µm. White signal: DAPI; Cyan signal: Normal duct cells and PanIN cells stained with anti‐CK19 antibody; Red signal: αSMA + fibroblasts stained with anti‐αSMA antibody; Green dot signal: Activin A mRNA with dig‐labeled antisense mRNA probes. Values were presented as mean ± SD, PanIN lesion n = 29 from 12 KC mice. J) Sera from 4‐week‐old control or KC mice were used to perform Activin A ELISA analysis. N = 7 mice; two duplicate experiments for each mouse, n = total 14 repeated measurements. Each dot represents the datum from one measurement. Values were presented as mean ± SD. *, P < 0.05 (two‐tailed Student's t‐test).

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Isolation, Control, Ab Array, Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Infection, Two Tailed Test, Expressing, Multiplex Assay, Staining, Labeling

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: The proposed model for the cooperation of oncogenic Kras G12D/+ , Muc4 overexpression, and fibroblast activation for PanIN initiation. A–E) 3‐week‐old KC mice were administered either Saline solution or a dose of 1 µg k −1 g follistatin (FST) for a duration of two weeks. After the two‐week treatment period, we performed integrated 3D/2D pancreas histology to assess fibroblast recruitment and PanIN formation. A) Representative images depicting 3D and 2D histology of the pancreas. In the 3D staining images, green color represents CK19 staining, indicating PanIN or ductal cells; red color represents αSMA staining, indicating activated fibroblasts; and white color represents DAPI staining, indicating nuclei. In the 2D IHC images, brown color represents αSMA staining, indicating activated fibroblasts. Scale bar: 100 µm. B) The percentage of lesions associated with fibroblast. Each dot represents the datum of one mouse. Values were presented as mean ± SD. Mouse number and lesion number are denoted by N and n, respectively. ***, P < 0.001 (two‐tailed Student's t‐test). C) The H score of αSMA + fibroblasts associated with lesion of specified mice. Values were presented as mean ± SD. Mouse number and lesion number are denoted by N and n, respectively. **, P < 0.01 (two‐tailed Student's t‐test). D) Representative lesion images (upper panel) and atrophy lesion quantification (lower panel) at KC mice treated with Saline solution or FST. Green color represents CK19 staining, indicating PanIN. Each dot represents the datum of one mouse. Values were presented as mean ± SD. Mouse number and lesion number are denoted by N and n, respectively. **, P < 0.01 (two‐tailed Student's t‐test). E) The percentage of lobe and lesions volume in KC mice treated with Saline solution or FST. Mouse number and lesion number are denoted by N and n, respectively. The total tissue volume calculated in the Saline solution group is 279.4 mm 3 , while in the FST group, it is 252.8 mm 3 . F) mPSCs were obtained from 4‐week‐old control mice and subjected to overnight infection with 10 MOI of the specified lentiviral shRNA. After one day of recovery from the virus infection, we selectively enriched lentiviral shRNA‐positive cells by applying puromycin selection at a concentration of 1 µg mL −1 for three days. Following a two‐day recovery period, the cells were subjected to Western blot analysis (upper panel) and cocultured with double EpCAM and Muc4‐positive pancreatic cells isolated from 4‐week‐old KC mice for the assessment of soft agar colony formation (lower panel). The number of colonies larger than 50 µm was quantified after a 14‐day coculture period. N indicates independent experiment, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. ****, P < 0.0001 (two‐tailed Student's t‐test). G) Proposed model of oncogenic Kras G12D ‐mediated PanIN initiation. The overexpression of Muc4 (Muc4/X) in Kras G12D/+ pancreatic cells enhances cell transformation and stimulates Activin A secretion, which in turn activates and recruits' fibroblasts. The activated fibroblasts further promotes Activin A secretion, elevates pancreatic cell transforming status, and enhances cancer stemness properties, thereby contributing to PanIN initiation. The inhibition of Activin A signaling using Follistatin (FST), an Activin A antagonist, effectively suppresses fibroblast recruitment and inhibits PanIN formation. Additionally, reducing Activin A expression in mPSCs through the use of lentiviral Inhba shRNA also impedes PanIN formation.

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Over Expression, Activation Assay, Saline, Staining, Two Tailed Test, Control, Infection, shRNA, Virus, Selection, Concentration Assay, Western Blot, Isolation, Transformation Assay, Inhibition, Expressing

Journal: Journal of Translational Medicine

Article Title: IκBα kinase inhibitor BAY 11-7082 promotes anti-tumor effect in RAS-driven cancers

doi: 10.1186/s12967-024-05384-4

Figure Lengend Snippet: BAY 11-7082 treatment alters the expression of multiple genes in NRAS, KRAS, and HRAS mutant cancer cells. (A) Heatmaps showing the top 50 upregulated or downregulated genes in SKMEL-103, AsPC1, and RH-36 cells upon treatment with BAY 11-7082 (5 µM) for 48 h as compared with DMSO-treated cells. (B) Volcano plot showing top 15 genes upregulated or downregulated after 48-h treatment with BAY 11-7082 (5 µM) in RAS mutant cells

Article Snippet: RAS mutant cancer cell lines (NRAS: M245, SKMEL-103, SKMEL-2; KRAS: PANC1, AsPC1, SU.86.86) were purchased from American Type Culture Collection (ATCC) as listed in Supplementary Tables and maintained in a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s modified Eagle medium (Life Technologies, Carlsbad, CA, USA) or Roswell Park Memorial Institute-1640 Medium (Life Technologies), each supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (both from Life Technologies).

Techniques: Expressing, Mutagenesis

Journal: Journal of Translational Medicine

Article Title: IκBα kinase inhibitor BAY 11-7082 promotes anti-tumor effect in RAS-driven cancers

doi: 10.1186/s12967-024-05384-4

Figure Lengend Snippet: Model showing the new IκBα kinase Inhibitor BAY 11-7082 for Treating RAS-driven cancers. A model depicting various cancer types driven by oncogenic NRAS, KRAS, and HRAS mutations overexpress IκBα kinase. IκBα kinase can be effectively targeted by small molecule inhibitor BAY 11-7082, leading to downregulation of several prooncogenic signaling pathways, including PI3K-AKT signaling cascade and upregulation of apoptosis that contributes to tumor growth inhibition observed in RAS-driven cancers

Article Snippet: RAS mutant cancer cell lines (NRAS: M245, SKMEL-103, SKMEL-2; KRAS: PANC1, AsPC1, SU.86.86) were purchased from American Type Culture Collection (ATCC) as listed in Supplementary Tables and maintained in a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s modified Eagle medium (Life Technologies, Carlsbad, CA, USA) or Roswell Park Memorial Institute-1640 Medium (Life Technologies), each supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (both from Life Technologies).

Techniques: Protein-Protein interactions, Inhibition

Journal: Communications Biology

Article Title: Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles

doi: 10.1038/s42003-021-01882-z

Figure Lengend Snippet: a Venn diagram of proteins identified in SW620 cell-derived sMB-R (sMV-HD), sMV-LD and Exos. b Heatmap illustration of proteins identified in sMB-R (sMV-HD), sMV-LD and Exos. Proteins present in higher abundance in sMB-R (red) as compared to sMV-LD and Exos include conserved cytokinetic proteins as well as additional cytokinetic proteins. *Proteins uniquely identified in sMB-Rs. **Proteins enriched (fold change >2) in sMB-R compared to sMV-LD and Exos. c STRING-based protein-protein interaction network analysis of 928 enriched proteins in sMB-Rs (sMV-HD) compared to sMV-LD and Exos. The interactions were “evidence”-based, with “experiments” as active interaction source and interaction threshold set at 0.900 (highest confidence). Disconnected nodes in the network are hidden. Proteins identified under biological processes or molecular processes (Gene Ontology) are indicated. Centralspindlin complex components (RACGAP1 and KIF23/MKLP1) are also indicated. d EnrichmentMap of Reactome pathways enriched in 456 proteins commonly identified in SW620 cell-derived sMB-R proteome (2300 proteins) with the proteome of MB-Rs shed by Hela cells reported recently by Peterman et al. 2019 . e Mass spectrometry-based identification of KRAS peptides (UniProtKB ID RASK_HUMAN) in sMB-Rs. Two peptides (TEYKLVVVGAGGVGK and LVVVGAGGVGK) spanning Gly-12/ Val-12 substitution in KRAS protein. Peptide spectral profiles are displayed on the right. f Immunofluorescence microscopy of SW620 cells using anti-MKLP1 and anti-KRAS G12V antibodies. Nuclei (blue) were stained with Hoechst stain. White arrows indicate the position of MB and MB-Rs. Inset represents higher magnification. Scale bar, 10 µm. g Western blot analysis of exosomes, crude 10,000 x g sMVs, and isopycnic (iodixanol-density) gradient centrifugation fractions of sMV-LD and -HD/sMB-Rs using anti-KRAS G12V antibody.

Article Snippet: Cells were then incubated with primary antibodies (1:100) (mouse anti-MKLP1 (Santa Cruz Biotechnology), mouse anti-RACGAP1 (Santa Cruz), rabbit anti-KRAS G12V mutant specific (Cell Signalling), rabbit anti-RAB7 (Abcam) and rabbit anti-β-tubulin (Cell Signalling) in blocking solution for 1 h at room temperature.

Techniques: Derivative Assay, Mass Spectrometry, Immunofluorescence, Microscopy, Staining, Western Blot, Gradient Centrifugation

Journal: Communications Biology

Article Title: Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles

doi: 10.1038/s42003-021-01882-z

Figure Lengend Snippet: a Uptake of sMB-Rs by fibroblasts. Fluorescence microscopy analysis of NIH3T3 fibroblasts incubated with/without SW620 cell-derived sMB-Rs or Exos (50 µg ml −1 ) for 2 h using anti-MKLP1 and anit-RACGAP1 antibodies. b Uptake and accumulation of sMB-Rs in NIH3T3 fibroblasts was quantified by counting MKLP1 + puncta per cell; data represented as mean ± s.e.m. Nuclei (blue) were stained with Hoechst. Scale bar, 10 µm. c Internalisation of sMB-Rs by fibroblasts. Confocal microscopy of NIH3T3 fibroblasts incubated with sMB-Rs using anti-MKLP1 (in green) and anti-RAB7 (in red) antibodies. Confocal microscopy analysis along Z-axis (inset) reveal internalisation of sMB-Rs following uptake. Scale bar, 10 µm. d Intercellular transfer of sMB-R KRAS G12V into NIH3T3 cells. Fluorescence microscopy of NIH3T3 fibroblasts incubated with SW620 cell-derived sMB-Rs (5 µg) for 2 h using anti-KRAS G12V antibodies. Nuclei were stained with Hoechst stain (blue). Right panel represents fluorescence signals from left panel overlaid onto bright-field images. Inset represents enlarged image. Scale bar, 10 µm.

Article Snippet: Cells were then incubated with primary antibodies (1:100) (mouse anti-MKLP1 (Santa Cruz Biotechnology), mouse anti-RACGAP1 (Santa Cruz), rabbit anti-KRAS G12V mutant specific (Cell Signalling), rabbit anti-RAB7 (Abcam) and rabbit anti-β-tubulin (Cell Signalling) in blocking solution for 1 h at room temperature.

Techniques: Fluorescence, Microscopy, Incubation, Derivative Assay, Staining, Confocal Microscopy

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: The expression of GSTP1 and p53 in PC specimens. (A) Low and high GSTP1 expression in two normal pancreas samples. (B) Low and high GSTP1 expression in two PC samples. (C) Low and high p53 expression in two PC samples. (D) High GSTP1 and mtp53 expression in one PC sample. (E) Low GSTP1 and mtp53 expression in one PC sample. (F) High GSTP1 and wtp53 expression in one serial section of PC tissue. (G) Kaplan–Meier analysis of high and low expression of GSTP1 in PC patients. (H) Kaplan–Meier analysis of Mtp53 and wtp53 expression in PC patients. (I) Kaplan–Meier analysis of high and low expression of GSTP1 in wtp53 PC patients.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: Expressing

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: The relationship between GSTP1 and p53 in vitro. (A) GSTP1 and p53 protein expression in six PC cell lines. (B) GSTP1 mRNA expression in six PC cell lines. (C) GSTP1 and p53 expression in GSTP1‐ or p53‐silenced mtp53 BxPC‐3 cells. (D) GSTP1 and p53 expression in GSTP1‐ or p53‐silenced mtp53 PANC‐1 cells. (E) GSTP1 and p53 expression in GSTP1‐ or p53‐silenced wtp53 Capan‐2 cells. (F) GSTP1 and p53 expression in the GFP, GSTP1‐GFP, GSTP1‐GFP plus si‐p53, and si‐p53 groups of SW1990 cells with wtp53. 1: sictrl group; 2: si1‐GSTP1 group; 3: si2‐GSTP1 group; 4: si‐p53 group; 5: GFP group; 6: GSTP1‐GFP group; 7: GSTP1‐GFP + si‐p53 group; 8: Si‐p53 group. The experiments were performed in triplicate. Error bars ± SDs. * p < 0.05 and ** p < 0.01 compared with the control.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: In Vitro, Expressing, Control

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: GSTP1 inhibited cell proliferation and drug resistance in a wtp53‐dependent manner in vitro. (A) MTT assays in si1‐GSTP1 and sictrl transfected wtp53 Capan‐2 cells cultured within 4 days. (B) MTT assays of GFP, GSTP1‐GFP and GSTP1‐GFP plus si‐p53 transfected SW1990 cells with wtp53 cultured within 4 days. (C, D) MTT assays in si1‐GSTP1 and sictrl transfected wtp53 Capan‐2 cells under OXA (C) or GEM (D) treatment for 2 days. (E, F) MTT assays in GFP, GSTP1‐GFP and GSTP1‐GFP plus si‐p53 transfected SW1990 cells with wtp53 under OXA (E) or GEM (F) treatment for 2 days. The experiments were performed in triplicate. Error bars ± SDs. * p < 0.05 and ** p < 0.01 compared with the control.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: In Vitro, Transfection, Cell Culture, Control

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: GSTP1 regulated cell proliferation and drug resistance via p53/p21‐ and Bax/Bcl2‐mediated signaling pathways. (A, B) p53/p21 and Bax/Bcl2 protein expression in si1‐GSTP1 and sictrl transfected wtp53 Capan‐2 cells with or without OXA (A) or GEM (B) treatment for 2 days. (C, D) p53/p21 and Bax/Bcl2 protein expression in GFP and GSTP1‐GFP transfected SW1990 cells with wtp53 with or without OXA (C) or GEM (D) treatment for 2 days. 1: Sictrl group; 2: si1‐GSTP1 group; 3: Sictrl with OXA (A) or GEM (B) treatment group; 4: si1‐GSTP1 with OXA (A) or GEM (B) treatment group; 5: GFP group; 6: GSTP1‐GFP group; 7: GFP with OXA (C) or GEM (D) treatment group; 8: GSTP1‐GFP with OXA (C) or GEM (D) treatment group. The experiments were performed in triplicate. Error bars ± SDs. * p < 0.05 and ** p < 0.01 compared with the control.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: Protein-Protein interactions, Expressing, Transfection, Control

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: GSTP1 mediated cell invasion and migration via partially regulating wtp53 mediated EMT signaling. (A) Cell invasion and migration in sictrl, si1‐GSTP1 and si2‐GSTP1 transfected wtp53 Capan‐2 cells. (B) Cell invasion and migration of GFP, GSTP1‐GFP and GSTP1‐GFP plus si‐p53 transfected SW1990 cells with wtp53. (C) Protein expression of EMT epithelial and mesenchymal biomarkers in sictrl, si1‐GSTP1 and si2‐GSTP1 transfected wtp53 Capan‐2 cells. (D) Protein expression of EMT epithelial and mesenchymal biomarkers in GFP and GSTP1‐GFP transfected SW1990 cells with wtp53. 1: Sictrl group; 2: si1‐GSTP1 group; 3: si2‐GSTP1 group; 4: GFP group; 5: GSTP1‐GFP group. The experiments were performed in triplicate. Error bars ± SDs. * p < 0.05 and ** p < 0.01 compared with the control.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: Migration, Transfection, Expressing, Control

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: The feedback regulation of GSTP1 and wtp53 mediated the malignant progression of PC in vitro. (A) GSTP1 was not coimmunoprecipitated with p53 in mtp53 PANC‐1 or wtp53 Capan‐2 cells. (B) P53 protein could bind to the GSTP1 DNA promoter in wtp53 Capan‐2 or SW1990 cells. (C) P53 protein could not bind to the GSTP1 DNA promoter in mtp53 BxPC‐3 or PANC‐1 cells. (D) IF staining of p53 (TRITC, red) and nuclei (Hoechst, blue) in si1‐GSTP1 and sictrl transfected wtp53 Capan‐2 cells. (E) IF staining of p53 (TRITC, red) and nuclei (Hoechst, blue) in GFP and GSTP1‐GFP transfected SW1990 cells with wtp53. (F) IF staining of p53 (TRITC, red) and nuclei (Hoechst, blue) in si1‐GSTP1 and sictrl transfected mtp53 BxPC‐3 cells. (G) IF staining of p53 (TRITC, red) and nuclei (Hoechst, blue) in si1‐GSTP1 and sictrl transfected mtp53 PANC‐1 cells.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: In Vitro, Staining, Transfection

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: GSTP1 inhibited tumor growth in coordination with wtp53 in vivo. (A) Subcutaneous tumor volumes in GFP and GSTP1‐GFP transfected SW1990 cells with wtp53 in 5 nude mice. (B) HE staining of the subcutaneous tumor. (C) GSTP1 and p53 expression in subcutaneous tumors in the GFP and GSTP1‐GFP groups, as determined by WB. (D) ki67 expression in the subcutaneous tumors of the GFP and GSTP1‐GFP groups was compared via IHC. (E) Liver metastases in GFP and GSTP1‐GFP transfected SW1990 cells with wtp53 in 5 nude mice. (F) HE staining of liver metastases. (G) Representative images from the IHC assays of GSTP1 and p53 protein expression in the GFP and GSTP1‐GFP groups in vivo. (H) Statistical data from the IHC assays of GSTP1 and p53 protein expression in the GFP and GSTP1‐GFP groups in vivo. Error bars ± SDs. * p < 0.05 and ** p < 0.01 compared with the control.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: In Vivo, Transfection, Staining, Expressing, Control

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: LAI-1 and lysosomes subcellular colocalization in A549 cell line. Live cell imaging fluorescence microscopy was performed. ( A ) Differential interference contrast of A549 cells. ( B ) Lysotracker Green labelling lysosomes (green channel). ( C ) A total of 10 µM of LAI-1 treatment after 3 h (red channel). ( D ) Merged channel showing lysosomes and LAI-1 colocalization (orange). Images are representative of three independent experiments. Scale bar 30 µm. Colocalization between Lysotracker and LAI-1 was quantified using Pearson’s correlation coefficient (PCC) and Mander’s overlap coefficient (MOC).

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Live Cell Imaging, Fluorescence, Microscopy

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Cell viability dose–response curves, evaluated with MTT assay, for all cell lines (A549, SW900 and DMS53) after 24 h incubation with chloroquine (CQ), 3-Methyladenine (3-MA) and LAI-1 treatment at different concentrations. Each point represents the mean value ± SD.

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: MTT Assay, Incubation

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Inhibitory concentration (IC); IC 25 , IC 50 and IC 75 values after LAI-1 treatment for A549, SW900 and DMS53 cell lines. Data show mean value ± SD.

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Concentration Assay

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Autophagy markers after treatment with different autophagy inhibitors. ( A ) Western blot showing autophagy-related proteins LC3 and p62/SQSTM1 after 24 h with chloroquine (CQ, 150 µM), 3-Methyladenine (3-MA, 10 mM) and LAI-1 (10 µM) treatment in A549 cells. ( B ) LC3-I and LC3-II protein levels and their LC3-II/I ratios. ( C ) p62/SQSTM1 protein expression. Protein expression was normalized using GAPDH as loading control. Fold induction against control group (CT) was calculated. Figure shows mean ± SEM. Statistical differences against CT are shown as *** p < 0.001, ** p < 0.01 and * p < 0.05. The whole western blot figures are showed in .

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Western Blot, Expressing, Control

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Autophagy modulation after LAI-1 treatment. ( A ) Dose–response Western blot showing autophagy-related proteins LC3 and p62/SQSTM1 after 24 h of LAI-1 treatment at different concentrations (10, 15 and 20 µM) in A549, SW900 and DMS53 cell lines. ( B ) LC3-I and LC3-II protein levels and their LC3-II/I ratios after dose–response assay. ( C ) p62/SQSTM1 protein expression after dose–response experiment. ( D ) Time-course Western blot showing autophagy-related proteins LC3 and p62/SQSTM1 after treating A549 cells with LAI-1 (10 µM) at different time points (0, 4, 8, 16, 24, 48 h). ( E ) LC3-I and LC3-II protein levels and their LC3-II/I ratios after time-course experiment. ( F ) p62/SQSTM1 protein expression after time-course assay. ( G ) Time-course Western blot showing autophagy activation proteins Akt and mTOR after treating A549 cells with LAI-1 (10 µM) at different time points (0, 4, 8 h). ( H ) Akt and phospho-Akt at S473 (pAkt) protein levels after time-course experiment. ( I ) mTOR and phospho-mTOR at S2778 (pmTOR) protein expression after time-course assay. Protein expression was normalized using GAPDH as loading control. Fold induction against control group (CT) was calculated. Figure shows mean ± SEM. Statistical differences against CT are shown as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. The whole western blot figures are showed in .

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Western Blot, Expressing, Activation Assay, Control

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Subcellular localization of LC3 and LAMP-1. A549 cells were harvested for 24 h on coverslips and then treated with LAI-1 (15 µM) and chloroquine (CQ, 50 µM) for different times. The fusion between autophagosomes, marked with LC3 (green), and lysosomes, marked with LAMP-1 (red), was studied in merged images. Zoomed images were used to better observe this process. The localization and intensity of LC3 staining were also analyzed. DAPI (blue) staining was used for nuclear localization. Images are representative of three independent experiments. Scale bar 30 µm.

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Staining

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Lysosomal and intracellular pH modifications using LAI-1 treatment. ( A ) A549 cells were treated for 1 h with LAI-1 or chloroquine (CQ) at different concentrations. After that, acridine orange staining (5 µg/mL) was performed for 30 min at room temperature. Images are representative of three independent experiments. Scale bar 50 µm. ( B ) Intracellular pH measurement in A549 cells treated with different LAI-1 concentrations for 1 h. Staining with pH Rodo Red AM staining kit and a calibration curve was conducted to quantify intracellular pH. Figure shows mean ± SEM. Statistical differences against control group (CT) are shown as ** p < 0.01.

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Staining, Control

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Cell death characterization using flow cytometry. The different cell lines (A549, SW900 and DMS53) were treated for 24 h with different LAI-1 concentrations. Cells were stained with Annexin-V APC/ Sytox Green kit. Early apoptotic cells were defined as Annexin-positive and Sytox-negative, whereas late apoptotic or necrotic cells were defined as Annexin- and Sytox-positive. ( A ) Representative plots. ( B ) Quantification of % of cells in early apoptosis and in late apoptosis or necrosis after different treatment. Statistical differences against control group (CT) are shown as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Flow Cytometry, Staining, Control

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Expression of apoptotic and cell-cycle related proteins. The different cell lines (A549, SW900 and DMS53) were treated for 24 h with different LAI-1 concentrations and several proteins were assessed with Western blot ( A ). Quantification of procaspase 3 ( B ), PARP ( C ), cleaved-PARP ( D ), p21 ( E ) and p53 ( F ) protein expression after Western blot analysis was performed using GAPDH expression as loading control. Fold induction against control group (CT) was calculated. Figures show mean ± SEM. Statistical differences against CT are shown as * p < 0.05, ** p < 0.01 and *** p < 0.001. The whole western blot figures are showed in .

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Expressing, Western Blot, Control

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Cell viability, evaluated with MTT assay, for A549, SW900 and DMS53 lung cancer cell lines after 24 h incubation with cisplatin (CisPt, 40 µM), LAI-1 (15 µM) and the combination. Figure shows mean ± SEM. Statistical differences are shown as **** p < 0.0001 ** p < 0.01 and * p < 0.05.

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: MTT Assay, Incubation