human cell line aspc1  (ATCC)

Journal: Journal of visualized experiments : JoVE

Article Title: Mapping Dysfunctional Protein-Protein Interactions in Disease

doi: 10.3791/69197

Figure Lengend Snippet: ( A ) Biological specificity and lot-to-lot consistency. MDA-MB-468 (epichaperome-high) and ASPC1 (epichaperome-low) cells were lysed in native buffer (20 mM Tris, pH 7.4; 20 mM KCl; 5 mM MgCl 2 ; 0.01% NP-40; protease/phosphatase inhibitors). For each capture, 40 μL of PU-bead slurry was incubated with 250 μg of total protein (1 μg/μL) for 3 h at 4 °C with rotation. Beads were washed in native buffer; complexes were denatured/eluted in ~100 μL of SDS sample buffer. 5-10 μL of each eluate was resolved by SDS-PAGE and immunoblotted for HSP90, HSC70, and HOP. Input lysates are shown for reference. Two PU-bead lots (Batch 1, fresh; Batch 2, aged) yield strong enrichment in MDA-MB-468 and minimal signal in ASPC1, demonstrating preserved specificity and lot consistency. ( B ) Global cargo versus control beads. MDA-MB-468 lysates were processed as in (A) with either PU-beads or matched control beads. ~20 μL of each eluate was loaded, separated by SDS-PAGE, and Coomassie-stained. PU-beads recover a complex, high-MW cargo characteristic of epichaperome-bound assemblies, whereas control beads show minimal background. Molecular-weight markers (kDa) are indicated. Please click here to view a larger version of this figure .

Article Snippet: Human cell line: ASPC1 (RRID:CVCL_0152) , ATCC , CRL-1682 , Control cell line, epichaperome low/chaperone high.

Techniques: Incubation, SDS Page, Control, Staining, Molecular Weight

Journal: Cancers

Article Title: Mesothelin-Associated Anti-Senescence Through P53 in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers17122058

Figure Lengend Snippet: Figure 1. A total of 105 tissue samples from PDAC patients and 44 normal tissue samples from CPTAC were retrieved for an MSLN-related gene set enrichment analysis (GSEA). (A) A WikiPathways cancer analysis revealed ten MSLN-positive-related and five MSLN-negative-related categories. (B) A Reactome pathway analysis revealed ten MSLN-positive-related and ten MSLN-negative-related categories. (C,D) Two representatives of DNA damage/DNA repair pathway enrichment plot show that the running scores of these signaling pathways are <0, indicating that MSLN may participate in those biological processes by inversely regulating corresponding pathways.

Article Snippet: Human PDAC cell lines AsPC1, CFPAC1, MIA Paca2, and Panc1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Yao Lab. Human PDAC cell line Panc28 was obtained from MD Anderson Dr. Craig Logsdon’s lab. Murine PDAC Panc02 cell line was obtained as a gift from Dr. Sabry EL-Naggar, the Medical University of South Carolina [20].

Techniques: Protein-Protein interactions

Journal: Cancers

Article Title: Mesothelin-Associated Anti-Senescence Through P53 in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers17122058

Figure Lengend Snippet: Figure 2. Cell senescence was induced with MSLN loss in PDAC cells. Senescence-associated β- galactosidase (SA-β-gal) staining was performed in MSLN-KO or scramble MIA PaCa2 (A) and Panc28 cells (B) and observed with 10× objectives, respectively. The proportion of senescence cells in A and B was quantified (C). (D) SA-β-gal staining was performed in MSLN-KO or scramble Panc02 cells. The Panc02 MSLN-KO and scramble PDAC cells were observed with 10×, 20×, and 40× objectives, respectively. *** p < 0.001; unpaired two-tailed t-test.

Article Snippet: Human PDAC cell lines AsPC1, CFPAC1, MIA Paca2, and Panc1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Yao Lab. Human PDAC cell line Panc28 was obtained from MD Anderson Dr. Craig Logsdon’s lab. Murine PDAC Panc02 cell line was obtained as a gift from Dr. Sabry EL-Naggar, the Medical University of South Carolina [20].

Techniques: Staining, Two Tailed Test

Journal: Cancers

Article Title: Mesothelin-Associated Anti-Senescence Through P53 in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers17122058

Figure Lengend Snippet: Figure 3. MSLN expressions are negatively correlated with senescence regulators. MSLN KO resulted in elevated levels of senescence markers P16, P21, and P53 in two mouse cell lines: KPC cells (A) and Panc02 cells (B). MSLN KD resulted in elevated levels of senescence markers P16, P21, and P53 in two human cell lines: ASPC1 and CFPAC1 cells (C). MSLN OE resulted in reduced levels of senescence markers P16, P21, and P53 in two human cell lines: Panc1 and Panc28 cells (D). (E–J) The expression levels of MSLN, P53, P21, and P16 in different manipulated mouse and human cells shown above were quantified by using ImageJ software and are presented. Quantification was carried out using triplicate scans and normalized onto GAPDH, and the results are presented in the bar graphs. The original Western blot figures can be found in Supplementary File S1.

Article Snippet: Human PDAC cell lines AsPC1, CFPAC1, MIA Paca2, and Panc1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Yao Lab. Human PDAC cell line Panc28 was obtained from MD Anderson Dr. Craig Logsdon’s lab. Murine PDAC Panc02 cell line was obtained as a gift from Dr. Sabry EL-Naggar, the Medical University of South Carolina [20].

Techniques: Expressing, Software, Western Blot

Journal: Cancers

Article Title: Mesothelin-Associated Anti-Senescence Through P53 in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers17122058

Figure Lengend Snippet: Figure 4. Mesothelin deficiency induced γH2A.X expression indicative of DNA damage and resulted in a moderate but significant reduction in cell viability in PDAC cells. (A) MSLN knockdown in CFPAC1 cells resulted in increased expression of DNA damage response protein γH2A.X. (B) MSLN knockout in Panc28 cells resulted in increased expression of DNA damage response protein γH2A.X. Antibodies are anti-MSLN (1:1000 dilution), anti-H2A.X (1:1000 dilution), anti-γH2A.X. (1:1000 dilution), and anti-GAPDH (1:1000 dilution). Blots shown are representative of three independent biological replicates. (C,D) MSLN knockdown or knockout reduces cell viability in PDAC cells, as determined by Trypan Blue exclusion assay. Representative brightfield images of PDAC cells stained with 0.2% Trypan Blue solution. Cell viability was quantified by calculating ratio of number of unstained cells to total number of cells. All photos were taken with 10× objective. Bars represent mean ± SD from three independent biological replicates. ⃝, scramble control cells; □, MSLN-KO or MSLN-KD cells. p < 0.05 indicates statistical significance. The original Western blot figures can be found in Supplementary File S1.

Article Snippet: Human PDAC cell lines AsPC1, CFPAC1, MIA Paca2, and Panc1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Yao Lab. Human PDAC cell line Panc28 was obtained from MD Anderson Dr. Craig Logsdon’s lab. Murine PDAC Panc02 cell line was obtained as a gift from Dr. Sabry EL-Naggar, the Medical University of South Carolina [20].

Techniques: Expressing, Knockdown, Knock-Out, Trypan Blue Exclusion Assay, Staining, Control, Western Blot

Journal: Cancers

Article Title: Mesothelin-Associated Anti-Senescence Through P53 in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers17122058

Figure Lengend Snippet: Figure 5. MSLN suppresses SASP production. Senescence-associated secretory phenotype of IL-8 se- cretion by PDACs was quantified by ELISA. Media from MSLN knockdown/overexpression/control PDAC cell culturing system was collected and applied to ELISA with IL-8 ELISA Kit. (A) ASPC1 cells; (B) CFPAC1 cells; (C) Panc1 cells; (D) Panc28 cells. p < 0.05 indicates statistical significance based on t-test.

Article Snippet: Human PDAC cell lines AsPC1, CFPAC1, MIA Paca2, and Panc1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Yao Lab. Human PDAC cell line Panc28 was obtained from MD Anderson Dr. Craig Logsdon’s lab. Murine PDAC Panc02 cell line was obtained as a gift from Dr. Sabry EL-Naggar, the Medical University of South Carolina [20].

Techniques: Enzyme-linked Immunosorbent Assay, Knockdown, Over Expression, Control, Cell Culture

Journal: Cancers

Article Title: Mesothelin-Associated Anti-Senescence Through P53 in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers17122058

Figure Lengend Snippet: Figure 6. The proposed model of the mesothelin (MSLN)-mediated anti-senescence mechanism (MAAS) in pancreatic ductal adenocarcinoma (PDAC). (A) In PDAC cells with high MSLN expres- sion, oncogenic signaling pathways such as PI3K/AKT/mTOR, ERK/MAPK, and FAK/SRC/NF-κB are activated. These promote cell proliferation, survival, and migration while suppressing cellular senescence and apoptosis. (B) In MSLN-deficient PDAC cells, the loss of MSLN leads to the accu- mulation of DNA damage and the activation of the DNA damage response (DDR), as evidenced by increased γH2AX expression. This triggers the upregulation of p53, p21waf1, and p16ink4a, re- sulting in cell cycle arrest and the acquisition of senescence phenotypes. Senescent PDAC cells also produce elevated levels of IL-8, a key component of the senescence-associated secretory phenotype (SASP). Collectively, this model illustrates how MSLN suppresses senescence and facilitates tumor progression, representing a novel mechanism termed mesothelin-associated anti-senescence (MAAS).

Article Snippet: Human PDAC cell lines AsPC1, CFPAC1, MIA Paca2, and Panc1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Yao Lab. Human PDAC cell line Panc28 was obtained from MD Anderson Dr. Craig Logsdon’s lab. Murine PDAC Panc02 cell line was obtained as a gift from Dr. Sabry EL-Naggar, the Medical University of South Carolina [20].

Techniques: Protein-Protein interactions, Migration, Activation Assay, Expressing