rabbit anti mesothelin  (R&D Systems)

Journal: Cell Reports Medicine

Article Title: Immune targeting of triple-negative breast cancer through a clinically actionable STING agonist-CAR T cell platform

doi: 10.1016/j.xcrm.2025.102198

Figure Lengend Snippet: TNBC tumor cell STING signaling primes response to CAR T cell therapy (A) Immunoblot of mesothelin (MSLN) in PTEN WT (HCC1143, HCC1806, and MDAMB231) and PTEN-null (MDAMB468, HCC1937, and HCC70) TNBC cell lines. (B) Flow cytometry of MSLN expression in HCC1806 and HCC70 using HCC1143 as a control (representative of 2 independent experiments). (C) Schematic of CAR constructs with retroviral and lentiviral vectors. SP, signal peptide; ScFv, single-chain variable fragment; EC, extracellular; TM, transmembrane. (D and E) Cell lines were co-cultured in 2D with CD19 or MSLN CAR T cells for 24 h at indicated effector:target (E:T) ratios and assessed by CTG assay ( n = 3). Data are representative of three independent experiments. (F and G) Representative images of HCC1806 and HCC70 co-cultured as 3D spheroids with CD19 or MSLN CAR T cells in a microfluidics device for 72 h. E:T ratio of 1:1. Staining as indicated for live and dead cells. Scale bar, 100 μm. (H) Quantification of 3D tumor spheroid cell death using DRAQ7/Hoechst staining ( n = 2–6). Ctrl indicates tumor cells only. (I) CM from 3D co-culture was analyzed for IFNγ, granzyme B, and TNF-α by ELISA ( n = 2–6). (J) Representative images of MSLN CAR T cell migration with untreated HCC70 and HCC1806 spheroids utilizing the 3D microfluidics device. MSLN CAR T cells fully migrate toward HCC70 (PTEN null) by 48 h, in contrast to HCC1806 cells (PTEN WT), denoted by a black arrow. E:T ratio of 1:1. Live and dead cells were stained as indicated. Scale bar, 100 μm. Quantification of migrated CAR T cells and percent tumor cell death are shown ( n = 5–6). (K) Representative images of MSLN CAR T cell migration with HCC1806 Scr and Rab7 KO spheroids utilizing the 3D microfluidic device after 48 h. E:T ratio of 1:1. Scale bar, 100 μm. Spheroids were pretreated with 50 μM ADU or ctrl (PBS) for 24 h before loading. Quantification of migrated CAR T cells and percent tumor cell death are shown ( n = 4–7). CM was analyzed for IFNγ and granzyme B by ELISA ( n = 3–5). Quantitative data are represented as mean ± SEM. p values were calculated by two-way ANOVA followed by Sidak’s post hoc test (D and E), one-way (K) and two-way (H and I) ANOVA followed by Tukey’s post hoc test, and unpaired Student’s t test (J). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Membranes were blocked for 1 h with the Odyssey blocking buffer (LI-COR Biosciences, Cat.# 927–60001) and probed for various proteins overnight in blocking buffer or HIKARI Signal Enhancer Solution 1 (Nacalai USA, Cat.# NU00102) using the following antibodies: STING (Cell Signaling, Cat.# 13647S), PTEN (Cell Signaling, Cat.# 9559S), Rab7 (Cell Signaling, Cat.# 9367S), pSTING (Cell Signaling, Cat.# 50907S), pIRF3 (Cell Signaling, Cat.# 4947S), IRF3 (Cell Signaling, Cat.# 4302S), pTBK1 (Cell Signaling, Cat.# 5483S), TBK1 (Cell Signaling, Cat.# 3504S), pSTAT1 (Cell Signaling, Cat.# 9167S), STAT1 (Cell Signaling, Cat.# 9172S), Mesothelin (Cell Signaling, Cat.# 65367S) and β-actin (Cell Signaling, Cat.# 3700S).

Techniques: Western Blot, Flow Cytometry, Expressing, Control, Construct, Retroviral, Cell Culture, CTG Assay, Staining, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Migration

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: A Western blot assay was used to detect MSLN, while senescent markers and DNA damage response markers were determined with antibodies anti-MSLN (99966), anti-P53 (13684), anti-P21 (8242), anti-P16 (3033), anti-H2A.X (7631S) (Cell Signaling Technology, Danvers, MA, USA), anti-γH2A.X. (AB2893, Abcam, Cam- bridge, UK), and anti-GAPDH (G8795, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.

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 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: A Western blot assay was used to detect MSLN, while senescent markers and DNA damage response markers were determined with antibodies anti-MSLN (99966), anti-P53 (13684), anti-P21 (8242), anti-P16 (3033), anti-H2A.X (7631S) (Cell Signaling Technology, Danvers, MA, USA), anti-γH2A.X. (AB2893, Abcam, Cam- bridge, UK), and anti-GAPDH (G8795, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.

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: A Western blot assay was used to detect MSLN, while senescent markers and DNA damage response markers were determined with antibodies anti-MSLN (99966), anti-P53 (13684), anti-P21 (8242), anti-P16 (3033), anti-H2A.X (7631S) (Cell Signaling Technology, Danvers, MA, USA), anti-γH2A.X. (AB2893, Abcam, Cam- bridge, UK), and anti-GAPDH (G8795, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.

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 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: A Western blot assay was used to detect MSLN, while senescent markers and DNA damage response markers were determined with antibodies anti-MSLN (99966), anti-P53 (13684), anti-P21 (8242), anti-P16 (3033), anti-H2A.X (7631S) (Cell Signaling Technology, Danvers, MA, USA), anti-γH2A.X. (AB2893, Abcam, Cam- bridge, UK), and anti-GAPDH (G8795, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.

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