Journal: PLoS ONE

Article Title: Methylation-Associated Partial Down-Regulation of Mesothelin Causes Resistance to Anti-Mesothelin Immunotoxins in a Pancreatic Cancer Cell Line

doi: 10.1371/journal.pone.0122462

Figure Lengend Snippet: A : KLM-1 and resistant KLM-1 (KLM-1-R) cells were incubated for 72 hrs with the anti-mesothelin SS1-LR-GGS, RG7787 or anti-CD25 LMB-2 as a control. Growth inhibition was evaluated with an ATP cell viability assay. With IC 50 s below 10 ng/ml, KLM-1 is sensitive to the anti-mesothelin RITs, which is not the case for KLM-1-R (IC 50 s > 1 μg/ml). 1 μg/ml LMB-2 decreased cell viability, indicating that this RIT concentration induces non-specific uptake. B : KLM-1 and KLM-1-R cells were incubated for 72 hrs with 100 or 1000 ng/ml RG7787. Apoptosis was evaluated with the Annexin V-PE Apoptosis Detection Kit I. RG7787 induces a significant increase in apoptotic KLM-1 cells, whereas KLM-1-R cells shows no meaningful increase in apoptosis. C : KLM-1 and KLM-1-R cells were incubated for 72 hrs with HB21(Fv)-PE40. Growth inhibition was evaluated with an ATP cell viability assay. Both cell lines are highly sensitive to this RIT.

Article Snippet: Soluble mesothelin levels in cell line medium were measured in duplicate with a mesothelin Meso Scale Assay (Morphotek, Inc.), using the electrochemiluminescence technology of Meso Scale Discovery.

Techniques: Incubation, Inhibition, Viability Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Methylation-Associated Partial Down-Regulation of Mesothelin Causes Resistance to Anti-Mesothelin Immunotoxins in a Pancreatic Cancer Cell Line

doi: 10.1371/journal.pone.0122462

Figure Lengend Snippet: A : Surface mesothelin levels are 5-fold lower in resistant KLM-1 (KLM-1-R) compared to KLM-1. Mesothelin expression of KLM-1, KLM-1-R and mesothelin-negative A431 cells (negative control) were evaluated using flow cytometry. Filled histograms are secondary antibody controls. B : Whole mesothelin protein level is decreased in KLM-1-R. Precursor (72 kDa) and cleaved mature mesothelin (37 kDa) were present in KLM-1. In KLM-1-R, the cleaved portion was detected at low levels. Protein levels were probed in untreated KLM-1 and KLM-1-R cell lysate by Western blot. β-actin acts as loading control. C : At each time point, cellular uptake of RG7787-Alexa647 in KLM-1 is significantly higher than in KLM-1-R, and significantly lower than in KLM-1-R- Msln (transfected with mesothelin). Uptake was evaluated at 30, 75 and 150 min. Average geomean fluorescence intensities converted into amount of RG7787 molecules.

Article Snippet: Soluble mesothelin levels in cell line medium were measured in duplicate with a mesothelin Meso Scale Assay (Morphotek, Inc.), using the electrochemiluminescence technology of Meso Scale Discovery.

Techniques: Expressing, Negative Control, Flow Cytometry, Western Blot, Transfection, Fluorescence

Journal: PLoS ONE

Article Title: Methylation-Associated Partial Down-Regulation of Mesothelin Causes Resistance to Anti-Mesothelin Immunotoxins in a Pancreatic Cancer Cell Line

doi: 10.1371/journal.pone.0122462

Figure Lengend Snippet: A : AZA leads to a 2.8-fold increase of mesothelin surface expression in resistant KLM-1 (KLM-1-R). Flow cytometry histogram of KLM-1, KLM-1-R, and KLM-1-R-AZA. Filled histograms represent secondary antibody controls. B : Three weeks of incubation with AZA, a DNA methyltransferase inhibitor, increases sensitivity to RG7787. KLM-1, KLM-1-R, and the AZA-treated cells (KLM-1-AZA and KLM-1-R-AZA) were treated for 72 hrs with RG7787. Growth inhibition was evaluated with an ATP cell viability assay. Dotted lines represent AZA-treated cells. C : CpGs in a region upstream of the mesothelin transcription start site are more methylated in KLM-1-R than in KLM-1. Three weeks of incubation with AZA decreases methylation in KLM-1-R cells. The analyzed region is located at chr16:808890-808742 and spans 147 bp and seven CpGs. D : Mesothelin transfection in KLM-1-R results in significant overexpression of mesothelin compared to KLM-1 (5-fold) and KLM-1-R (23-fold). Flow cytometry surface mesothelin levels in KLM-1, KLM-1-R and mesothelin-transfected resistant cells (KLM-1-R- Msln ). E : Mesothelin overexpression in KLM-1-R restores sensitivity to RG7787. KLM-1 and KLM-1-R- Msln cells were incubated for 72 hrs with RG7787. Growth inhibition was evaluated with an ATP cell viability assay.

Article Snippet: Soluble mesothelin levels in cell line medium were measured in duplicate with a mesothelin Meso Scale Assay (Morphotek, Inc.), using the electrochemiluminescence technology of Meso Scale Discovery.

Techniques: Expressing, Flow Cytometry, Incubation, Inhibition, Viability Assay, Methylation, Transfection, Over Expression

Journal: bioRxiv

Article Title: A genetic screen in enteroendocrine cells reveals mechanisms that control protein sensing and GLP-1 release

doi: 10.64898/2025.11.30.691441

Figure Lengend Snippet: a, Expression levels of CaMPARI and ZIM3-KRAB-dCas9 in each isolated single clones. Clone 2C6 is selected for all the CaMPARI screens and validation shown in this manuscript. b, CRISPRi efficiency by qPCR. Left, Knockdown efficiency for two candidate genes shown in . Right, Knockdown efficiency for all tested target genes. c, FACS screen gating strategy. Top and bottom 35% of CaMPARI photoconversion ratio (red/green) was collected. d, Heatmap showing log/fold change) for each individual sgRNA (5 per gene) targeting the top 50 hits from the tryptone screen. e-g, Phenotype scores for all library genes, comparing FACS screen with interal survival screen control. e, Tryptone screen. f, KCI screen. g, Phenylalanine screen. Pearson correlation coefficient is shown on the plot.

Article Snippet: To perform a pilot screen as outlined in , we first obtained a medium-scale mouse sgRNA library (CRISPRi_v2 sublibrary m1; Addgene #83989).

Techniques: Expressing, Isolation, Clone Assay, Biomarker Discovery, Knockdown, Control

Journal: bioRxiv

Article Title: A genetic screen in enteroendocrine cells reveals mechanisms that control protein sensing and GLP-1 release

doi: 10.64898/2025.11.30.691441

Figure Lengend Snippet: a, Composition of custom sgRNA library for large-scale CRISPRi screening. b-c, Volcano plot and rank plot for custom library screen. b, Significant hits (FDR < 0.05) are highlighted. c, Top 10 hits with highest phenotype scrore [logifold change) x -log 10 {pvalue)] are highlighted. d, Functional protein-protein interaction network for all positive hits by STRING. Line thickness indicates the strength of data support for interaction. Genes with mitochondrial annotation (GO:0005739) are highlighted in red. e, Hit distribution for two most critical mitochondrial energy metabolism pathways, TCA cycle and OXPHOS. Strong hits with FDR< 0.05 are highlighted in black, and weak hits with FDR < 0.1 are labeled in ’gray50’. Non-hit genes with FDR≥ 0.1 are ’gray1O’.

Article Snippet: To perform a pilot screen as outlined in , we first obtained a medium-scale mouse sgRNA library (CRISPRi_v2 sublibrary m1; Addgene #83989).

Techniques: Functional Assay, Labeling

Journal: bioRxiv

Article Title: A genetic screen in enteroendocrine cells reveals mechanisms that control protein sensing and GLP-1 release

doi: 10.64898/2025.11.30.691441

Figure Lengend Snippet: a-c, Valiation of top hits in mitochondrial respiration pathways by CRISPRi KD. a, Schematic for experimental design. b, Integrated calcium activity in STC-1 stably expressing non-targeting control (NTC) or sgRNA targeting top hit genes. c, Relative GLP-1 secretion in STC-1 after CRISPRi KD. d-f, Validation of the role of mitochondrial respiration in amino acid sensing by pharmacological inhibition of OXPHOS Complex I. d, Schematic for experimental design. e, Integrated calcium activity in STC-1 cells pretreated with vehicle or IACS010759. f, Relative GLP-1 secretion in STC-1 after stimulation, with vehicle or IACS010759. g-i, Stimulating OXPHOS boosts EEC activity and GLP-1 secretion. g, Schematic for experimental design. h, Integrat-ed calcium activity in STC-1 stably expressing NTC or sgRNA targeting Luc7I2, an inhibitor of OXPHOS. i, Relative GLP-1 secretion in STC-1 with the indicated perturbation and stimulation. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: To perform a pilot screen as outlined in , we first obtained a medium-scale mouse sgRNA library (CRISPRi_v2 sublibrary m1; Addgene #83989).

Techniques: Activity Assay, Stable Transfection, Expressing, Control, Biomarker Discovery, Inhibition

Journal: bioRxiv

Article Title: A genetic screen in enteroendocrine cells reveals mechanisms that control protein sensing and GLP-1 release

doi: 10.64898/2025.11.30.691441

Figure Lengend Snippet: a-b, Amino acid metabolism and entry into the TCA cycle is required for EEC sensing. a, Gls is a key enzyme required for glutamine metabolism and its entry into TCA cycle, but not for praline or glutamate. b, Integrated calcium activity in STC-1 stably expressing NTC or sgRNA targeting Gls. c-d, Restoring NADH and redox is not sufficient for amino acid sensing when OXPHOS is inhibited. c, Schematic for experimental design. d, Integrated calcium activity in STC-1 with the indicated treatments and stimulation.Cells were pre-treated with vehicle/lACS and/or pyruvate for 1 h before stimulation. e-h, KATPchannel is dispensable for amino acid sensing in STC-1. e, Schematic of KATPchannel, composed of Kcnj11 and Abcc8. f, Gene expression levels of Abcc8 and Kcnj11 in STC-1 vs. NIH3T3 by RNA-seq. g, Rank plot showed neither Abcc8 nor Kcnj11 is a hit from the custom library screen by tryoptone. h, pharmacological inhibition of KATP channel in STC-1 does not increase baseline calcium activity, but moderatly increase acitivty with strong stimulation (5 mg/ml tryptone or KCI). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: To perform a pilot screen as outlined in , we first obtained a medium-scale mouse sgRNA library (CRISPRi_v2 sublibrary m1; Addgene #83989).

Techniques: Activity Assay, Stable Transfection, Expressing, Gene Expression, RNA Sequencing, Inhibition