Journal: microPublication Biology

Article Title: Quantification of calreticulin redistribution in LNCaP prostate cancer cells using a fixed-cell imaging workflow

doi: 10.17912/micropub.biology.002112

Figure Lengend Snippet:

Article Snippet: ERp57 antibody , Proteintech , 15967-1-AP.

Techniques: Plasmid Preparation, Cell-Signaling

Journal: microPublication Biology

Article Title: Quantification of calreticulin redistribution in LNCaP prostate cancer cells using a fixed-cell imaging workflow

doi: 10.17912/micropub.biology.002112

Figure Lengend Snippet: (A) The Redistribution Factor (RF), defined as ectoCRT/(ectoCRT + endoCRT) quantifies the fraction of CRT localized to the cell surface, normalizing for the large intracellular pool of calreticulin. Surface GFP-tagged CRT analysis included approximately 23 cells per condition across three independent experiments, while intracellular GFP-tagged CRT analysis included approximately 99 cells per condition across three independent experiments. Each data point represents an individual cell. Fluorescence intensity values were obtained using automated image segmentation under identical imaging conditions. (B) Comparison of treatment-induced redistribution factor (RF) between endogenous CRT and GFP-CRT workflows. Both approaches demonstrated similar treatment-dependent increases in RF, with parallel trends across TG and TSA conditions. The close concordance between endogenous and GFP-tagged CRT supports that the GFP reporter successfully recapitulates treatment-induced CRT redistribution, validating the GFP-CRT imaging workflow for quantitative analysis. Endogenous surface calreticulin analysis included approximately 30 cells per condition across 3 independent experiments, while endogenous intracellular CRT analysis included approximately 50 cells per condition across three independent experiments. (C) Quantification of endogenous surface-exposed calreticulin (ectoCRT) under non-permeabilized conditions. Parental LNCaP cells were treated with vehicle (0.1% DMSO), thapsigargin (TG), or trichostatin A (TSA). Cells were incubated with anti-CRT antibody before fixation to detect surface-accessible endogenous CRT. Mean fluorescence intensity (arbitrary units, a.u.) increased following TG and TSA treatment, indicating enhanced surface exposure of endogenous CRT. Statistical comparisons between groups are indicated (P values calculated using unpaired T-Test). Surface and intracellular fluorescence were quantified by automated image segmentation under identical image acquisition and analysis parameters for all treatment groups. Endogenous surface calreticulin analysis included approximately 30 cells per condition across 3 independent experiments. (D) Quantification of surface-exposed CRT in LNCaP cells expressing GFP-tagged wild-type calreticulin (pMSCV-IRES-GFP/CALR wt). Under identical treatment conditions, TG and TSA increased cell-surface CRT fluorescence compared with vehicle-treated controls. Mean fluorescence intensity (arbitrary units, a.u.) increased following TG and TSA treatment, indicating enhanced surface exposure of GFP-tagged CRT. Statistical comparisons between groups are indicated (P values calculated using unpaired T-Test). Surface GFP-tagged CRT analysis included approximately 23 cells per condition across three independent experiments. (E) Quantification of endogenous intracellular calreticulin following permeabilization. Cells were permeabilized with 1% Triton X-100 prior to immunostaining to measure intracellular CRT. Mean fluorescence intensity decreased following TG and TSA treatment relative to vehicle-treated cells, consistent with redistribution of CRT from intracellular compartments to the plasma membrane. Statistical comparisons between groups are indicated (P values calculated using unpaired T-Test). Endogenous intracellular CRT analysis included approximately 50 cells per condition across three independent experiments. (F) Quantification of intracellular GFP-CRT following permeabilization. Intracellular fluorescence decreased after TG and TSA treatment, mirroring the redistribution observed for endogenous CRT. Intracellular GFP-tagged CRT analysis included approximately 99 cells per condition across three independent experiments. (G) Representative immunofluorescence images of endogenous CRT. The left panels show surface CRT staining, while lower panels show intracellular CRT detected under permeabilized conditions. Images illustrate reduced intracellular CRT and increased surface CRT following TG and TSA treatment. Scale bars = 10 μm. (H) Comparison of redistribution factor (RF) between an exposure time of 250ms and 1000ms. Both exposures showed strong agreement across a four-fold exposure range indicating the RF is largely independent of exposure settings in this large range, supporting its robustness across acquisition settings. (I) Schematic illustrating calreticulin redistribution during immunogenic cell death. Under baseline conditions CRT is predominantly retained within the endoplasmic reticulum. ER stress induces CRT translocation to the plasma membrane while a fraction remains intracellular. The redistribution factor (RF) provides quantitative value for the proportion of total detectable CRT localized to the cell surface.

Article Snippet: ERp57 antibody , Proteintech , 15967-1-AP.

Techniques: Biomarker Discovery, Analysis, Fluorescence, Automated, Imaging, Comparison, Incubation, Expressing, Immunostaining, Clinical Proteomics, Membrane, Immunofluorescence, Staining, Translocation Assay

Journal: Oncology Letters

Article Title: Thrombospondin-1 triggers calreticulin expression in human mucoepidermoid carcinoma MC-3 cells via the PERK/CHOP pathway

doi: 10.3892/ol.2026.15589

Figure Lengend Snippet: TSP-1 promotes the translocation of CRT to the cell surface in MC-3 cells. Cells were stained with an APC-conjugated anti-CRT antibody, and CRT expression on the cell membrane is presented as mean fluorescence intensity. At the 72 h point: **P<0.01 vs. the control group; # P<0.05 vs. the TSP-1 group; and ▲ P<0.05 vs. the TSP-1 + ISRIB group. MFI, mean fluorescence intensity; CRT, calreticulin; TSP-1, thrombospondin-1.

Article Snippet: Primary antibodies, including anti-PERK (1:1,000; cat. no. MA8131; Abmart Pharmaceutical Technology Co., Ltd.), anti-CHOP (1:1,000; cat. no. WL00880; Wanleibio Co., Ltd.), anti-CRT (1:800; cat. no. bs-5913R; BIOSS) and anti-β-actin (1:5,000; cat. no. 66009-1-Ig; Proteintech Group Inc.), were diluted in 5% BSA and incubated with the membranes overnight at 4°C.

Techniques: Translocation Assay, Staining, Expressing, Membrane, Fluorescence, Control

Journal: Oncology Letters

Article Title: Thrombospondin-1 triggers calreticulin expression in human mucoepidermoid carcinoma MC-3 cells via the PERK/CHOP pathway

doi: 10.3892/ol.2026.15589

Figure Lengend Snippet: TSP-1 effects on cell surface CRT expression at 4 h. Cells were stained with an APC-conjugated anti-CRT antibody, and CRT expression on the cell membrane is presented as mean fluorescence intensity. At the 4 h time point. *P<0.05 compared with control group; # P<0.05 compared with TSP-1 action group; and ▲ P<0.05 compared with TSP-1 + ISRIB group. TSP-1, thrombospondin-1; CRT, calreticulin.

Article Snippet: Primary antibodies, including anti-PERK (1:1,000; cat. no. MA8131; Abmart Pharmaceutical Technology Co., Ltd.), anti-CHOP (1:1,000; cat. no. WL00880; Wanleibio Co., Ltd.), anti-CRT (1:800; cat. no. bs-5913R; BIOSS) and anti-β-actin (1:5,000; cat. no. 66009-1-Ig; Proteintech Group Inc.), were diluted in 5% BSA and incubated with the membranes overnight at 4°C.

Techniques: Expressing, Staining, Membrane, Fluorescence, Control