Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Macrophages release neuraminidase and cleaved calreticulin for programmed cell removal.

doi: 10.1073/pnas.2426644122

Figure Lengend Snippet: Fig. 1. LPS activation promotes CALR secretion by macrophages. (A) CD14+ monocytes were isolated from healthy donor PBMCs and were differentiated into macrophages with human serum. 10 ng/mL was added on day 6 of differentiation for activation. Macrophages were analyzed on day 7. (B) Flow cytometry analysis of CALR on control (no stim) or macrophages stimulated with LPS overnight. A representative histogram of CALR MFI (Left) and fold change of surface CALR expression (Right) (n = 5 healthy donors). (C) Immunofluorescence staining of macrophage cell surfaces using anti-CALR and anti-PHA-L. (D) ELISA showing levels of CALR secretion in culture media of control or stimulated macrophages (n = 5 healthy donors). (E) mRNA levels of CALR in control or LPS-stimulated macrophages (n = 3 healthy donors). Data are presented as mean ± SEM. Statistical significance was determined using Student’s t test; **P < 0.01.

Article Snippet: The membrane was incubated overnight at 4 °C with primary antibodies against CALR (Cell Signaling D3E6 XP, #12238), NEU1 (Thermo Fisher Scientific, #PA5- 42552), β- Tubulin (Cell Signaling, #2128) diluted in blocking buffer.

Techniques: Activation Assay, Isolation, Flow Cytometry, Control, Expressing, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Macrophages release neuraminidase and cleaved calreticulin for programmed cell removal.

doi: 10.1073/pnas.2426644122

Figure Lengend Snippet: Fig. 2. CALR translocation and secretion are regulated by ER–Golgi trafficking. (A) Flow cytometry analysis of CALR surface MFI. Macrophages were untreated, stimulated with LPS, or treated with both LPS and BFA (n = 5 biological replicates). (B) Immunofluorescence of CALR and PHA-L on the surface of macrophages. (C) ELISA of secreted CALR following treatment with BFA. (n = 5 biological replicates). (D) Immunofluorescence of macrophages showing colocalization of CALR and KDEL receptor at the cell surface. Macrophages were stained with antibodies against the KDEL receptor, the N terminus of CALR, and the C-terminus of CALR. Data are presented as mean ± SEM. Statistical significance was determined using Student’s t test; *P < 0.05, **P < 0.01.

Article Snippet: The membrane was incubated overnight at 4 °C with primary antibodies against CALR (Cell Signaling D3E6 XP, #12238), NEU1 (Thermo Fisher Scientific, #PA5- 42552), β- Tubulin (Cell Signaling, #2128) diluted in blocking buffer.

Techniques: Translocation Assay, Flow Cytometry, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Macrophages release neuraminidase and cleaved calreticulin for programmed cell removal.

doi: 10.1073/pnas.2426644122

Figure Lengend Snippet: Fig. 5. Modification of sialic acids on target cells by macrophage neuraminidase. (A) Immunofluorescence of NEU1 in LPS-activated macrophages. (B) Western blotting of whole cell macrophage lysates for NEU1 (anti-NEU1) and loading control β-tubulin. Each lane represents a biological replicate (n = 3 biological replicates). (C) Amplex red assay for detection of NEU activity in the macrophage cell culture supernatant (n = 5 biological replicates). (D and E) Immunofluorescence and flow cytometry analysis of WGA and PHA-L staining on SW620 cells. SW620 cells were incubated for 8 h with control media or macrophage-conditioned media. Cells were then stained with WGA and PHA-L for (D) imaging or (E) flow cytometry analysis. For flow cytometry, SW620 cells were treated with control media, macrophage-conditioned media, or recombinant NEU enzyme prior to staining (n = 5 technical replicates. (F) Flow cytometry analysis of phagocytosis. SW620s were first treated with recombinant NEU or heat-inactivated NEU (ΔNEU). Cells were then incubated with PBS control or 50 μg recombinant CALR-ΔC. Macrophages were then cocultured with SW620s and phagocytosis was determined by flow cytometry (n = 5 biological replicates). (G and H) LPS-stimulated macrophages were pretreated overnight with the NEU inhibitor C9-BA-DANA. After inhibitor washout, macrophages were cocultured with SW620 tumor cells in a transwell system for 16 h to allow exchange of soluble factors. Macrophages and SW620 cells were then combined for 2 h to allow phagocytosis. (G) Flow cytometry analysis of surface CALR on SW620 cells following coculture (n = 5 biological replicates). (H) Quantification of phagocytosis of SW620 cells by macrophages after NEU1 inhibition (n = 5 biological replicates). Data are presented as mean ± SEM. Statistical significance was determined using Student’s t test; *P < 0.05, **P < 0.01.

Article Snippet: The membrane was incubated overnight at 4 °C with primary antibodies against CALR (Cell Signaling D3E6 XP, #12238), NEU1 (Thermo Fisher Scientific, #PA5- 42552), β- Tubulin (Cell Signaling, #2128) diluted in blocking buffer.

Techniques: Modification, Immunofluorescence, Western Blot, Control, Amplex Red Assay, Activity Assay, Cell Culture, Flow Cytometry, Staining, Incubation, Imaging, Recombinant, Inhibition

Journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

Article Title: Follistatin Alleviates Hepatic Steatosis in NAFLD via the mTOR Dependent Pathway

doi: 10.2147/dmso.s380053

Figure Lengend Snippet: Figure 2 FST expression increased in the FFA-treated human liver (LO2) cells. (A) CCK-8 assay results showed the viability of LO2 cells treated with different concentrations of FFA. (B and C) Oil red O staining and analysis result of LO2 cells with the control (CON) and 1 mM FFA treatment (400×, scale bar: 50 µm). (D and E) The protein expression and analysis results of FST, ACC1, FASN, SREBP1, and ChREBP in LO2 cells with the control and 1 mM FFA treatment. β-actin was used as a loading control. Notes: NC group was LO2 cells without FFA treatment. control (CON) group was LO2 cells treated with BSA. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 vs NC group or CON group. Abbreviations: FST, follistatin; FFA, free fatty acid; BSA, bovine serum albumin (fatty acid free); SREBP1, sterol regulatory element-binding protein1; ChREBP, carbohydrate- responsive element-binding protein; ACC1, acetyl-CoA carboxylase1; FASN, fatty acid synthase.

Article Snippet: Next, the sections were incubated with 5% BSA for 1 h at 37°C and primary antibody against FST (#60060-1-Ig, Proteintech, China) at 4°C overnight and then washed with phosphate-buffered saline three times.

Techniques: Expressing, CCK-8 Assay, Staining, Control, Binding Assay

Journal: Frontiers in Immunology

Article Title: Immunogenic cell death after combined treatment with radiation and ATR inhibitors is dually regulated by apoptotic caspases

doi: 10.3389/fimmu.2023.1138920

Figure Lengend Snippet: Surface-presentation of calreticulin is increased upon irradiation, but is not further increased by the addition of ATR inhibitors. (A) Procedure of Hoechst 33342-based barcoding, in which a live mock sample was stained with permeable Hoechst 33342, before it was divided equally to differently treated live-cell samples. Each of the barcoded samples were thereafter split into two aliquots; one for primary anti-calreticulin (anti-CALR) staining and one for secondary antibody control staining. The two aliquots were thereafter stained with propidium iodide (PI) for discrimination of live and dead cells. (B) The gating hierarchy employed for the flow cytometry analysis. Debris was excluded and cells were gated in a forward-scatter area (FSC-A) versus side-scatter area (SSC-A) plot. Cell singlets were gated in a side-scatter width (SSC-W) versus SSC-A plot. The barcoded populations were separated in a Hoechst-A versus SSC-A plot, in which the Hoechst 33342-stained mock population is shifted upwards the Hoechst-A axis. Live cells were gated in PI-A versus SSC-A plots, in which dead (PI + ) cells are shifted upwards the PI-A axis. Finally, histograms of the ecto-CALR signals (Alexa Fluor 488) are obtained from the live cells in both of the barcoded populations, and the median value of ecto-CALR signal is obtained from each histogram. Similar gating hierarchy and analysis was done for the secondary antibody control samples. (Demonstrated in H1975 cells). (C) Results from experiments performed as in (A, B) for H1975, SW900 and H460 cells after treatment with ionizing radiation and ATR inhibitors. Bar charts show ecto-CALR signals normalized to the barcoded mock population of each sample. Note that results from both a 24 hours time-point (light grey) and 72 hours time-point (dark grey) are shown for H1975, whereas results from the 72 hours time-point are shown for SW900 and H460. * p ≤ 0.05, ** p ≤ 0.01.

Article Snippet: 500 × g , and the cell pellets were resuspended in 100 µl medium (10% FBS) with primary anti-CALR antibodies (Abcam, ab2907, 1:250), or plain medium (10% FBS) for secondary antibody controls, and incubated on ice for 30 minutes.

Techniques: Irradiation, Staining, Flow Cytometry

Journal: Journal of Nanobiotechnology

Article Title: Injectable celastrol-loading emulsion hydrogel for immunotherapy of low-immunogenic cancer

doi: 10.1186/s12951-025-03154-y

Figure Lengend Snippet: Cytotoxicity and inducing immune activation of Cel in vitro. (A) Cell viability of H22-luc cells and Hep1-6 cells examined post 48 h incubation with different concentrations Cel. (B) Flow cytometry and (C) quantitative apoptotic cell analysis of H22-luc cells and Hep1-6 cells after treatment with PBS or Cel. (D) Expression of CRT was detected by flow cytometry and (F , G) corresponding quantification in H22-luc cells and Hep1-6 cells. ( E) PD-L1 expression was detected by flow cytometry and (H , I) corresponding quantification in H22-luc cells and Hep1-6 cells. Statistical significance was calculated via t-test. Data represent means ± S.E.M. * p < 0.05, ** p < 0.01, and *** p < 0.001

Article Snippet: H22-luc cells, Hep1-6 cells, or KPC cells were separately seeded in six-well plates and incubated with PBS or Cel for 48 h. After different treatments, the cells were stained with CRT primary antibody (A00894, Boster, 1:50, diluted in PBS) for 30 min, followed by incubation with donkey anti-rabbit IgG (Clone poly4064, Biolegend, 1:200, diluted in PBS) for 30 min. Then, cells were rinsed and resuspended with 100ul PBS, analyzed using a flow cytometer (BD Fortessa X-20).

Techniques: Activation Assay, In Vitro, Incubation, Flow Cytometry, Cell Analysis, Expressing