Journal: Function

Article Title: Calcium Signaling in Pancreatic Immune Cells In situ

doi: 10.1093/function/zqaa026

Figure Lengend Snippet: Immunostaining after Recording ATP-elicited Ca 2+ Signals in pancreatic macrophages (PMs). (A). Representative images of pancreatic lobule loaded with Fluo-4AM before (Ai) and after ATP (10 µM) application (Aii), the arrow indicates the position of the ( n = 8). A corresponding Fluo 4 trace from a PM is shown in Aiii . Corresponding immunostaining of this lobule with antibodies F4/80 Alexa Fluo 647 is shown below ( Aiv) . Hoechst 33342 staining of the same area is shown in Av . Arrow points to ear-like shape of PM nucleus. Overlay of antibody and Hoechst 33342 staining is shown in Avi . Scale bar is 10µm. (B). Immunostaining of another area in a pancreatic lobule with monoclonal F4/80 antibodies labeled with Alexa Fluor 647 (Bi) . Staining of nuclei in the same lobule with Hoechst 33342 (Bii) . Overlay of B i with Bii is shown in Biii . Scale bar is 10µm. (C). Representative images of a pancreatic lobule loaded with Fluo-4AM before (Ci) and after ATP (10 µM) application (Cii) , the arrow indicates the position of the PM. Corresponding Fluo 4 trace is shown in Ciii. Immunostaining of the same area with monoclonal CD11b antibody conjugated with Alexa Fluor 647 ( n = 8) is shown in Civ . Overlay of Cii and Civ is shown in Cv . Scale bar is 10µm.

Article Snippet: Mouse F4/80 monoclonal rat Antibody (CI-A3-1) [Alexa Fluor ® 647] and mouse CD11b/Integrin alpha M Alexa Fluor ® 647-conjugated monoclonal rat antibodies were obtained from Novus Biologicals Europe and R&D Systems Bio-techne, respectively.

Techniques: Immunostaining, Staining, Labeling

Journal: Function

Article Title: Calcium Signaling in Pancreatic Immune Cells In situ

doi: 10.1093/function/zqaa026

Figure Lengend Snippet: IgG-elicited Ca 2+ Spikes in PMs . (A). Single short Ca 2+ spike occurring after application of IgG (0.1–0.25 mg/mL) in a PM from a control pancreatic lobule. This was an infrequent observation (5 out of 29 cells tested) and is most likely not an IgG-elicited Ca 2+ signal as such single spikes have been also observed in 3 out of 15 cells in the absence of IgG stimulation. (B) . Representative trace of IgG (0.1–0.25 mg/mL)-induced Ca 2+ signals in PMs in pancreatic lobules isolated from mice with AP (FAEE-AP model—48 h). Such oscillations were observed in 9 out of 31 cells. Single short spikes have been observed in 4 out of 31 cells. No oscillations were observed in the absence of stimulation with IgG ( n = 14), while single short spikes have been observed in 2 out of 14 cells. (C). Average Ca 2+ spike frequencies in PMs displaying Ca 2+ signals under the conditions indicated. The frequencies in control PMs, both stimulated with IgG (blue bar) and unstimulated (green), as well as in unstimulated PMs from the FAEE-AP model (48 h, orange bar) were much lower than in PMs from the FAEE-AP model stimulated with IgG (red bar, P < 0.007). (D) . Average Ca 2+ spike duration in PMs displaying Ca 2+ signals under the conditions indicated. Although the average spike duration was longer in the PMs from the FAEE-AP mice stimulated with IgG than under the other conditions, the difference was not statistically different ( P > 0.2). (E). Representative images of immunostaining of PMs in lobules using antibodies F4/80 conjugated with Alexa Fluor 647. Lobules were isolated from control and FAEE-AP 3-day mice (72 h in vivo FAEE-AP model). Scale bar is 20µm. (F). Comparison of the average density of PMs in lobules from control and FAEE-AP 2-day and 3-day mice (48 h and 72 h in vivo FAEE-AP model, respectively). Control, 2.36 ± 0.6 SEM, n = 14; FAEE-AP 2 day, 9.56 ± 1.86 SEM, * P < 0.033, n = 16; FAEE-AP 3 days, 15.37 ± 1.51 SEM, * P < 0.038 as compared to FAEE-AP 2-day, n = 35. The difference between control and FAEE-AP 3-day was very highly significant (**** P < 0.0001).

Article Snippet: Mouse F4/80 monoclonal rat Antibody (CI-A3-1) [Alexa Fluor ® 647] and mouse CD11b/Integrin alpha M Alexa Fluor ® 647-conjugated monoclonal rat antibodies were obtained from Novus Biologicals Europe and R&D Systems Bio-techne, respectively.

Techniques: Control, Isolation, Immunostaining, In Vivo, Comparison

Journal: Molecular Cancer Research

Article Title: β8 Integrin Mediates Pancreatic Cancer Cell Radiochemoresistance

doi: 10.1158/1541-7786.mcr-18-1352

Figure Lengend Snippet: Figure 5. b8 integrin interactome and subcellular colocalization. A, Sequential immunoprecipitation-mass spectrometry–based analysis of b8 integrin interactome was performed in unirradiated and 6-Gy X-rays irradiated Patu8902 cells. Time point of analysis after irradiation was 2 hours. B, Comparative changes in the b8 integrin interactome between control and irradiation upon categorization into different molecular functions using the PANTHER classification system. C, Immunofluorescence costaining of b8 integrin with GM130 (Golgi), mitochondria, aV integrin, APPL2, and Caveolin 1. Scale bars, 10 mm. D, Pearson correlation analysis of C. Data show mean SD (n ¼ 3).

Article Snippet: The antibodies against b1 integrin (WB 1:1,000, Abcam, ab179471), b8 integrin (IF 1:200, WB 1:1,000; Abcam, ab80673, Rb, recognizes aa 614-663 (this antibody was generally used in this study); Abnova, H00003696-M01, Mo, recognizes aa 392–503 (this antibody was used only where specifically indicated), GM130 (IF 1:100, WB 1:1,000, BD, 610822), MEK1/2 (WB 1:1,000, Cell Signaling Technology, 4694s), gH2AX (WB 1:1,000, Cell Signaling Technology, 9718s), aV integrin (IF 1:250, Novus, NB100-2618), APPL2 (IF 1:100, Sigma-Aldrich, SAB1400605), Caveolin 1 (IF 1:100, BD, 610407), LC3B (IF 1:500, Sigma-Aldrich, SAB4200361), b-actin (WB 1:10,000, Sigma-Aldrich, A5441), HRP-conjugated secondary antibody (GE, Rb NXA931, Mo NXA931), Alexa Fluor 488– or Alexa Fluor 594–conjugated secondary antibodies (1:500, Life Technologies) were purchased and used as indicated.

Techniques: Immunoprecipitation, Mass Spectrometry, Irradiation, Control