Journal: Frontiers in Immunology

Article Title: GJA5 and ATP1A1 perturbations recapitulate inflammation-related beat irregularities in iPSC-based atrial myocardium tissue model

doi: 10.3389/fimmu.2025.1719392

Figure Lengend Snippet: Fabrication of 3D condensed tissues incorporating M1 macrophages. (A) Schematic of 3D tissue formation protocol, consisting of aCM, cfb, and monocyte preculture for 3 days (d-10), tissue seeding in 96 well plates (d-7) subsequent culture and M1 maturation for 7 days (d-7 to d0) and activation of M1 (d0). (B) Brightfield image of the injection molded pillar device used for seeding tissues. (C) Annotated representative brightfield image of condensed 3D tissue consisting of aCM+cfb. 9 D) Sharp electrode recordings of single cell aCMs in 3D tissues consisting of aCM+cfb (n=17/N=4) compared to 2D monolayer of aCM for (n=31/N=3), demonstrating significant increase in upstroke velocity for 3D tissues (Mann-Whitney test). (E) IF staining for cardiac marker cTnT and cardiac tissue resident macrophage marker CX3CR1 in aCM+cfb+M1 tissue. Zoomed-in image showing area marked by white rectangle, white arrows indicate macrophages. ****P<0.0001.

Article Snippet: Fixed aCM in suspension were co-stained with MLC2a REAfinityTM conjugated with APC (1:10, Miltenyi Biotec) and cTnT REAfinityTM conjugated with FITC (1:10, Miltenyi Biotec) antibodies and incubated for 15 min at RT.

Techniques: Activation Assay, Injection, Single Cell, MANN-WHITNEY, Staining, Marker

Journal: Frontiers in Immunology

Article Title: GJA5 and ATP1A1 perturbations recapitulate inflammation-related beat irregularities in iPSC-based atrial myocardium tissue model

doi: 10.3389/fimmu.2025.1719392

Figure Lengend Snippet: Fabrication of 3D condensed tissues incorporating M1 macrophages. (A) Schematic of 3D tissue formation protocol, consisting of aCM, cfb, and monocyte preculture for 3 days (d-10), tissue seeding in 96 well plates (d-7) subsequent culture and M1 maturation for 7 days (d-7 to d0) and activation of M1 (d0). (B) Brightfield image of the injection molded pillar device used for seeding tissues. (C) Annotated representative brightfield image of condensed 3D tissue consisting of aCM+cfb. 9 D) Sharp electrode recordings of single cell aCMs in 3D tissues consisting of aCM+cfb (n=17/N=4) compared to 2D monolayer of aCM for (n=31/N=3), demonstrating significant increase in upstroke velocity for 3D tissues (Mann-Whitney test). (E) IF staining for cardiac marker cTnT and cardiac tissue resident macrophage marker CX3CR1 in aCM+cfb+M1 tissue. Zoomed-in image showing area marked by white rectangle, white arrows indicate macrophages. ****P<0.0001.

Article Snippet: Tissues were incubated with primary antibodies (cTnT REAfinity conjugated FITC (1:100, Miltenyi Biotec)) and CX3CR1 rabbit anti-human (1H14L7) (1:250, Invitrogen) or Collagen I Polyclonal Antibody (1:100, ThermoFisher Scientific) overnight at 4 °C on a plate shaker.

Techniques: Activation Assay, Injection, Single Cell, MANN-WHITNEY, Staining, Marker

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: Infiltration of CD68-positive activated microglia/macrophages co-expressing PSAP and PGRN into the SFO and surrounding tissues a – b) Double immunofluorescent staining of PGRN (green) and CD68 (red) around the SFO in 10-month-old-female WT and SAP-D −/− mice. b , Magnified images of the indicated white squares in a . ⅰ: SFO, ⅱ: Fornix, and ⅲ: Perivascular, bv: blood vessel. c ) Quantification of PGRN- and/or CD68-staining in the SFO and surrounding areas in WT and SAP-D −/− mice. Data are shown as the mean ± SD (n = 3). The left panel presents a stacked bar chart, whereas the right panel shows the individual data values in a bar chart format. d – h ) Triple immunofluorescent staining of PSAP (red), PGRN (green), and CD68 (cyan) around the SFO in 10-month-old-female SAP-D −/− mice. e) Magnified images of the indicated white squares in d) ⅳ: Boundary, ⅴ: Fornix, and ⅵ: Perivascular. White arrowheads indicate triple co-staining with PSAP, PGRN, and CD68. Open arrowheads indicate PGRN signals alone. Co-localization rates of CD68 positive areas in PSAP ( f ), PGRN ( g ), and PSAP-PGRN staining areas ( h ) around the SFO of SAP-D −/− mice, respectively. f-h ) The left panel presents a stacked bar chart, whereas the right panel presents the individual data values in a bar chart format. Data are shown as mean ± SD (n = 3). Nuclei are labeled by DAPI (blue) staining. All scale bars, 50 μm.

Article Snippet: The primary antibodies used were a rabbit antibody against PSAP (dilution 1:100, 10801-1-AP, Proteintech Group Inc., IL, USA), a sheep antibody against PGRN (dilution 1:100, AF 2557, R&D Systems Inc., MN, USA), a guinea pig antibody against c-Fos (dilution 1:500, 226308, Synaptic Systems GmbH, Göttingen, Germany), a rat antibody against CD68-FITC conjugated (dilution 1:500, MCA1957FA, BIO-RAD Laboratories Inc., CA, USA), and a rat antibody against LAMP1 (dilution 1:100, ab25245, Abcam, Cambridge, UK).

Techniques: Expressing, Staining, Labeling

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: c-Fos expression was induced in the SFO of SAP-D −/− mice without dehydration. a) Expression levels of c-Fos , Gpr37 , and Cd68 in the SFO and fornix were quantified via RT-qPCR. For RNA extraction, tissue samples were microdissected from the brains of 6-month-old female WT mice (n = 10) and SAP-D −/− mice (n = 6) under a stereomicroscope. After cDNA synthesis, RT-qPCR was performed, and the data were normalized to Gapdh expression. A significant increase in Cd68 expression was observed in SAP-D −/− mice, consistent with immunostaining results, confirming accurate sampling of the SFO and surrounding fornix. The results of Student's t-tests for each panel are as follows: left panel, p = 0.0046, Cohen's d = 1.74 (95 % CI: 0.0027, 0.0122); center panel, p = 0.0352, Cohen's d = 1.20 (95 % CI: 0.00091, 0.02191); and right panel, p = 0.0007, Cohen's d = 2.41 (95 % CI: 0.0079, 0.0233). b) Immunofluorescent staining of c-Fos (green) in the SFO of 10-month-old female WT and SAP-D −/− mice. WT and SAP-D −/− mice had free access to drinking water (indicated as FD) or 24 h water deprivation (indicated as DH). The white dotted lines enclose the SFO. Nuclei are labeled by DAPI (blue) staining. Scale bars, 50 μm. c) Percentage of c-Fos positive cells among all DAPI stained cells in the SFO (%). Data are shown as the mean ± SD (n = 7). , , , and indicate the individual values in each group. Two-way ANOVA showed significant main effects of water deprivation, F(1,24) = 35.13, p < 0.0001, ηp 2 = 0.13, 95 % CI (−8.67, −4.19), and genotype, F(1,24) = 130.40, p < 0.0001, ηp 2 = 0.36, 95 % CI (−14.64, −10.16), as well as a significant water deprivation × genotype interaction, F(1,24) = 38.60, p < 0.0001, ηp 2 = 0.14, 95 % CI (−17.98, −9.01). Post-hoc Tukey's tests showed significant differences between WT-FD versus WT-DH ( p < 0.0001, Cohen's d = 5.83 [95 % CI: −17.42, −8.94]), WT-FD versus SAP-D −/− -FD ( p < 0.0001, Cohen's d = 6.91 [95 % CI: −23.38, −14.91]), and WT-DH versus SAP-D −/− -DH ( p = 0.006, Cohen's d = 1.90 [95 % CI: −9.89, −1.41]), but no significant difference between SAP-D −/− -FD versus SAP-D −/− -DH ( p = 0.997, Cohen's d = 0.09 [95 % CI: −3.92, 4.54]). ns: no significant difference. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: The primary antibodies used were a rabbit antibody against PSAP (dilution 1:100, 10801-1-AP, Proteintech Group Inc., IL, USA), a sheep antibody against PGRN (dilution 1:100, AF 2557, R&D Systems Inc., MN, USA), a guinea pig antibody against c-Fos (dilution 1:500, 226308, Synaptic Systems GmbH, Göttingen, Germany), a rat antibody against CD68-FITC conjugated (dilution 1:500, MCA1957FA, BIO-RAD Laboratories Inc., CA, USA), and a rat antibody against LAMP1 (dilution 1:100, ab25245, Abcam, Cambridge, UK).

Techniques: Expressing, Quantitative RT-PCR, RNA Extraction, cDNA Synthesis, Immunostaining, Sampling, Staining, Labeling