Journal: Research

Article Title: Novel Combination of Irreversible Electroporation and Allogenic Chimeric Antigen Receptor T-Cell Therapy Synergizes Therapeutic Outcomes in a Preclinical Human Pancreatic Cancer Mouse Model

doi: 10.34133/research.1105

Figure Lengend Snippet: Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression in AsPC-1 cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.

Article Snippet: Cell suspensions were centrifuged at 500 × g, with the liquid aspirated and resuspended in 500 μl of flow cytometry buffer (Thermo Fisher, 00-4222-26).

Techniques: Binding Assay, Electroporation, Membrane, Flow Cytometry, Control, Expressing, Imaging, Cell Characterization

Journal: Inflammation Research

Article Title: Genetic depletion of the early autophagy protein ATG13 impairs mitochondrial energy metabolism, augments oxidative stress, induces the polarization of macrophages to the M1 inflammatory mode, and compromises myelin integrity in skeletal muscle

doi: 10.1007/s00011-025-02158-6

Figure Lengend Snippet: Autophagy impairment in the splenic Mφs of Tg +/-ATG13 mice. A Dual IF analysis of LC3 (rabbit anti-LC3; Cat#; ProteinTech; dilution 1:100) and CD11b (mouse anti-CD11b; Cat#; Invitrogen; dilution 1:100) in 5 μm thick paraffin-embedded sections of 10–12-week-old nontransgenic and hemizygous atg13 knockout mice ( n = 6/group). ( Inset ) Magnified dual IF images of (a) NTg and (b) Tg +/-ATG13 . Dual flow cytometry of PE-labeled LC3 and FITC-labeled CD11b in purified Mφs isolated from 10- to 12-week-old B NTg and C Tg +/- ATG13 mice (male). The total number of gated events was 20,000/group. The cells under the enclosed area represent a distinct population of CD11b-ir cells, which are also LC3-positive. D Histogram analyses to quantify the CD11b + LC3 + cells among the NTg and Tg +/-ATG13 Mφs. E Immunoblot analyses of LC3II revealed two distinct bands corresponding to autophagy-active LC3BII (lower) and autophagy-inactive LC3BI (upper). IB analyses of WDFY3 (~ 100 kD) and p62 (> 60 kD) were also performed in splenic Mφs of NTg and Tg +/-ATG13 mice. Beta-actin immunoblotting was performed as a loading control. F The relative densitometric analysis results were plotted after normalization to the respective β-actin bands. The white and gray bars represent the LC3B band densities of the NTg and Tg +/-ATG13 mice, respectively. (An unpaired t test was performed to test the significance of the difference in the means between groups; * p < 0.05 vs. the LC3BI of Tg +/-ATG13 . NS = not significant. G DAB immunostaining of splenic sections of WDFY3 (rabbit; cat# Invitrogen, dilution 1:100) ( n = 5/group) counterstained with hematoxylin. (inset) Enclosed zones are magnified (a = NTg, b = Tg). H WDFY3 + cells were counted in 5 different images from 5 mice/group following the quantification per sq.mm. of sections (unpaired t test; *** p < 0.005 = 0.002844221). I & J Representative flow cytometry analyses of WDFY3 (PE-tagged) and CD11b (FITC-tagged) cells (10000 gated events). K Histogram of the number of gated cells in the circled population. L The scatter bar graph shows the counts of CD11b + WDFY3 + cells in six analyses per group (unpaired t test shows **** p < 0.0005). The results are presented as the mean ± SD of three experiments

Article Snippet: Briefly, cells were suspended in eBioscienceTM Flow Cytometry Staining Buffer (Ref# 00-4222-57; ThermoFisher, Inc.), stained with FITC-, PE-, or APC-conjugated antibodies for 30 min at room temperature, washed (3X), and then subjected to analysis via a flow cytometer.

Techniques: Knock-Out, Flow Cytometry, Labeling, Purification, Isolation, Western Blot, Control, Immunostaining

Journal: Inflammation Research

Article Title: Genetic depletion of the early autophagy protein ATG13 impairs mitochondrial energy metabolism, augments oxidative stress, induces the polarization of macrophages to the M1 inflammatory mode, and compromises myelin integrity in skeletal muscle

doi: 10.1007/s00011-025-02158-6

Figure Lengend Snippet: Polarization of M1Mφ cells in the spleens of Tg+/-ATG13 mice. A Dual IF analysis of CD40 (rabbit anti-CD40; Cat#; ProteinTech; dilution 1:100) and IBA1 (mouse anti-IBA1; Cat#; Invitrogen; dilution 1:100) in 5 μm thick paraffin-embedded spleen sections from 10- to 12-week-old male NTg and Tg+/-ATG13 mice (n=6/group). B Quantification of CD40-ir cells in the 50 μm radius of blood vessels in the red pulp zone was performed. An unpaired t test was performed to verify the significance of the difference in the means between groups, and the results are shown as ****p<0.0005. The results were confirmed after counting 7 independent images per group. C IB followed by D β-actin-normalized densitometric analyses; ***p<0.005 (unpaired t test) between groups. Dual flow cytometry of APC-labeled CD40 and FITC-labeled CD11b in purified Mφs isolated from 10- to 12-week-old E NTg and F Tg+/- ATG13 mice (n=6 male). G The quantification analysis of CD11b+ CD40+ population was performed followed by measuring the significance by unpaired t-test (***p<0.005) . The total number of gated events was 20,000/group. H & I Similarly, dual flow cytometry analysis of CD86 (PE-tagged; dilution 1:100) and CD11b (FITC-tagged; dilution 1:100) followed by J quantification (n=7 analysis/group) was performed (****p<0.0005; unpaired t test). K & L Dual flow cytometry analysis of CD163 (APC-tagged; dilution 1:100) and CD11b (FITC-tagged; dilution 1:100) followed by M quantification (n=7 analysis; ****p<0.0005 by unpaired t test) was performed on purified Mφ cells. The results are presented as the mean ± SD of three experiments

Article Snippet: Briefly, cells were suspended in eBioscienceTM Flow Cytometry Staining Buffer (Ref# 00-4222-57; ThermoFisher, Inc.), stained with FITC-, PE-, or APC-conjugated antibodies for 30 min at room temperature, washed (3X), and then subjected to analysis via a flow cytometer.

Techniques: Flow Cytometry, Labeling, Purification, Isolation

Journal: Inflammation Research

Article Title: Genetic depletion of the early autophagy protein ATG13 impairs mitochondrial energy metabolism, augments oxidative stress, induces the polarization of macrophages to the M1 inflammatory mode, and compromises myelin integrity in skeletal muscle

doi: 10.1007/s00011-025-02158-6

Figure Lengend Snippet: Functional characterization of the M2Mφ phenotype in splenic Mφ cells from Tg +/−ATG13 mice. A Dual IF analysis of the M2Mφ functional marker arginase-1 (APC-tagged) and the M2Mφ surface marker CD163 (FITC-tagged). The enclosed subsets show CD163 + arginase 1 + cells among the purified splenic Mφs. Total gated events = 10,000. B Scatter histogram displaying the number of CD163 + Arginase 1 + cells in n = 6 analyses/group. An unpaired t test was used to determine the significance of the difference in the means between groups, *** p < 0.005. C Paraffin-embedded splenic tissue sections of NTg and Tg +/- ATG13 were immunostained with IBA1 (green) and Arginase-1(red). Nuclei were stained with DAPI. D Phagocytosis assay of live Mφ cells by immunocytochemistry with pHrodo™ green conjugated BioParticles™ (Cat#P35381; ThermoFisher; 10 6 cells in 10,000 Mφ cells). E Quantification of phagocytic cells in n = 6 images/group followed by an unpaired t test to verify the significance of the difference; *** p < 0.005. F Dual flow cytometry of CD206 (APC-tagged) and Bioparticles™ (pHrodogreen-tagged) followed by G quantification of Bioparticles™ (%)-conjugated CD206+ cells ( n = 6 analyses). An unpaired t test was used to verify the significance of the mean, and the results are indicated by * p < 0.05. Total gated events = 20,000. H Dual flow cytometry of CD163 (APC-tagged) and LysoTracker™ (red-DND99-tagged detected in PE filter; ThermoFisher; Cat# pHrodogreen-tagged) followed by I quantification of LysoTracker™ (%)-conjugated CD163 + ve cells ( n = 6 analyses). An unpaired t test was used to verify the significance of the mean, and the results are indicated by * p < 0.05. Total gate = 50,000. P62 IF analysis in J NTg and K Tg Mφs treated with 100 nM Bafilomycin A1 for 2 h under control and serum-starved condition (24 h). L Numbers and M size for p62-ir puncta were measured in 12 independent cells per group by ImageJ software, displayed in histogram analyses. The significance of mean was calculated by Kruskal-Wallis test, resulting in * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001. The results are presented as the mean ± SD of three experiments

Article Snippet: Briefly, cells were suspended in eBioscienceTM Flow Cytometry Staining Buffer (Ref# 00-4222-57; ThermoFisher, Inc.), stained with FITC-, PE-, or APC-conjugated antibodies for 30 min at room temperature, washed (3X), and then subjected to analysis via a flow cytometer.

Techniques: Functional Assay, Marker, Purification, Staining, Phagocytosis Assay, Immunocytochemistry, Flow Cytometry, Control, Software