Fig 1: Increase in surface expression of DRA and ASMase activity in response to ALI. (A) Cartoon showing the role of ASMase in changing membrane ceramide amount and fusion of lipid rafts aggregates due to the increase in membrane ceramide content. (B) Immunofluorescence detection of ASMase (red) and Flag-DRA (green) in the apical membrane of unpermeabilized, well-differentiated Caco-2/BBe cells grown as submerged cultures or followed by 2-day ALI modifications 12–14 days post-confluency. Co-localization of endogenous ASMase and Flag-DRA detected in cells grown as submerged cultures and enhanced by 2-day ALI modifications. A single plane (XY) at the apical surface of the Z-stack section is shown. Manders’ overlap coefficient showing 90% spatial colocalization of Flag-DRA and ASMase inside enclosed areas. Scale bar: 50 µm, n = 3 repetitions of the experiment were performed. (C) Left: representative immunoblot and densitometric analysis of total and cell surface biotinylation of DRA (above) and CFTR (below) in Caco-2/BBe cells grown as submerged or 2-day ALI modifications after 12–14 days post-confluency. Right: quantitation of surface-to-total ratio normalized to GAPDH and expressed as a percent of control. Results are Mean ± SEM, n = 3 independent experiments. (D) Normalized ASMase activity in 12–14 days post-confluent Caco-2/BBE cells and changes in activity after 2-day ALI modifications. Results are Mean ± SEM, n = 3–8 independent experiments. NS = nonsignificant; p-values represent unpaired Student’s t-test.
Fig 2: Increase in surface DRA expression due to ALI blocked by the ASMase inhibitor, desipramine. The effect of the ASMase inhibitor, desipramine, was determined by the apical plasma membrane localization of DRA in Caco-2/BBe cells grown as a submerged culture for 12 days or followed by ALI 2-day cultures. Flag-DRA was transduced into Caco-2/BBe cells, 24 h before ALI initiation. Transduced cells were exposed to ALI 2-day cultures or were pretreated with 13 µM of desipramine 1 hr before ALI was initiated and during ALI treatment. (A) A representative confocal image after immunostaining for Flag-DRA (red), ASMase (green), and nuclei (white). A single plane of a multi-Z-stack is shown. All panels represent the XY projection near the top of the cell; the right panel includes the XZ projection on the top. Scale bar 20 µm. (B) Normalized ASMase activity in Caco-2/BBe cells expressing Flag-DRA shown for four conditions in A. Pretreating cells with 13 µM of desipramine reduces ASMase activity below the control level, suggesting there is some ASMase activity under basal conditions. Results are Mean ± SEM; n = 3–8 independent experiments. Statistical analysis was performed using ANOVA.
Fig 3: CD161 engagement blocks calcium influx in CTLs through ASM activation and ceramide generation. A, Representative immunofluorescence staining for CD161 and ASM in TIL-derived CD161+ CTLs treated with microbeads loaded with control IgG or CD161 cross-linking mAb (CD161 activation). Scale bar, 5 µm. Right, quantification of colocalization percentage of ASM and CD161 signals. B, Relative intracellular ceramide level of CD161+ CTLs treated with control or CD161 stimulating microbeads. C, Representative fluorescent images for calcium entry of Fura-3 loaded CD161+ CTLs pretreated with control or CD161 cross-linking microbeads upon TCR activation by CD3 stimulating mAb (OKT3). Scale bar, 10 µm. D, Representative tracings for intracellular Ca2+ concentrations in Fura-2–loaded CD161+ CTLs pretreated with control or CD161 cross-linking microbeads and activated by CD3 mAb. E, Representative fluorescent images for calcium influx of CD161+ CTLs edited with different shRNAs upon TCR stimulation. Scale bar, 10 µm. F and G, Tumoricidal effects of TIL-derived CD161+ CTLs edited with different shRNAs (F) or treated with vehicle or imipramine (G) against autologous tumor cells. H, Tumoricidal effects of shASM-1–treated CD161+ CTLs with or without ASM reconstitution. See the quantification of G, H in Supplementary Fig. S5F and S5H. Shown are representative or mean ± SEM of n = 4 (A, B, F–H) or n = 3 (C–E) different patients. ***, P < 0.001 by the Student t test (A, B, G) or two-sided one-way ANOVA with the Tukey test (F, H).
Fig 4: DRA expression and activity were further enhanced by exposing differentiated human colonoids to ALI culture. Human colonoids were grown as confluent epithelial monolayers on permeable inserts. Post-confluency monolayers were exposed to either differentiation medium (DF) for 6 days or to 4 days DF followed by 2-day ALI modifications. (A) Changes in TEER in response to differentiation or ALI modification are shown. Points are Means ± SEM, n = 4. (B) Relative mRNA expression of DRA by quantitative PCR. Messenger RNA levels were normalized to 18S ribosomal RNA expression. The results are normalized to UD set as 1 and expressed as fold change. Results are Mean ± SEM from n = 3 independent experiments. NS=nonsignificant; * p < 0.05 vs. UD control, # p < 0.05 vs. 6-day DF control. p-values were analyzed using two-tailed Student’s t-test with Welch’s correction. (C) A representative Western blot (above) and densitometry analysis Means ± SEM (below) from multiple experiments (n = 3) showing an increase in DRA protein expression in 4-day DF + 2-day ALI compared to 6-day DF condition. p-values represent unpaired Student’s t-test. (D) A representative trace of basal Cl-/HCO3- exchange activity (left) and Means ± SEM of n = 3 independent experiments (right) and (E) basal DRA activity and forskolin (10 µM, 10 min) stimulation comparing 6-day DF and 4-day DF + 2-day ALI conditions, shown as DRA activity and as a percent of control, n = 3. Statistical analysis was performed using ANOVA. (F) Confocal fluorescence microscopy of DRA (red) and nucleus stained with DAPI (blue) expression in colonoids; upper XZ and lower XY project at the level of the apical domain. Scale bar: 10 µm. (G) Normalized ASMase activity in colonoids comparing UD, 6-day DF, and 4-day DF + 2-day ALI. Results are Means ± SEM, n = 3 independent experiments. p-values represent unpaired Student’s t-test.
Fig 5: ALI increased differentiation of human colonoids. Human intestinal colonic organoids (colonoids) derived from biopsies were grown as confluent epithelial monolayers on permeable inserts. Post-confluency monolayers studied in all these experiments and compared: (1) UD; (2) UD + ALI; (3) 5-day DF; (4) 5-day DF + ALI. (A) Changes in TEER in response to differentiation or ALI modifications are shown. Results are Means ± SEM of n = 3–4 experiments. p-value compared with the UD control. (B) Relative mRNA levels of genes used to evaluate proliferation and differentiation, including DRA by qRT-PCR. Messenger RNA levels are normalized to 18S ribosomal RNA expression. Results are normalized to UD set as 1 and expressed as Log2 fold change. Data were analyzed using a two-tailed Student’s t-test with Welch’s correction. * p < 0.05 compared with UD control. Results are Means ± SEM of n = 3–4 independent experiments. Atoh, atonal homolog 1; Lgr5, leucine-rich repeat-containing G protein-coupled receptor; Muc2, mucin 2; ki67, nuclear protein ki67; SPDEF, SAM pointed domain containing ETS transcription factor. (C) Methanol–Carnoy’s fixed colonoid monolayers stained with anti-MUC2 (green) and nucleus stained with DAPI (blue). Representative confocal XZ (above) and 3D-XYZ (below) projections depicting the MUC2 layer in colonoid monolayers are shown. Scale bar 10 µm. (D) Representative Western blot and densitometry analyses from multiple experiments show changes in DRA protein expression. Results are Means ± SEM of n = 3 experiments. (E) Confocal fluorescence microscopy of DRA (red) expression in colonoids. Upper: XZ projection; lower: XY projection at the level of the apical domain. Scale bar 10 µm. (F) Normalized ASMase activity in colonoids. Results are means ± SEM of 3–5 independent experiments. NS = nonsignificant; p-value is compared with the UD control. p-values represent unpaired Student’s t-test.
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