Fig 2: Mouse endothelial cells poorly support bridging molecule-mediated endocytosis.(A and B) Endocytosis of C. glabrata coated with either human or mouse serum by the indicated endothelial cells after 45 min (A) and 180 min (B). (C) Endocytosis of C. glabrata coated with fresh human serum by mouse liver endothelial cells expressing human gC1qR, integrin αv, or integrin β5. Data are the mean ± SD of 3 experiments each performed in triplicate. HUVEC, human umbilical vein endothelial cell; orgs/HPF, organisms per high power field; ns, not significant; **P < 0.01, ****P < 0.0001. ***P < 0.001, ****P < 0.0001 by ANOVA with the Dunnett’s test for multiple comparisons.

Fig 3: P32-T130M reduces OXPHOS activity and maintains efficient differentiation in HT29-MTX cells. (A) HT29-MTX cells derive from HT29 cells following differentiation induced by methotrexate (MTX). (B) Representative image of HT29-MTX cell growth characteristics displaying mucous vacuoles. (C) Protein level of p32 was compared between HT29 and HT29-MTX cells by Western blot experiments using primary antibodies against p32 (clone EPR8871) or β-actin. (D) Amount of p32 was quantified densitometrically using Western Blots of HT29 and HT29-MTX cell lysates. (E) Western blot experiment with whole protein extracts of transient HT29-MTX + p32-wt, HT29-MTX + p32-T130M and HT29-MTX + mock transfectants was performed using the anti-p32 antibody clone EPR8871. (F) Cell count of transient HT29-MTX transfectants was determined after 72 h of incubation and normalized to mock transfected HT29-MTX cells. (G) Time-dependent measurement of oxygen consumption of transient HT29-MTX transfectants. (H) The AUC was calculated for each single experiment and each cell line. (I) L-lactate production was measured in cell culture supernatants from transient HT29-MTX transfectants after 72 h of incubation and normalized to the cell count. Mean ± SD of HT29-MTX + mock transfectants is depicted by dotted gray lines. (J) Western blot experiment with whole protein extracts of transient HT29-MTX transfectants was performed using primary antibodies against phospho-AMPKα, AMPKα or β-actin. (K) Schematic representation of energy metabolism of HT29-MTX cells overexpressing p32-wt or p32-T130M using data displayed in (H, I). Transfectants from the same experiment are connected by a dashed line. (L) Western blot analyses were performed with whole protein lysates of transient HT29-MTX transfectants using primary antibodies against KLF4 or β-actin. (M) For relative quantification bands were analyzed densitometrically using ImageJ and the amount of KLF4 was normalized to the amount of β-actin. (N, O) Expression of (N) ATOH1 and (O) SPDEF1 mRNA was quantified in transient HT29-MTX transfectants by qPCR. (P) Schematic model for goblet cell differentiation in the colonic crypt. Statistical analysis of significance for (D, N, I) was performed using an unpaired or paired t-test, respectively. For (F, H, M, O) statistical significance was determined using a one-way ANOVA followed by Tukey’s multiple comparison test. *p ≤ 0.05, ***p ≤ 0.001.

Fig 4: Caspase-1 activation enhances glycolysis and impairs cell differentiation. (A) Schematic model of PMA induced differentiation of THP-1 monocytes into macrophages. This figure was generated using pictures provided by the Servier Medical Art homepage https://smart.servier.com/. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License. (B) QPCR analyses were performed to quantify mRNA expression level of C1qbp, Fis1, Slc2a1, Ldha, and Ki67. (C) Western blot analysis of whole protein extracts from PMA-induced differentiated THP-1 macrophages using gC1qR-directed antibodies specific for epitopes located in exon 1, 3, or 6. (D) Secretion of IL-1β (left panel), gC1qR (middle panel) or lactate (right panel) was determined using specific ELISA using supernatants from untreated or LPS stimulated PMA-differentiated THP-1 cells in the presence or absence of caspase-1 inhibitor (10 μg/ml; Ac-YVAD-cmk from InvivoGen). (E) GC1qR protein expression in mitochondrial/ cell membrane protein fractions was quantified by Western blot experiments. PMA-differentiated THP-1 cells were stimulated with LPS in the presence or absence of caspase-1 inhibitor (10 μg/ml; Ac-YVAD-cmk) or were left untreated. Densitometry was performed using the software ImageJ (right panel). (F) Schematic model of butyrate induced goblet cell differentiation of HT29-MTX cells (left panel). Oxygen consumption rate (OCR) as well as extracellular acidification rate (ECAR) of HT29-MTX cells stimulated with 1.25 mM butyrate for 24 h or left untreated were determined using the Seahorse XF Cell Mito Stress Test (right panel). (G) HT29-MTX cells were incubated in the absence or presence of 1.25 mM butyrate for 72 h. After incubation, cells were counted. (H) HT29-MTX cells were transiently transfected for 96 h with plasmids encoding full-length human caspase-1 (C), human ASC (A), human NLRP3 (N) or with a mock plasmid in the presence or absence of 1.25 mM butyrate. Whole protein extracts were separated by SDS-PAGE under reducing conditions and Western blot experiments were performed using indicated primary antibodies. Results are presented as mean ± SEM from at least three independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Fig 5: P32-T130M does not affect proliferation of HAP1 cells and growth of HAP1 cell-derived spheroids. (A) HAP1 transfectants were incubated for 72 h and cell viability was determined by a neutral red assay, measuring the optical density (OD) at 540 nm. (B) Graphical setup of HAP1 transfectants grown as spheroids in hanging drops. (C) Representative pictures of HAP1-p32-wt, HAP1-p32-T130M and HAP1-mock spheroids after 8 days of incubation (brightfield, 2.5× magnification). (D) Mean area of the HAP1 spheroids was determined using ImageJ. The three independent experiments comprise 10 to 12 spheroids each. For (A) and (D) statistical significance was determined using a one-way ANOVA followed by Tukey’s multiple comparison test. **p ≤ 0.01, ***p ≤ 0.001.