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Additional file 5. Raw-data-Western blotting.

Additional file 4: Figure S4. Anterograde transport of secretory cargo proteins is not significantly altered in ACBD3-depleted cells. In order to investigate whether ACBD3 depletion might have affected anterograde transport between the ER and the Golgi, secretion of three different cargo proteins was tested, including TfR-RM4-SNAP (A), VSVG-tsO45-GFP (B), endogenous MMP-2 (C) and YFP-GL-GPI (D). (A-B) Briefly, plasmids encoding the indicated constructs were transiently transfected into control cells or ACBD3-depleted HeLa cells for 18 hours. Cells were then treated with cycloheximide for 2 hours, prior to induction of synchronized protein secretion by shifting temperature from 40.5 to 32 °C. (VSVG-tsO45-GFP) or treatment with D/D solubilizer drug (TfR-FM4-SNAP) for the indicated times. At the indicated timepoints, the cells were placed on ice and subjected to surface biotinylation using sulfo-NHS-LC-biotin for 30 min. The cells were then lysed, subjected to pulldown with streptavidin-agarose and analyzed by western blot. (C) For MMP2 measurement, the conditioned media from control HT1080 or ACBD3-KO HT1080 cells were collected after 18 hours incubation and added to Total MMP2 Quantikine ELISA kit for quantification, as described in the methods. (D) After 18 hrs transfection of YFP-GL-GPI, HeLa WT and ACBD3-KO cells were stained for indicated antibodies and then examined by confocal microscopy. Line profiles through regions of interest were analyzed by Fiji. (Scale bars = 10 μm) (E-F) HeLa-WT or HeLa-ACBD3-KO cells were transfected with sialyltransferase-RFP (ST-RFP, a Golgi marker) and His-tagged Shiga toxin B fragment (2.5 mg/ml final concentration in DMEM+1�S) was added to cells for 45min at 4°C. After the withdrawal of unbound toxin by washing for three times in ice-cold PBS, cells were incubated with DMEM+10�S at 37°C for indicated time points. Then cells were stained using anti-His-tag and anti-calnexin (as an ER marker) antibodies. The results show that plasma membrane-to-Golgi transport of His-tagged Shiga toxin B fragment is not altered in ACBD3-KO cells, while the Golgi-to-ER transport of His-tagged Shiga toxin B fragment is accelerated in ACBD3-KO cells. Scale bars = 10 μm.

Additional file 3: Figure S3. (A) 3D-SIM images showing that β-COP co-localized most extensively with ArfGAP3 and endogenously tagged KDELR1, followed by ArfGAP1. ACBD3 and β-COP didn’t show a significant overlap. Line profiles through regions of interest were analyzed by Fiji. Scale bars = 2 μm. Co-localization (Pearson’s R) was determined and subjected to two-tailed, unpaired t tests (n = 20 cells/combination, mean and SD, ****, p < 0.0001). (B) 3D-SIM images showing moderate co-localization between endogenous ACBD3 and endogenous ARFGAP1/3. No co-localization between endogenous ACBD3 and Golgin97, which serves as a negative control. Line profiles through regions of interest were analyzed by Fiji. Scale bars = 2 μm. Co-localization (Pearson’s R) was determined and subjected to two-tailed, unpaired t tests (n = 20 cells/combination, mean and SD, ****, p < 0.0001).

Additional file 1: Figure S1. (A) Characterization of HeLa cells expressing KDELR1 tagged with myc-BirA*. HeLa cells expressing KDELR1-myc-BirA* for 18 hours were grown in media containing biotin (50 μM) for 6 h and double labelled with streptavidin-488 (green) and anti-myc (red). (B) Lists of identified interaction partners in KDELR1-BioID with a number of identified peptides (all and unique) with their sequence coverage. (C) Immunoblotting of KDELR1-BioID samples confirmed biotinylation of the candidate interaction partners identified in mass spectrometry. Anti-GM130 and anti-Golgin97 blots were included as negative controls here. (D - J) Confocal micrographs of HeLa cells expressing KDELR1-mCherry, showing that depletion of Giantin, GRASP55, Golgin160 and ARFGAP1/2/3 do not result in ER re-distribution of KDELR1-mCherry. (K) Western blots showing depletion of Giantin, GRASP55, Golgin160, ACBD3 and ArfGAP1/2/3, respectively, by RNA interference. Scale bars = 10 μm.

Additional file 2: Figure S2. (A) Confocal micrographs of HeLa cells expressing KDELR1-mCherry showing that depletion of ACBD3 results in re-distribution of KDELR1-mCherry from the Golgi to the ER in vivo (Calnexin; ER marker). Expression of RNAi-resistant form of EGFP-ACBD3 restores perinuclear localization of KDEL receptor in the ACBD3 Knockdown cells. (B-C) ACBD3 knock-out by CRISPR/Cas9 technique in HT1080 cells result in re-distribution of KDELR1-mCherry to the ER. Confocal micrographs of WT and ACBD3-knockout HT1080 cells expressing KDELR1-mCherry showing that knockout of ACBD3 results in relocating KDELR1-mCherry from the Golgi to the ER in vivo. Calnexin was used as an ER-marker in these experiments. (D) Confocal results showing that hGH-GFP-KDEL expression induces exogenously expressed KDELR1-mCherry to re-distribute KDEL receptor to the ER (calnexin-positive compartment). The bar graph shows the summary of the confocal experiments. (***, p < 0.001) (E-F) ACBD3 depletion does not influence Golgi localization of other cycling proteins, such as GPP130 and CI-MPR. (G-H) ACBD3 depletion does not influence Golgi localization of a Golgi resident glycosyltransferase ManII nor secretion of ER-resident chaperone ERP29. scale bar = 10 μm.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune reactions. While TREM-1-amplified responses likely aid an improved detection and elimination of pathogens, excessive production of cytokines and oxygen radicals can also severely harm the host. Studies addressing the pathogenic role of TREM-1 during endotoxin-induced shock or microbial sepsis have so far mostly relied on the administration of TREM-1 fusion proteins or peptides representing part of the extracellular domain of TREM-1. However, binding of these agents to the yet unidentified TREM-1 ligand could also impact signaling through alternative receptors. More importantly, controversial results have been obtained regarding the requirement of TREM-1 for microbial control. To unambiguously investigate the role of TREM-1 in homeostasis and disease, we have generated mice deficient in Trem1. Trem1−/− mice are viable, fertile and show no altered hematopoietic compartment. In CD4+ T cell- and dextran sodium sulfate-induced models of colitis, Trem1−/− mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines. Trem1−/− mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major. Furthermore, reduced morbidity was observed for influenza virus-infected Trem1−/− mice. Importantly, while immune-associated pathologies were significantly reduced, Trem1−/− mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Trem1+/+ controls. Our results not only demonstrate an unanticipated pathogenic impact of TREM-1 during a viral and parasitic infection, but also indicate that therapeutic blocking of TREM-1 in distinct inflammatory disorders holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control., PLoS Pathogens, 10 (1), ISSN:1553-7374, ISSN:1553-7366

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune reactions. While TREM-1-amplified responses likely aid an improved detection and elimination of pathogens, excessive production of cytokines and oxygen radicals can also severely harm the host. Studies addressing the pathogenic role of TREM-1 during endotoxin-induced shock or microbial sepsis have so far mostly relied on the administration of TREM-1 fusion proteins or peptides representing part of the extracellular domain of TREM-1. However, binding of these agents to the yet unidentified TREM-1 ligand could also impact signaling through alternative receptors. More importantly, controversial results have been obtained regarding the requirement of TREM-1 for microbial control. To unambiguously investigate the role of TREM-1 in homeostasis and disease, we have generated mice deficient in Trem1. Trem1(-/-) mice are viable, fertile and show no altered hematopoietic compartment. In CD4(+) T cell- and dextran sodium sulfate-induced models of colitis, Trem1(-/-) mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines. Trem1(-/-) mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major. Furthermore, reduced morbidity was observed for influenza virus-infected Trem1(-/-) mice. Importantly, while immune-associated pathologies were significantly reduced, Trem1(-/-) mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Trem1(+/+) controls. Our results not only demonstrate an unanticipated pathogenic impact of TREM-1 during a viral and parasitic infection, but also indicate that therapeutic blocking of TREM-1 in distinct inflammatory disorders holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.