Fig 2: Identification of CerS2 as major ceramide synthase in RAW264.7 mouse macrophages. (A) The reaction catalyzed by ceramide synthase. (B) Ceramide synthase in vitro activity assays on membrane lysates from RAW264.7 mouse macrophages treated with fumonisin (5 µM, 30 min) or DMSO, showing robust inhibition of ceramide synthase activity following fumonisin treatment. As a control, denatured membrane proteomes were used. Data represent mean ± SEM for three biological replicates per group. (C) Western blot analysis confirming abundant expression of CerS2 in RAW264.7 membrane lysates. As a positive control, membrane lysates from HEK293T were used. A total of 40 µg of lysate was loaded for all samples in this analysis, and actin was used as a loading control. The Western blot analysis was performed on five biological replicates with reproducible results.

Fig 3: ABCB1 expression and activity are dependent on CerS. CerS siRNA transfection efficiency. CerS2 or CerS6 siRNA (10 nM) was applied for up to 72 h to A498 cells, and efficiency was determined by (A) quantitative PCR (n = 3–5) or (B) immunoblotting (n = 6). C, CerS6 siRNA was transfected for 72 h, and CerS6 and ABCB1 were assessed by immunoblotting (n = 4). HPCT and renal cancer cell lines were transiently transfected with CerS-specific siRNA (10 nM, 72 h). ABCB1 and CerS immunoblots (D, D') were performed (n = 2–9). E, heterologous expression of hemagglutinin (HA)-tagged CerS increased ABCB1. Immunoblot is representative of n = 3 to 4. F, F', surface immunofluorescence staining and threshold analysis of ABCB1 in nonfixed nonpermeabilized A498 cells (siRNA 10 nM, 72 h). The scale bar represents 10 µm. Images (n = 8–10 per treatment) were analyzed from two independent experiments. G, rhodamine 123+ efflux assay in A498 cells transfected with CerS-specific siRNA (10 nM) for 72 h (n = 5–9). Statistical analyses compare CerS siRNA to control siRNA at each time point. H, MTT assay of A498 cells transfected with CerS6 siRNA (10 nM, 48 h) and exposed to 1 µM DOX for 24 h (n = 3). I, electrical impedance measurements of A498 cells transfected with CerS isoform-specific siRNA (10 nM, 48 h) and plated with 1 µM DOX into ECIS arrays. Capacitance at 40 kHz was monitored for 4 days. Slope analyses were performed from the steepest portion of each curve (n = 6). CerS, ceramide synthase; DOX, doxorubicin; ECIS, Electric cell–substrate impedance sensing; HPCT, human proximal convoluted tubule; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Fig 4: ER stress and unfolded protein response by CerS downregulationactivates ATF6a in MDR cells. Cells were transiently transfected with CerS-specific siRNA for 72 h or treated with SERCA inhibitor thapsigargin (TG) (3 µM for 6 h) as positive control. A, analysis of GRP78 by immunoblotting in A498 cells. A', densitometry analysis of CerS-specific siRNA transfected A498 cells after 72 h (n = 5–6). B, quantitative PCR analysis of ABCB1 and CHOP mRNA in A498 cells (n = 6). Immunoblots and densitometry analyses for full length (FL, 90 kDa) and cleaved (50 kDa) ATF6a in CerS siRNA (C, C') and CerS6 overexpressing (D) cells after 72 h (representative immunoblots of n = 7–9). Values indicate densitometry analysis with correction to GAPDH. Immunoblots in C for CerS2, CerS6, and GAPDH are reused from Figure 2D because target proteins were analyzed from the same set of samples. E, E', immunofluorescence staining and quantification of nuclear ATF6a using threshold analysis in CerS siRNA cells after 72 h in fixed A498 cells. A minimum of 500 nuclei per treatment were analyzed from thresholded images using Fiji/ImageJ from two independent experiments. The scale bar represents 10 µm. ATF6a, activating transcription factor 6a; CerS, ceramide synthase; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78 kDa; MDR, multidrug resistance; SERCA, sarco-/endoplasmic reticulum calcium ATPase.

Fig 5: Characterization of CerS2 activity during phagosomal maturation. (A) Western blot analysis confirming the enrichment of CerS2 on EPs. As controls, EEA1 and LAMP1 were used to assess the purity of EP and LP preparations, respectively. In each case, 25 µg of lysate was loaded for all samples in this analysis, and actin was used as a loading control. The Western blot analysis was performed on eight biological replicates with reproducible results. (B) Ceramide synthase in vitro activity assay on EPs and LPs from RAW264.7 mouse macrophages, showing heightened ceramide synthase activity on EPs. Data represents mean ± SEM for three biological replicates per group. (C) Levels of C17:1 containing ceramide on EPs and LPs, following feeding of RAW264.7 mouse macrophages with 1 mM C17:1 FFA (4 h, 37 °C). Data represent mean ± SEM for five biological replicates. ***P < 0.001 for LP group versus EP group by Student’s t test.