Fig 2: D10 cells treated with 4µ8c produce IL-5, but they cannot secrete it. D10 cells were treated as in Fig. 3a. a At 20 h, the cells were treated with monensin for an additional 4 hours and intracellular staining was performed for IL-5 and IL-13. b The supernatants were isolated at 20 h from the cells shown in A and an ELISA was performed in duplicate for IL-4, IL-5, and IL-13. The data is representative of six experiments. c The average percent of cells positive for IL-5 and IL-13 are graphed from six experiments. The error bars represent the standard error of the mean. Hypothesis testing was done by Student’s T test unpaired, Welch’s correction (p value < 0.05). d A cytokine secretion assay was performed on D10 cells treated as above. The data shown is a representative of the percent IL-5 secreting cells from each sample group. The experiment was done with duplicate samples three times. e The percent IL-5 secreting cells for each treatment that was performed in duplicate was averaged and graphed for the three individual experiments performed. f Protein was isolated from cells treated as in 3A and immunoblotted with IL-5, IL-13, and ß-actin antibody. The data is representative of two experiments

Fig 3: IL-5 is reduced in established mouse TH2 cells upon treatment with 4µ8c. D10 cells were rested in complete T cell media for 24 h at 37 °C. The cells were then left un-stimulated (NS) or stimulated with PMA and ionomycin (PI) or plate-bound a-CD3 and a-CD28 in the presence or absence (-) of 4µ8c for 24 h. a As a control the level of spliced xbp1 mRNA was measured by qRT-PCR, as 4µ8c blocks the ability of IRE1a to cleave xbp1. The data shown is the fold change in reduction of treated vs. untreated after normalizing to the ns control for five experiments. The supernatants were harvested, and ELISA was performed from these samples as shown in B and C. b The data shown is from six experiments where cells were re-stimulated with PMA and ionomycin in the presence or absence (-) of 4µ8c. c The data shown is for five experiments where the cells were re-stimulated with plate-bound antibodies in the presence or absence (-) of 4µ8c. The standard error, upper and lower bars, and the mean, middle bar, is shown in all graphs. Hypothesis testing was done by Student’s T test unpaired, Welch’s correction (p value < 0.05)

Fig 4: IL-5 is reduced in established human TH2 cells upon treatment with 4µ8c. a Blood was collected from seven individual volunteers in total. Cells were harvested from the blood using Ficoll, and CD4+ cells were isolated using Dynabeads. The cells were activated with plate-bound a-CD3 and a-CD28 for eleven days under TH2 conditions (IL-2, IL-4, a-IFN?, and a-IL-12). The cells were rested for 24 h and then re-stimulated with plate-bound antibodies for 24 h in the presence or absence (-) of 4µ8c. An ELISA was performed on the supernatants. Of note, for TH2 cells differentiated from one individual, we were unable to detect IL-5 after differentiating the cells for 11 days, and that sample was removed from the IL-5 analysis, leaving us with an N of six. One of the samples from an individual was lost prior to running the ELISA for IL-4, leaving us with an N of six. The data shown are the results of six individual human samples for IL-4 and IL-5 and seven individual human samples for IL-13. The standard error, upper and lower bars, and the mean, middle bar, is shown in all graphs. Hypothesis testing was done by Student’s T test unpaired, Welch’s correction (p value < 0.05). b CD4+ cells were isolated from human blood as in A, activated under TH2 or TH1 conditions (IL-2, IL-12, and a-IL-4) for three days, and then stimulated with PMA and ionomycin in the presence of monensin for four hours. Intracellular staining was performed. The results are representative of six samples for IL-4 and three samples for IL-5. c The results for the percent positive and mean fluorescence intensity (MFI) d for IL-4 and IL-5 in treated and untreated cells differentiated three days in the presence of 4µ8c is shown for all intracellular flow experiments performed