Fig 2: In vitro differentiated INS mutant cells presented increased ER-stress associated with reduced proliferation and insulin content.(A) Immunohistochemistry for ER-stress markers (BIP, GRP170, MANF) and proliferation marker KI67 along with INS in Stage 7 cells. Scale bars = 100 µm. (B) Quantification of (A). Percentage of Stage 7 insulin positive cells that express BIP, GRP170, MANF and KI67 (n = 3–7 independent differentiation experiments per genotype). (C) qRT-PCR of beta-cell and ER-stress markers (n = 5–6 independent differentiation experiments per genotype). (D) Sensitivity to ER-stress-induced apoptosis of Stage 7 cells. Percentage of insulin positive cells that are labeled by TUNEL assay in control conditions and after treatment with ER-stress inducers brefeldin A (BFA), thapsigargin (TGA) and tunicamycin (TM). (E) Static sequentially stimulated insulin secretion of Stage 7 cells, presented as fractional secretion of total INS content. Low G = 3.3 mM glucose; Hi G = 20 mM glucose; TOL = tolbutamide 100 µM; KCl = 30 mM KCl; FSK = 1 µM forskolin. (n = 8–12 independent stimulations per genotype). (F) Human insulin content in Stage 7 cells. Cell mass normalized by DNA content (n = 8–12 independent stimulations per cell genotype). Data represent mean ± SEM. Student’s t test, *p < 0.05, **p < 0.01.

Fig 3: Validation of the SILAC/MS results over a time course of infection by Western blot analysis.(A) A549 cells and (B) HEK-293 cells were infected with DENV-2 (DV2) at a m.o.i. of 3 or mock (Mo) infected. At the indicated times p.i. (24, 48, 72 and 96 h) the cells were harvested and lysed in 2X SDS-PAGE sample buffer to produce a total cell lysate. The levels of the viral NS5 protein and the cellular proteins CTSL1, ERC1, KPNA2, MFN1, PRAF2, UBE2S, HYOU1 and GAPDH (used as a loading control) in each lysate (ten μg of protein loaded per lane) were analyzed by Western blotting using specific anti-sera. The results shown are typical of at least two independent Western blotting experiments. The positions of relevant molecular mass markers are shown in kDa.

Fig 4: Validation of the SILAC/MS results by Western blot analysis.SILAC labeled A549 cells that had been either DENV-2 (DV2) or mock (Mo) infected were harvested at 24 hours p.i. and used for the production of cytoplasmic (Cyt) and nuclear (Nuc) extracts that were subsequently used for MS analysis. The levels of the cellular proteins CTSL1, ERC1, KPNA2, MFN1, PRAF2, UBE2S, HYOU1 and GAPDH (used as a loading control) in each fraction (ten μg of protein loaded per lane) were analyzed by Western blotting using specific anti-sera. The results shown are typical of two independent Western blotting experiments. The positions of relevant molecular mass markers are shown in kDa. The intensity of the bands was determined using ImageJ and normalized to the intensity of the GAPDH bands in each experiment. The average values of the intensities were used to determine the Western blot ratios (DV2/Mo) of each protein in the cytoplasmic and nuclear fractions. NaN = one or more peptides corresponding to the proteins were identified but they were not used for quantitation; ND = no peptides corresponding to the protein were identified.