Fig 2: Exposure of preeclamptic pMSCs to Hinokitiol rescues FNglycosylation and matrix deposition. (A) Western blot for FN and Con A following immunoprecipitation of FN in TC and PE pMSCs. n = 3 separate pMSC isolations. (B) Intracellular Fe2+ content in TC and PE pMSCs. n = 4 separate pMSC isolations performed in duplicate. (C) Western blot and densitometry for FN in PE pMSCs upon exposure to 1 µM Hinokitiol. n = 4 separate pMSC isolations. (D) Western blot for FN and Con A following immunoprecipitation of FN in PE pMSCs exposed to 1 µM Hinokitiol. Representative of two separate pMSC isolations. (E) IF for FN (green) and the organelle markers CALR (ER), STX6 (Golgi), and LAMP1 (lysosomes) in red (top, middle, and bottom panels, respectively) following 1 µM Hinokitiol exposure. In IF images, arrowheads indicate FN fibrillar deposition while stars show FN clustering. Nuclei were counterstained with DAPI. Scale bars at 63× magnification represent 7 µM. Data are presented as mean ± SEM. *P = 0.05 (Unpaired Student’s t-test).

Fig 3: Golgi marker syntaxin-6 mediates nuclear translocation of EGFRvIII. A: Co-localization results of EGFRvIII and syntxain-6 with or without 24h serum-free starvation culture in U251EGFRvIII and C6EGFRvIII respectively by immunofluorescence staining. Yellow signal indicates the co-localization of the two proteins. Lower histogram is a comparison of co-localization coefficients. Here, we declare that all the statistics of co-localization coefficients in this article are made by Image-Pro Plus 6.0 software. Scale bar are respectively 50µm and 25µm. B: Western blot results after reciprocal immunoprecipitation of EGFRvIII and Syntaxin6. Column below represents the relative densities of bands. C: Co-localization results of EGFRvIII and GM130 with or without 24h serum-free starvation culture in U251EGFRvIII and C6EGFRvIII respectively by immunofluorescence staining. Yellow signal indicates the co-localization of the two proteins. Lower histogram is a comparison of co-localization coefficients. Scale bar are respectively 25µm and 10µm. D: U251EGFRvIII cells were transfected with control shRNA and two shRNA targeting syntaxin-6 (STX6) respectively by lentivirus infection, and after 48h immunoblotting for STX6 were analyzed. The lower histogram was the results of relative densities. E: U251EGFRvIII cells were treated with or without 24h serum-free starvation culture for 24h after STX6 shRNA treatment for 48h, and immunoblotting results of EGFRvIII's nuclear translocation were analyzed in control group and shRNA knockdown group. The lower histogram was the relative densities of nuclear EGFRvIII. F: U251EGFRvIII cells were treated with or without 24h serum-free starvation culture for 24h after STX6 shRNA treatment for 48h, and imunofluorescence results of EGFRvIII's nuclear translocation were analyzed in control group and shRNA knockdown group. Scale bar are respectively 50µm and 25µm. Lower histogram was the cell numbers of EGFRvIII's nuclear translocation in 100 cells.

Fig 4: CellProfiler analysis pipeline of Stx6-positive vesicle recruitment to rBCVs A confocal micrograph inset (10.5 µm2 area) from a BMM infected with wild-type DsRedm-expressing B. abortus and stained for endogenous Syntaxin 6 (AlexaFluor™488-Stx6) was selected in the DsRed channel (Brucella) and input into CellProfiler for analysis. Scale bars, 10 and 2 µm.Color to gray module split the red (Brucella) and green (Stx6) channels and reverted the images to gray scale.Identify primary objects module identified individual Brucella and Stx6-positive vesicles based on their size.Expand or shrink objects module expanded the size of individual Brucella by 6 pixels to encompass whole rBCVs and associated vesicles.Relate objects module identified Stx6-positive vesicles within the rBCV area and filtered out non-associated vesicles.Classify objects module counted the number of vesicles associated with each rBCV (expanded Brucella). X- and y-axes represent pixels coordinates. The output data counted the number of vesicles associated with each Brucella, which was derived to determine the percentage of Stx6-positive rBCVs.

Fig 5: H-Ras and K-Ras4a contain lysine fatty acylation.(a) Amino acid sequences of the HVRs of Ras proteins. (b) Scheme showing the Alk14 metabolic labeling method to study lysine fatty acylation. (c) In-gel fluorescence detection of the fatty acylation levels of Ras proteins, RalA and STX6 in HEK293T cells (top panel), and quantification of the relative fatty acylation levels (bottom panel). The fatty acylation level of H-Ras without NH2OH treatment was set to 1. (d) In-gel fluorescence showing the fatty acylation levels of H-Ras-WT and -3KR mutant without or with NH2OH treatment. (e) Scheme showing the lysine to arginine mutants (4KR, 3KR, and 7KR) used to identify potential fatty acylation sites. (f) In-gel fluorescence showing the fatty acylation levels of K-Ras4a-WT and KR mutants without or with NH2OH treatment (top panel) and quantification of fatty acylation levels with NH2OH treatment relative to that of K-Ras4a-WT (bottom panel). (g, h) Tandem mass (MS/MS) spectrum of triply charged H-Ras (g) and K-Ras4a (h) peptides with Alk14-modification on K170 and K182, respectively. The b- and y-ions are shown along with the peptide sequence. The cysteine residues were carbamidomethylated due to iodoacetamide alkylation during sample preparation and methionine was oxidized. FL, fluorescence; WB, western blot. Statistical evaluation was by unpaired two-tailed Student’s t test. Error bars represent SEM in three biological replicates. ***p<0.001; ns, not significant. Representative images from three independent experiments are shown.