Journal: bioRxiv

Article Title: Tumor-derived Extracellular Vesicles Induce ER Stress to Drive Tolerogenic Dendritic Cell Development in the Tumor Microenvironment

doi: 10.64898/2026.02.10.705213

Figure Lengend Snippet: Western blot analysis of nuclear extracts from BMDCs treated with PBS (Ctrl) or tumor-derived EVs (20 µg/ml; 24h). Immunoblots show the nuclear enrichment of the mature transcription factors SREBP2 (A) and ATF4 (B) . Lamin B1 serves as the loading control for the nuclear fraction. Each representative of three independent experiments. (C) Western blot analysis of nuclear SREBP2 levels in BMDCs treated with PBS (Ctrl), EVs (20 µg/ml; 24h), or EVs (20 µg/ml; 24h) with a PERK inhibitor (GSK2606414; 500 nM, preincubation time 1h; 24 h). Densitometric quantification of the SREBP2 band intensity normalized to Lamin B1 band intensity performed using ImageJ. Representative of three independent experiments. (D) Relative Srebp2 expression levels between EV-uptaking (Emerald Green + ) and bystander (Emerald Green - ) DC subsets. DCs were isolated from BRAF V600E PTEN -/- tumors expressing CD81-Emerald Green using FACS (n=3). Representative of two independent experiments. (F) Flow cytometry analysis of neutral lipid accumulation in DCs. Cells were treated with PBS (Ctrl), EVs (20 µg/ml; 24h), or EVs in the presence of the SREBP2 inhibitor Fatostatin (FS). Shown are representative histograms of BODIPY 493/503 fluorescence ( left ) and the quantification of mean fluorescence intensity (MFI) normalized to the Ctrl group ( right ) (n=3). Representative of three independent experiments. (G) Heatmap displaying the relative mRNA expression of tolerogenic genes ( Cd274, Socs1, Il4ra, Ccl22, Cxcl16, Cd40 ) in BMDCs generated from WT or DC-restricted Srebp2-knockout (KO) mice (CD11c- Srebp2 -/- ). Cells were treated with PBS (Ctrl) or EVs (20 µg/ml) for 24 h. The color scale represents row-normalized gene expression (Z-score). Representative of two independent experiments. (H) Quantification of the frequency of CD11c + CD63 + DCs in BMDC cultures treated with PBS (Ctrl) or EVs (10 µg/ml) for 24 h (n=3). Representative of two independent experiments. (I) Impact of tumor EVs on mregDC development in vivo . TDLNs were harvested from mice harboring BRAF V600E PTEN -/- -NTC or BRAF V600E PTEN -/- -shRab27a melanomas (as described in ). Left , normalized frequency of CD11c + MHCII hi CD63 + cDCs. Right , Relative CCR7 expression by mregDCs (n=7–11). Data aggregated from two independent experiments. All data are reported as mean ± SEM. * p <0.05, ** p <0.01, **** p <0.0001. Data analyzed by unpaired, two-tailed Student’s t-test (D), Kruskal-Wallis test followed by Dunn’s multiple comparisons test (F), unpaired two-tailed Student’s t-test (H), and one-tailed Student’s t-test (I). cDC , conventional dendritic cell; Ctrl , control; DC , dendritic cell; EV , extracellular vesicle; FACS , fluorescence-activated cell sorting; FS , Fatostatin; KO , knockout; MFI , mean fluorescence intensity; mregDC , migratory regulatory dendritic cell; mRNA , messenger RNA; NTC , non-targeting control; PERKi , PERK inhibitor; shRNA , short hairpin RNA; TDLN , tumor-draining lymph node; WT , wild-type.

Article Snippet: For PERK inhibition experiments, BMDCs were pre-incubated with the PERK inhibitor GSK2606414 (500 μM; MedChemExpress, Cat# HY-18072) for 90 min prior to EV stimulation (20 μg/ml, 24 hrs).

Techniques: Western Blot, Derivative Assay, Control, Expressing, Isolation, Flow Cytometry, Fluorescence, Generated, Knock-Out, Gene Expression, In Vivo, Two Tailed Test, One-tailed Test, FACS, shRNA

Journal: bioRxiv

Article Title: The active secretion of a subunit of IL-12 by tissue cells is regulated by Valosin-Containing Protein and intracellular calcium redistribution

doi: 10.64898/2026.01.28.702376

Figure Lengend Snippet: (A) Percentage of UPR+ 7-AAD-L cells (GFP+ for XBP-1 binding to Unfolded Protein Response Element) after treatment with vehicle (DMSO), 3.12μM ionomycin, or 1.56μM CB-5083 then analyzed with flow cytometry. (B) Luciferase assay of supernatants from p35NL cells pre-treated with PERK inhibitor AMG PERK 44 for 2 hours then stimulated with 3.12 μM ionomycin for 24 hours. (C) Luciferase assay of supernatants from p35NL cells pre-treated with PERK inhibitor AMG PERK 44 for 2 hours then stimulated with 1.56μM CB-5083 for 24 hours. (D) Luciferase assay of supernatants from p35NL cells pre-treated with IRE1 inhibitor MKC8866 for 2 hours then stimulated with 3.12 μM ionomycin for 24 hours. (E) Luciferase assay of supernatants from p35NL cells pre-treated with IRE1 inhibitor MKC8866 for 2 hours then stimulated with 1.56μM CB-5083 for 24 hours. (F) Luciferase assay of supernatants from p35NL cells pre-treated with ATF6 inhibitor Ceapin-A7 for 2 hours then stimulated with 3.12 μM ionomycin for 24 hours. (G) Luciferase assay of supernatants from p35NL cells pre-treated with ATF6 inhibitor Ceapin-A7 for 2 hours then stimulated with 1.56μM CB-5083 for 24 hours. (n=3). Data is represented as raw Relative Luminescence Units (RLUs). Significant changes in luminescence were determined through ordinary one-way ANOVAs of cells stimulated with ionomycin only or CB-5083 only (control). *, p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant

Article Snippet: The following inhibitors were used to target specific arms of the UPR response: AMG PERK 44 for PERK, MKC8866 for IRE1α, and Ceapin-A7 for ATF6 (Med Chem Express).

Techniques: Binding Assay, Flow Cytometry, Luciferase, Control