2021 n a dpbs gibco 2037539 tbs t n a n a dapi thermo fisher scientific d1306 poly l lysine sigma aldrich a 005 c acrylamide sigma aldrich a4058  (Thermo Fisher)

Journal: bioRxiv

Article Title: Divergent Cell-Type Specific Hypoxia Responses in Human Stem Cell–Derived and Primary Islets

doi: 10.1101/2025.07.16.665006

Figure Lengend Snippet: Divergent functional, metabolic, and cellular responses of primary and SC-islets to acute hypoxia. Functional, metabolic, and immunohistochemical analyses of primary islets (top row, A-E) and SC-islets (bottom row, F-J) after exposure to 1% O 2 for 0, 6, 24, or 48 hours. (A, F) Representative immunohistochemical images of (A) primary and (F) SC-islet sections at each timepoint. Panels show co-staining for DAPI (blue), C-peptide (Cpep, green), and PDX1 (red); DAPI, glucagon (GCG, green), and somatostatin (SST, cyan); and DAPI and BNIP3L (red). Scale bars = 200 µm. (B, G) Absolute insulin secretion in response to low (2 mM, grey bars) and high (20 mM, dark bars) glucose for (B) primary and (G) SC-islets. (C, H) Oxygen consumption rate (OCR) of (C) primary and (H) SC-islets, measured via Seahorse metabolic flux analysis. Different colored lines represent islets recovered after the indicated duration of hypoxia. The traces show metabolic responses to sequential injections (indicated by dashed lines) of glucose (20 mM), oligomycin (1.5 µM), FCCP (0.5 µM), and finally rotenone/antimycin A (0.5 µM). (D, I) Glucose-stimulated insulin secretion (GSIS) stimulation index (SI) for (D) primary and (I) SC-islets, calculated as the ratio of high-to-low glucose secretion. (E, J) Extracellular acidification rate (ECAR) of (E) primary and (J) SC-islets, measured concurrently with OCR. *Data in (B, D, G, I) are presented as mean ± SEM of n=3 (primary) or n=4 (SC-islets) biological replicates; individual data points are shown as open circles. Data in (B, E, G, J) are representative traces from a single experiment, with lines showing the mean ± SEM of technical replicates. For insulin secretion (B, G) and stimulation index (D, I), statistical significance was assessed using repeated measures ANOVA followed by pairwise t-tests with Bonferroni correction for post-hoc analysis. Significance is denoted as *p < 0.05, **p < 0.01.

Article Snippet: DAPI (ThermoFisher; D1306) was used for nuclear staining.

Techniques: Functional Assay, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Fatty Acid Synthase binds endogenous dsRNAs and dampens the innate immune response to exogenous dsRNAs by limiting their accumulation

doi: 10.1101/2025.07.16.662511

Figure Lengend Snippet: A) Schematic representation of the ADAR1 CRISPR/Cas9 KO (ADAR1 KO) in HCT116 cells. Two gRNAs targeting the ADAR1 exon 2 (in pink and in blue, respectively) were used to delete a region of 414 bp. Indel event due to Cas9 cleavages in the KO is represented in orange. B) Western blot on ADAR1 and TUBULIN on lysates from ADAR1 KO and WT HCT116 with or without IFN-I treatment (1000U/mL, 24 hours treatments). C) ADAR1 KO and WT HCT116 cell viability monitored by MTT assay in control and 500nM 5-AZA treated cells over three days. Results represent the mean ± standard deviation (SD) of three biological replicates. Statistical analysis was performed using two-way ANOVA test with multiple comparison to WT NT (*= pval<0.05; **** = pval<0.00005). D) Representative confocal immunofluorescence images from WT and ADAR1 KO HCT116 cells treated (5-AZA) or not (NT) with 500nM 5-AZA. Samples were treated with E. coli RNase III (RNase III +) or mock-treated (RNase III -). Staining with mouse J2 anti-dsRNA antibody (in yellow) and with DAPI (in cyan) is shown. Scale bar: 10µM. E ) Relative integrated density was quantified in 20 areas per conditions using the Fiji software. Statistical analysis was performed using one-way ANOVA test (pval<0.05).

Article Snippet: DAPI (D1306, Invitrogen, Thermo Fisher Scientific) staining was performed for 5 minutes to reveal the nuclei.

Techniques: CRISPR, Western Blot, MTT Assay, Control, Standard Deviation, Comparison, Immunofluorescence, Staining, Software

Journal: bioRxiv

Article Title: Fatty Acid Synthase binds endogenous dsRNAs and dampens the innate immune response to exogenous dsRNAs by limiting their accumulation

doi: 10.1101/2025.07.16.662511

Figure Lengend Snippet: A) Schematic representation of the FASN CRISPR/Cas9 KO (FASN KO) in HCT116 cells. One gRNA targeting the FASN exon 7 (in blue) was used to generate an INDEL which produces a substitution of G-to-A and an insertion of T (INDEL in red) causing the appearance of a premature STOP codon. B) Western blot on FASN, MAVS and GAPDH on lysates from CTRL HCT116 cells treated or not with IFN-I (1000U/mL) for 24 hours (left panel) and on CTRL and FASN KO HCT116 (right panel). C) Representative confocal co-immunostaining images using mouse J2 anti-dsRNAs (in yellow) and rabbit anti-FASN (in magenta) primary antibodies in CTRL and FASN KO HCT116 cells. DNA was stained with DAPI (cyan). The right panel corresponds to a zoom into the area indicated by a yellow square in the MERGE panel. Scale bar: 10µM. D ) The relative integrated density of dsRNA staining was quantified in 20 areas per conditions using the Fiji software. Statistical analysis was performed using t-test (pval<0.005). E) Volcano plots displaying the differences in gene expression between CTRL and FASN KO HCT116 cells over three biological replicates. Genes with an FDR value < 0.01 and a log2 fold change > 1 and are displayed in red, genes with a log2 fold change < -1 are displayed in blue. All other genes are displayed in grey. Horizontal line represents FDR = 0.01, vertical lines represent a log2 fold change of -1 and 1. F) Type III IFN production in the supernatant of CTRL and FASN KO HCT116 cells monitored by measuring the optical density (OD) at 655 nm on HEK-Blue IFN-Lambda cells over three biological replicates. Statistical analysis was performed using t-test (pval<0.005) G) Representative confocal immunofluorescence analysis performed in CTRL and FASN KO HCT116 cells using mouse anti-MAVS antibody (in green) or MitoTracker dye (in magenta). Nuclei were stained with DAPI (in cyan). Scale bar: 10 μm. H) The relative integrated density of MAVS staining was quantified in 20 areas per conditions using the Image J software. Statistical analysis was performed using t-test (pval<0.00005).

Article Snippet: DAPI (D1306, Invitrogen, Thermo Fisher Scientific) staining was performed for 5 minutes to reveal the nuclei.

Techniques: CRISPR, Western Blot, Immunostaining, Staining, Software, Gene Expression, Immunofluorescence

Journal: bioRxiv

Article Title: Fatty Acid Synthase binds endogenous dsRNAs and dampens the innate immune response to exogenous dsRNAs by limiting their accumulation

doi: 10.1101/2025.07.16.662511

Figure Lengend Snippet: A) SINV-GFP infection kinetics in CTRL and FASN KO HCT116 cells. The relative GFP fluorescence area (expressed in percentage) as a function of time was measured after SINV GFP infection at an MOI of 0.1 every 6 h for 48 h with the CellcyteX automated cell counter and analyzer. Results represent the mean ± standard deviation (SD) of three biological replicates. B ) Western blot on lysates from CTRL and FASN KO HCT116 infected or not with SINV-GFP MOI 0.1, 24 hours) using rabbit anti-capsid antibody. C) RT-qPCR on SINV genome (SINV gRNA) or sub-genome (SINV sub-gRNA) on CTRL and FASN KO HCT116 infected or not (mock) with SINV-GFP MOI 0.1 for 24 hours. Results represent the mean ± standard deviation (SD) of three biological replicates. Gene expression was normalized over actin expression. Statistical analysis was performed using a t-test (** = pval<0.005, *** = pval<0.0005). D) Viral titers from supernatants of CTRL and FASN KO HCT116 cells infected with MOI 0.1 SINV-GFP for 24 hours as quantified by plaque assay. Results represent the mean ± standard deviation (SD) of three biological replicates. Statistical analysis was performed using a t-test (* = pval<0.05). E) RT-qPCR on four ISGs on CTRL and FASN KO HCT116 cells infected or not (mock) with SINV-GFP (MOI 0.1, 24 hours). Gene expression was normalized to actin expression. Results represent the mean ± standard deviation (SD) of three biological replicates. Statistical analysis was performed using a one-way ANOVA test with multiple comparison to CTRL mock (** = pval<0.005; **** = pval<0.0005). F ) Representative confocal co-immunostaining images using mouse J2 anti-dsRNAs (in yellow) and rabbit anti-FASN (in magenta) primary antibodies in CTRL and FASN KO HCT116 cells. DNA was stained with DAPI (cyan). Scale bar: 10µM. G) Epifluorescence microscopy analysis of SINV (+) RNA in SINV-infected CTRL and FASN KO HCT116 cells (MOI 0.1, 24 hpi) by RNA fluorescence in situ hybridization (FISH) (in red), DAPI staining (in cyan) and merge of the two channels are shown. Scale bar: 5 μm.

Article Snippet: DAPI (D1306, Invitrogen, Thermo Fisher Scientific) staining was performed for 5 minutes to reveal the nuclei.

Techniques: Infection, Fluorescence, Standard Deviation, Western Blot, Quantitative RT-PCR, Gene Expression, Expressing, Plaque Assay, Comparison, Immunostaining, Staining, Epifluorescence Microscopy, In Situ Hybridization