Journal: Molecular Therapy. Nucleic Acids

Article Title: Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation

doi: 10.1016/j.omtn.2022.08.003

Figure Lengend Snippet: In vitro validation of Lbp siRNAs and in vivo effect of Lbp UNA-siRNA delivered through lipid nanoparticles (A) Effect of several doses of chemically unmodified and modified (UNA) siRNA (si69107, si69108, si69109) against Lbp mRNA sequence in Hepa1-6 cell line. (B) Effect of chemically unmodified (1 and 3 mg/kg) and chemically modified (UNA, 1 and 3 mg/kg) LNP- Lbp siRNA on plasma LBP at days 3, 6, 10, and 12 after injection. (C) Effect of chemically modified (UNA, 1 and 3 mg/kg) LNP- Lbp siRNA on liver Lbp gene expression 3 days after second injection. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with 0 pmol/L or vehicle. (D) Effects of 6-month LNP- Lbp UNA-siRNA administration in HFHS-fed mice on liver, inguinal WAT (iWAT), and perigonadal WAT (pgWAT) Lbp mRNA levels and plasma Lbp concentration in male and female mice. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with CD; † p < 0.05, †† p < 0.01, and ††† p < 0.001 compared with HFHS.

Article Snippet: In humans, peptidylprolyl isomerase A (cyclophilin A) (4333763, PPIA as endogenous control); LBP ( LBP ; Hs01084621_m1); fatty acid synthase ( FASN ; Hs00188012_m1); acetyl-CoA carboxylase alpha ( ACACA ; Hs01046047_m1); sterol regulatory element binding transcription factor 1 ( SREBF1 ; Hs02561944_s1); stearoyl-CoA desaturase ( SCD1 ; Hs01682761_m1); peroxisome proliferator activated receptor alpha ( PPARA ; Hs00947536_m1); carnitine palmitoyltransferase 1A ( CPT1A ; Hs00912671_m1); interleukin-6 ( IL-6 ; Hs00985639_m1); and C-X-C motif chemokine ligand 8 ( CXCL8 or IL-8 ; Hs00174103_m1) were used.

Techniques: In Vitro, Biomarker Discovery, In Vivo, Modification, Sequencing, Clinical Proteomics, Injection, Gene Expression, Concentration Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation

doi: 10.1016/j.omtn.2022.08.003

Figure Lengend Snippet: Effect of LNP- Lbp UNA-siRNA treatment on liver lipid accumulation and lipogenesis induced by an HFHS diet (A–P) Effects of 6-months LNP- Lbp UNA-siRNA administration on representative standard Masson’s trichrome staining histological liver slides (20×), lipid droplet (LD) count and area (A and I), liver triglycerides (B and J), malonyl-CoA levels (C and K), expression of lipogenic (D and L) genes, FAS and ACC protein (E, F, M, and N), ACC activity (G and O), and MDA levels (H and P) in HFHS-fed male (A–H) and female (I–P) mice. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with CD; † p < 0.05, †† p < 0.01, and ††† p < 0.001 compared with HFHS.

Article Snippet: In humans, peptidylprolyl isomerase A (cyclophilin A) (4333763, PPIA as endogenous control); LBP ( LBP ; Hs01084621_m1); fatty acid synthase ( FASN ; Hs00188012_m1); acetyl-CoA carboxylase alpha ( ACACA ; Hs01046047_m1); sterol regulatory element binding transcription factor 1 ( SREBF1 ; Hs02561944_s1); stearoyl-CoA desaturase ( SCD1 ; Hs01682761_m1); peroxisome proliferator activated receptor alpha ( PPARA ; Hs00947536_m1); carnitine palmitoyltransferase 1A ( CPT1A ; Hs00912671_m1); interleukin-6 ( IL-6 ; Hs00985639_m1); and C-X-C motif chemokine ligand 8 ( CXCL8 or IL-8 ; Hs00174103_m1) were used.

Techniques: Staining, Expressing, Activity Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation

doi: 10.1016/j.omtn.2022.08.003

Figure Lengend Snippet: Result of LNP- Lbp UNA-siRNA treatment on liver lipid accumulation and lipogenesis in mice with established obesity (A–P) Effects of 8-weeks LNP- Lbp UNA-siRNA administration on Lbp mRNA levels (A and I), representative standard Masson’s trichrome staining histological liver slides (20×), LD count and area (B and J), liver triglycerides (C and K), malonyl-CoA levels (D and L), expression of lipogenic (E and M) genes, ACC protein (F and N), ACC activity (G and O), and MDA levels (H and P) in diet-induced obese male (A–H) and female (I–P) mice. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with vehicle-treated obese mice.

Article Snippet: In humans, peptidylprolyl isomerase A (cyclophilin A) (4333763, PPIA as endogenous control); LBP ( LBP ; Hs01084621_m1); fatty acid synthase ( FASN ; Hs00188012_m1); acetyl-CoA carboxylase alpha ( ACACA ; Hs01046047_m1); sterol regulatory element binding transcription factor 1 ( SREBF1 ; Hs02561944_s1); stearoyl-CoA desaturase ( SCD1 ; Hs01682761_m1); peroxisome proliferator activated receptor alpha ( PPARA ; Hs00947536_m1); carnitine palmitoyltransferase 1A ( CPT1A ; Hs00912671_m1); interleukin-6 ( IL-6 ; Hs00985639_m1); and C-X-C motif chemokine ligand 8 ( CXCL8 or IL-8 ; Hs00174103_m1) were used.

Techniques: Staining, Expressing, Activity Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation

doi: 10.1016/j.omtn.2022.08.003

Figure Lengend Snippet: Effect of LNP- Lbp UNA-siRNA treatment on insulin resistance during HFHS diet and in mice with established obesity (A–F) Effects of 6-months LNP- Lbp UNA-siRNA administration on insulin at 4, 8, and 25 weeks (A), insulin change between weeks 4 and 8 (B), and fasting glucose, HOMA IR , blood glucose during ITT, and glycemia (AUC) in ITT at the end of experiment (C–F) in HFHS-fed female and male mice. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with CD; † p < 0.05 and †† p < 0.01 compared with HFHS; ‡ p < 0.05, ‡‡ p < 0.01, and ‡‡‡ p < 0.001 compared with female mice. (G and H) Effect of 8-weeks LNP- Lbp UNA-siRNA administration on blood glucose during ITT (G) and glycemia (AUC) in ITT (H) in obese female and male mice. ∗p < 0.05 and ∗∗p < 0.01 compared with obese vehicle; ‡‡ p < 0.01 and ‡‡‡ p < 0.001 compared with female mice.

Article Snippet: In humans, peptidylprolyl isomerase A (cyclophilin A) (4333763, PPIA as endogenous control); LBP ( LBP ; Hs01084621_m1); fatty acid synthase ( FASN ; Hs00188012_m1); acetyl-CoA carboxylase alpha ( ACACA ; Hs01046047_m1); sterol regulatory element binding transcription factor 1 ( SREBF1 ; Hs02561944_s1); stearoyl-CoA desaturase ( SCD1 ; Hs01682761_m1); peroxisome proliferator activated receptor alpha ( PPARA ; Hs00947536_m1); carnitine palmitoyltransferase 1A ( CPT1A ; Hs00912671_m1); interleukin-6 ( IL-6 ; Hs00985639_m1); and C-X-C motif chemokine ligand 8 ( CXCL8 or IL-8 ; Hs00174103_m1) were used.

Techniques:

Journal: Molecular Therapy. Nucleic Acids

Article Title: Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation

doi: 10.1016/j.omtn.2022.08.003

Figure Lengend Snippet: LNP- Lbp UNA-siRNA treatment effects on liver lipogenesis and triglycerides in mice fed with standard diet (STD) and MCD (A–I) Effects of weekly LNP- Lbp UNA-siRNA administration in STD- and MCD-fed mice on liver Lbp and lipogenesis ( Fasn , Srebf1 , and Scd1 )-related gene expression (A and B), malonyl-CoA (C), FAS and ACC protein levels (D and E), ACC activity (F), NADPH production ( Idh1 , Me1 )-related gene expression (G), MDA (H), and triglycerides levels (I). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with vehicle; † p < 0.05, †† p < 0.01, and ††† p < 0.001 compared with STD. siC, weekly LNP-non-targeting UNA-siRNA (3 mg/kg)-treated mice; siLbp, weekly LNP- Lbp UNA-siRNA (3 mg/kg)-treated mice.

Article Snippet: In humans, peptidylprolyl isomerase A (cyclophilin A) (4333763, PPIA as endogenous control); LBP ( LBP ; Hs01084621_m1); fatty acid synthase ( FASN ; Hs00188012_m1); acetyl-CoA carboxylase alpha ( ACACA ; Hs01046047_m1); sterol regulatory element binding transcription factor 1 ( SREBF1 ; Hs02561944_s1); stearoyl-CoA desaturase ( SCD1 ; Hs01682761_m1); peroxisome proliferator activated receptor alpha ( PPARA ; Hs00947536_m1); carnitine palmitoyltransferase 1A ( CPT1A ; Hs00912671_m1); interleukin-6 ( IL-6 ; Hs00985639_m1); and C-X-C motif chemokine ligand 8 ( CXCL8 or IL-8 ; Hs00174103_m1) were used.

Techniques: Gene Expression, Activity Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation

doi: 10.1016/j.omtn.2022.08.003

Figure Lengend Snippet: Impact of Lbp gene knockdown in Hepa1-6 cells (A–G) Effects of Lbp gene knockdown in vehicle (BSA5%, 24 h)- and palmitate-treated (200 μM, 24 h) Hepa1-6 cells on Lbp (A), Srebf1 (B), and Scd1 (C) gene expression, ACC protein (D), ACC and AMPK activity (E and F), NADPH production-, antioxidant response-, and ER stress ( Syvn1 , Atf6 and Hspa5 )-related gene expression (G–I), and cellular viability and activity (MTT assay) (J). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with shC; † p < 0.05, †† p < 0.01, and ††† p < 0.001 compared with BSA.

Article Snippet: In humans, peptidylprolyl isomerase A (cyclophilin A) (4333763, PPIA as endogenous control); LBP ( LBP ; Hs01084621_m1); fatty acid synthase ( FASN ; Hs00188012_m1); acetyl-CoA carboxylase alpha ( ACACA ; Hs01046047_m1); sterol regulatory element binding transcription factor 1 ( SREBF1 ; Hs02561944_s1); stearoyl-CoA desaturase ( SCD1 ; Hs01682761_m1); peroxisome proliferator activated receptor alpha ( PPARA ; Hs00947536_m1); carnitine palmitoyltransferase 1A ( CPT1A ; Hs00912671_m1); interleukin-6 ( IL-6 ; Hs00985639_m1); and C-X-C motif chemokine ligand 8 ( CXCL8 or IL-8 ; Hs00174103_m1) were used.

Techniques: Knockdown, Gene Expression, Activity Assay, MTT Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation

doi: 10.1016/j.omtn.2022.08.003

Figure Lengend Snippet: Impact of Lbp gene knockdown in primary human hepatocytes (A–H) Effects of Lbp gene knockdown in human hepatocytes on Lbp mRNA (A), oil red O staining (B), and expression of Scd1 , CPT1A , PPARA , IL6 , and IL8 genes and AMPK activity (C–H). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with siC. siC, control scramble siRNA.

Article Snippet: In humans, peptidylprolyl isomerase A (cyclophilin A) (4333763, PPIA as endogenous control); LBP ( LBP ; Hs01084621_m1); fatty acid synthase ( FASN ; Hs00188012_m1); acetyl-CoA carboxylase alpha ( ACACA ; Hs01046047_m1); sterol regulatory element binding transcription factor 1 ( SREBF1 ; Hs02561944_s1); stearoyl-CoA desaturase ( SCD1 ; Hs01682761_m1); peroxisome proliferator activated receptor alpha ( PPARA ; Hs00947536_m1); carnitine palmitoyltransferase 1A ( CPT1A ; Hs00912671_m1); interleukin-6 ( IL-6 ; Hs00985639_m1); and C-X-C motif chemokine ligand 8 ( CXCL8 or IL-8 ; Hs00174103_m1) were used.

Techniques: Knockdown, Staining, Expressing, Activity Assay, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation

doi: 10.1016/j.omtn.2022.08.003

Figure Lengend Snippet: Association of liver LBP with expression of de novo lipogenesis genes in morbidly obese human subjects (A and B) Plasma LBP concentration (A) and liver, SAT, and VAT LBP mRNA levels (B) according to NAFLD. (C) Bivariate correlations between liver LBP mRNA levels and plasma LBP concentration in morbidly obese participants. (D–F) Bivariate correlations between liver LBP and SREBF1 (D), FASN (E), and ACACA (F) mRNA levels. (G) Bivariate correlations between plasma LBP concentration and liver FASN mRNA levels.

Article Snippet: In humans, peptidylprolyl isomerase A (cyclophilin A) (4333763, PPIA as endogenous control); LBP ( LBP ; Hs01084621_m1); fatty acid synthase ( FASN ; Hs00188012_m1); acetyl-CoA carboxylase alpha ( ACACA ; Hs01046047_m1); sterol regulatory element binding transcription factor 1 ( SREBF1 ; Hs02561944_s1); stearoyl-CoA desaturase ( SCD1 ; Hs01682761_m1); peroxisome proliferator activated receptor alpha ( PPARA ; Hs00947536_m1); carnitine palmitoyltransferase 1A ( CPT1A ; Hs00912671_m1); interleukin-6 ( IL-6 ; Hs00985639_m1); and C-X-C motif chemokine ligand 8 ( CXCL8 or IL-8 ; Hs00174103_m1) were used.

Techniques: Expressing, Clinical Proteomics, Concentration Assay

Journal: Cell Death & Disease

Article Title: Gut-derived lipopolysaccharide remodels tumoral microenvironment and synergizes with PD-L1 checkpoint blockade via TLR4/MyD88/AKT/NF-κB pathway in pancreatic cancer

doi: 10.1038/s41419-021-04293-4

Figure Lengend Snippet: A GEPIA analyzed TLR4 transcription data in PDAC tissues and normal tissues from the TCGA database. B western blot measured TLR4 protein expression in PDAC tumor tissues and adjacent normal tissues. C representative image of TLR4 expression in adjacent normal tissues and PDAC tissues. D LinkedOmics analyzed significantly enriched GO_BP annotations by GSEA of TLR4 in PDAC. E cBioportal tools analyzed the correlation between TLR4 and PD-L1 transcription in PDAC. F qRT-PCR measured TLR4 and PD-L1 mRNA expression in pancreatic cancer tissues. PDAC patient Serum LPS ( G ), serum LBP ( H ), and LPS/HDL ( I ) had a positive correlation with tumoral PD-L1 transcription. J representative image of TLR4 and PD-L1 co-immunofluorescence in PDAC tissues slice and CK19 staining in a continuous slice.

Article Snippet: Serum LPS-binding protein (LBP) concentration was detected by LBP enzyme-linked immunosorbent assay kit (Boster Biological Technology Co., Ltd), the procedures were followed the manual from the manufacturer.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: mBio

Article Title: Endotoxin Producers Overgrowing in Human Gut Microbiota as the Causative Agents for Nonalcoholic Fatty Liver Disease

doi: 10.1128/mBio.03263-19

Figure Lengend Snippet: E. cloacae B29 induction of systemic and local inflammation in HFD-fed NAFLD GF mice is dependent upon endotoxin expression. GF mice were fed either normal chow diet (NCD) or high-fat diet (HFD) and inoculated with either Luria-Bertani broth (LB), wild-type E. cloacae B29, or the endotoxin-lacking waaG mutant strain (data were collected at the end of 15 weeks after inoculation). (A) ELISA of serum LPS-binding protein (LBP). (B) ELISA of serum amyloid A (SAA-3). (C) RT-qPCR analysis of expression of the Tlr4 gene in the liver. (D to F) RT-qPCR analysis of expression of the Tnfα , Il1β , Mcp1 , Ikkε , and Reg3γ genes in the liver (D), ileum (E), and epididymal fat pad (F). All mRNA quantification data were normalized against the housekeeping gene. Gene expression levels were expressed as values relative to those of the control group (NCD+LB). Tlr4 , toll-like receptor 4 gene; Tnfα , tumor necrosis factor-α gene; Il1β , interleukin-1β gene; Mcp1 , monocyte chemoattractant protein-1 gene; Ikkε , I kappa B kinase epsilon gene; Reg3γ , c-type lectin regenerating islet-derived protein 3-γ. Data shown are means ± SEM ( n = 5 to 10). * , P < 0.05; * * , P < 0.01; ** * , P < 0.001.

Article Snippet: The serum LBP level was measured by a mouse lipopolysaccharide binding protein ELISA kit (HyCult Biotechnology, Uden, The Netherlands).

Techniques: Expressing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Binding Assay, Quantitative RT-PCR, Derivative Assay

Journal: mBio

Article Title: Endotoxin Producers Overgrowing in Human Gut Microbiota as the Causative Agents for Nonalcoholic Fatty Liver Disease

doi: 10.1128/mBio.03263-19

Figure Lengend Snippet: Absence of TLR4 in mice prevented NAFLD induced by the LPS-producing gut opportunistic pathobiont Enterobacter cloacae B29 (data were collected at the end of 15 weeks after inoculation). (A) Liver histology (hematoxylin and eosin stain) of TLR4 mutant (TLR4 −/− ) mice with or without B29 at the end of the trial. Scale bar, 100 μm. (B) NAFLD activity score. (C) Body weight. (D) Mass of epididymal, mesenteric, subcutaneous inguinal, and retroperitoneal fat pads of TLR4 −/− mice with or without B29. (E) Plasma insulin concentration before (fasting) and 30 min after oral glucose load in TLR4 −/− mice with or without B29. (F) ELISA of serum leptin in TLR4 −/− mice with or without B29 (after adjustment for body weight). (G) ELISA of serum LBP in TLR4 −/− mice with or without B29. (H) ELISA of serum amyloid A (SAA-3) in TLR4 −/− mice with or without B29. Data shown are means ± SEM ( n = 5 to 9). * , P < 0.05; * * , P < 0.01; ** * , P < 0.001.

Article Snippet: The serum LBP level was measured by a mouse lipopolysaccharide binding protein ELISA kit (HyCult Biotechnology, Uden, The Netherlands).

Techniques: H&E Stain, Mutagenesis, Activity Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Therapeutic effect of modified zengye decoction on primary Sjogren’s syndrome and its effect on plasma exosomal proteins

doi: 10.3389/fphar.2022.930638

Figure Lengend Snippet: The plasma exosomalDEPs before and after MZD treatment.

Article Snippet: The exosomal protein ceruloplasmin (CP), cartilage oligomeric matrix protein (COMP), insulin-like growth factor-binding protein 2 (IGFBP2), ficolin-2 (FCN2), and lipopolysaccharide-binding protein (LBP) concentrations were determined using ELISA kits supplied by EIAab Science Inc. (Wuhan, China) according to the instructions of the manufacturer.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Therapeutic effect of modified zengye decoction on primary Sjogren’s syndrome and its effect on plasma exosomal proteins

doi: 10.3389/fphar.2022.930638

Figure Lengend Snippet: The validation of the downregulated exosomal proteins associated with SS. The ELISA test results are shown using a bar graph, expressed as mean ± standard deviation. An unpaired t -test was used to compare the changes before and after treatment. The (A) COMP (B) CP, (C) FCN2 (D) GGH, (E) IGFBP2 (F) LBP, and (G) IGFBP5 concentrations in the plasma before and after treatment.

Article Snippet: The exosomal protein ceruloplasmin (CP), cartilage oligomeric matrix protein (COMP), insulin-like growth factor-binding protein 2 (IGFBP2), ficolin-2 (FCN2), and lipopolysaccharide-binding protein (LBP) concentrations were determined using ELISA kits supplied by EIAab Science Inc. (Wuhan, China) according to the instructions of the manufacturer.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Nature microbiology

Article Title: An RNA-Centric Dissection of Host Complexes Controlling Flavivirus Infection

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: a , Genome-scale CRISPR knockout screens for all four DENV serotypes (DENV-1 276RKI , DENV-2 429557 , DENV-3 Philippines/H871856 , and DENV-4 BC287/97 ) in Huh7.5.1 cells. The genetic screens were independently performed for each serotype, analyzed with MAGeCK, and combined to obtain significance scores (y-axis). The 50 most enriched genes were colored and grouped by function. b , Scatter plot depicting enrichment scores of high-confidence ChIRP-MS DENV hits (x-axis) and the 200 top scoring hits from DENV CRISPR genetic screens (y-axis). Common hits shared by both DENV genetic screens and DENV ChIRP-MS were colored in red (vigilin), blue (RRBP1), and purple (others). c , Western blot (WB) analysis of wild-type (WT) or clonal RRBP1 knock-out (KO) (upper panel) and vigilin-KO (lower panel) cells in Huh7.5.1 cells. Representative WB of n = 2 biologically independent replicates showing similar results. d , qRT-PCR analysis of DENV infected WT and RRBP1-KO Huh7.5.1 cells (48 hours post-infection (hpi), MOI of 0.1) or ZIKV PRVABC59 , POWV LB (48 hpi, MOI 0.1) and CHIKV 18½5 (24 hpi, MOI of 0.01) infected WT and RRBP1-KO Huh7.5.1 cells. e , qRT-PCR analysis as in ( d ) here with vigilin-KO. Note: The WT datasets for POWV in panel d and e derived from the same experiments. f , WB analysis of DENV-2 429557 infected (MOI of 0.1, 72 hpi) WT, RRBP1-KO, and vigilin-KO Huh7.5.1 cell lysates, probed with DENV prM and NS3 antibodies. Representative WB of n = 4 biologically independent replicates showing similar results. g , Titers of infectious particles production from WT, RRBP1-KO, and vigilin-KO Huh7.5.1 cells infected with DENV-2 429557 at MOI of 0.1 for 72 h. For panel d , e , and g, the datasets represent the mean with standard error of the mean (SEM) of n = 3 independent biological replicates, except for POWV, n = 4 independent biological replicates. All P -values were determined by two-tailed, unpaired t -test using GraphPad Prism (GraphPad Software), where * = P <0.05 and n.s. = non-significant.

Article Snippet: Western blot analysis from the two different fractions was then performed with antibodies against RRBP1, vigilin, the cytoplasmic markers GAPDH (Genetex GTX 627408) and tubulin (Abcam ab97872) and the ER marker RPN1 (Bethyl Laboratories A305–026A).

Techniques: CRISPR, Knock-Out, Western Blot, Quantitative RT-PCR, Infection, Derivative Assay, Two Tailed Test, Software

Journal: Nature microbiology

Article Title: An RNA-Centric Dissection of Host Complexes Controlling Flavivirus Infection

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: a, Single cell quantification and correlation between, RRBP1, vigilin, ER-GFP (an ER marker), and 4′,6-diamidino-2-phenylindole (DAPI) immunofluorescence signals. Total number of cells that were randomly chosen for each analysis, with mean and SEM are indicated. b , Western blot analysis of ER and cytosolic cell fractions probed with GAPDH or Tubulin (cytosolic markers), RPN1 (ER marker), RRBP1, and vigilin antibodies. Representative WB of n = 3 biologically independent replicates showing similar results. c , Western blot analysis of three independent co-IP experiments from non-infected Huh7.5.1 cells with RRBP1 as the bait showing similar results. Samples were treated with or without RNase A. d , and e , Representative IF of RRBP1 ( d ), vigilin ( e ) co-stained with RNA fluorescent in situ hybridization targeting (RNA-FISH) of DENV or ZIKV positive stranded RNA genomes. Representative images of n = 2 biologically independent replicates showing similar results. Scale bars, 10 μm.

Article Snippet: Western blot analysis from the two different fractions was then performed with antibodies against RRBP1, vigilin, the cytoplasmic markers GAPDH (Genetex GTX 627408) and tubulin (Abcam ab97872) and the ER marker RPN1 (Bethyl Laboratories A305–026A).

Techniques: Marker, Immunofluorescence, Western Blot, Co-Immunoprecipitation Assay, Infection, Staining, In Situ Hybridization

Journal: Nature microbiology

Article Title: An RNA-Centric Dissection of Host Complexes Controlling Flavivirus Infection

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: a , RRBP1 (left) and vigilin (right) irCLIP reverse transcriptase (RT) stop mapping statistics annotated to the human, DENV, or ZIKV genomes and the ribosomal RNAs (rRNA) from Huh7.5.1 cells infected with MOI of 0.1 for 48 h. b , Histogram of RT stops mapping to the rRNAs from the RRBP1 (top) and vigilin (bottom) irCLIP in uninfected Huh7.5.1 cells. The three cytosolic rRNAs are highlighted. Red dashed line denotes vigilin’s strongest binding site, which is adjacent to RRBP1’s. c , Annotation of peaks called from RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapping to functional elements of human mRNAs including 5’UTR, exons, 3’UTR, and introns. Enrichment values are calculated based on the size of each function domain relative to the human genome. d , RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapped at base resolution to the DENV genome. RT stop intensity was normalized to the total number of unique reads mapping to the viral genome. The 5’UTR and 3’UTR regions are highlighted in red and blue, respectively.

Article Snippet: Western blot analysis from the two different fractions was then performed with antibodies against RRBP1, vigilin, the cytoplasmic markers GAPDH (Genetex GTX 627408) and tubulin (Abcam ab97872) and the ER marker RPN1 (Bethyl Laboratories A305–026A).

Techniques: Reverse Transcription, Infection, Binding Assay, Functional Assay

Journal: Nature microbiology

Article Title: An RNA-Centric Dissection of Host Complexes Controlling Flavivirus Infection

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: a , and b , Time-course DENV-Luc infection assays. WT, RRBP1-KO, and RRBP1-KO + RRBP1 cDNA rescue ( a ) or WT, vigilin-KO, and vigilin-KO + vigilin cDNA rescue ( b ) HEK293FT cells were infected with DENV-Luc (MOI of 0.01) and harvested at indicated time points. Virus infectivity was then determined by measuring Renilla luciferase expression from infected cells. c , and d , Time-course CVB3-Luc infection assays. WT, RRBP1-KO, and RRBP1-KO + RRBP1 cDNA rescue ( c ) or WT, vigilin-KO, and vigilin-KO + vigilin cDNA rescue ( d ) HEK293FT cells were infected with CVB3-Luc (MOI of 1) and harvested at indicated time points. e , and f , Luciferase expression of luciferase-encoding DENV replicon RNA in WT and RRBP1-KO ( e ) or WT and vigilin-KO ( f ) HEK293FT cells over indicated time points post-electroporation of replicon RNA. The data in each panel ( a - f ) represent the mean with SEM of of n = 3 independent biological replicates. g , Luciferase expression 8 hours (left) post-DENV-Luc (MOI of 0.025) infection in the presence of the replication inhibitor MK0608 (50 μM final concentration) or 36 hours (right) post DENV-Luc infection in the presence of DMSO of WT, RRBP1-KO, and vigilin-KO HEK293FT cells. The data in each panel represents the mean with SEM of 10 biologically independent infections. Fold change between datasets is indicated. All P -values stated in this figure were determined by two-tailed, unpaired t -test using GraphPad Prism, where * = P < 0.05 and n.s. = non-significant.

Article Snippet: Western blot analysis from the two different fractions was then performed with antibodies against RRBP1, vigilin, the cytoplasmic markers GAPDH (Genetex GTX 627408) and tubulin (Abcam ab97872) and the ER marker RPN1 (Bethyl Laboratories A305–026A).

Techniques: Infection, Virus, Luciferase, Expressing, Electroporation, Concentration Assay, Two Tailed Test

Journal: Nature microbiology

Article Title: An RNA-Centric Dissection of Host Complexes Controlling Flavivirus Infection

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: a , Western blot analysis of WT Huh7.5.1, vigilin-KO, RRBP1-KO, and RRBP1-vigilin double-KO cells. Representative WB of n = 2 biologically independent replicates showing similar results. b , Luciferase expression at 8 hpi and 24 hpi upon DENV-Luc infection (MOI 0.01) of WT Huh7.5.1, vigilin-KO, RRBP1-KO, and RRBP1-vigilin double-KO cells. The data in each panel represent the mean and SEM of 9 biologically independent infections. The P -values were determined by two-tailed, unpaired t -test using GraphPad Prism, where * = P < 0.05 and n.s. = non-significant. c , Northern blot analysis of dengue genomic RNA extracted from WT Huh7.5.1 and RRBP1-vigilin double-KO cells that were first infected with DENV-2 16681 (MOI of 0.1) for 48 hours, followed by MK0608 replication inhibitor treatment for indicated time frames (top panel). Quantification of DENV genomic RNA (i.e. northern blot signal) from 3 independent experiments (error bars are SEM) as a percentage relative to time point 0 hour after MK0608 treatment (bottom).

Article Snippet: Western blot analysis from the two different fractions was then performed with antibodies against RRBP1, vigilin, the cytoplasmic markers GAPDH (Genetex GTX 627408) and tubulin (Abcam ab97872) and the ER marker RPN1 (Bethyl Laboratories A305–026A).

Techniques: Western Blot, Luciferase, Expressing, Infection, Two Tailed Test, Northern Blot