Journal: PLOS Pathogens

Article Title: Retinol binding protein 4 enhances cellular cholesterol uptake to facilitate influenza A virus infection

doi: 10.1371/journal.ppat.1013623

Figure Lengend Snippet: (A) Statistical analysis of the FPKM values of RBP4 in the blood of patients with influenza infection (n = 148) and healthy people (n = 82) obtained from four online GEO databases ( GSE155635 , GSE158592 , GSE196350 and GSE213168 ). (B) qPCR analysis of RBP4 mRNA levels in A549, HEK293T and PK-15 cells upon WSN (MOI = 0.1) infection at indicated periods. (C) ELISA of human RBP4 in supernatants of A549 and HEK293T cells infected with WSN (MOI = 0.1) for indicated periods. (D) Immunoblotting analysis of NP and M1 protein levels in HEK293T, PK-15 and 3D4/21 cells transfected with RBP4 or control plasmids for 12 hours, following infection with IAV WSN strain (MOI = 0.1) for various periods. (E, F) Immunoblotting (E) or qPCR analysis (F) of NP and M1 protein levels or mRNA expression in WT and RBP4-defecient PK-15 cells or HEK293T cells infected with IAV WSN strain (MOI = 0.1) for indicated periods. (G) Plaque assay of viral titers in WT and RBP4-deficient HEK293T and PK-15 cells infected with WSN (MOI = 0.1) for 24 hours. (H) Immunoblotting analysis of NP and M1 protein expression in WT and RBP4-deficient HEK293T cells infected with IAV PR8 strain (MOI = 0.1) for indicated periods. (I) Immunoblotting analysis of WT and RBP4-deficient PK-15 cells infected with IAV H3N2 (MOI = 0.1) or H9N2 (MOI = 0.1) for indicated periods. (J-L) RT-qPCR analysis of NP and M1 mRNA expression (J) or plaque assay of virus titers (L, left) in WT and RBP4-deficient HEK293T cells infected with IAV PR8 strain (MOI = 0.1) (J, L, left) or in WT and RBP4-deficient PK-15 cells infected with IAV H9N2 strain (K, left and L, middle) or H3N2 strain (K, right and L, right). (M) RT-qPCR analysis of mRNA levels of NP and M1 in RBP4-deficient PK-15 (RBP4-KO PK-15) cells infected with WSN (MOI = 0.1) for indicated periods. After 6 hours of WSN infection, cells were treated with or without recombinant porcine RBP4 (rpRBP4). Data are representative of two independent experiments (D, E, H, I) or pooled from at least three independent experiments (B, C, F, G, J-M, mean ± SD). p values were calculated using One-way ANOVA (B and C) or unpaired student’s t -test (A, F, G, J-M). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The primary antibodies used in this study were sourced from commercial suppliers as follows: Rabbit polyclonal antibodies against RBP4 (1 : 2,000, 11774–1-AP), CD36 (1 : 2,000, 18836–1-AP), STRA6 (1 : 3,000, 22001–1-AP), p65 (1 : 1,000, 10745–1-AP) and β-actin (1 : 20,000, 66009–1-lg) were purchased from Proteintech Group Inc. Rabbit polyclonal antibodies against CD36 (1 : 1,000, CY5796) and FLAG (1 : 10,000, AB0030) were purchased from Abways.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Control, Expressing, Plaque Assay, Quantitative RT-PCR, Virus, Recombinant

Journal: PLOS Pathogens

Article Title: Retinol binding protein 4 enhances cellular cholesterol uptake to facilitate influenza A virus infection

doi: 10.1371/journal.ppat.1013623

Figure Lengend Snippet: (A, B) Analysis of NP and M1 protein levels and mRNA expression or plaque assay of virus titers in BMDMs derived from WT and RBP4-deficient mice infected with IAV WSN strain (MOI = 0.5) at indicated time points. Representative immunoblotting results from two independent experiments (A) and pooled qPCR data and virus titers (B) from three independent experiments (mean ± SD) were shown, respectively. (C) Schematic diagram of the influenza virus infection model in mice. WT and RBP4-deficient mice were intranasally infected with 1 × 10 4 PFU of WSN. Lungs were collected 3 days post-infection, and body weight was monitored daily for 6 days. (D) Body weight changes in mice infected with IAV WSN as described in (C) (mean ± SEM, n = 6). (E) Immunoblotting analysis of NP and M1 protein expression in lungs of mice infected with IAV as in C (n = 3). (F) Immunofluorescence of NP proteins in lung tissues from WT and RBP4-deficient mice infected IAV as described in (C). Scale bars, 100 μm. Mean fluorescence intensity was calculated from two independent experiments (right). (G-J) Plaque assay analysis of virus titers (G), qPCR analysis of M1 copy number (H) and inflammatory cytokine expression (I) in lung tissues or histopathological analysis of lung tissues from WT and RBP4-deficient mice infected with WSN at day 3 post-infection. Each dot represents an individual mouse. Scale bars, 100 μm in J. Data are representative of two independent experiments (E-J, mean ± SEM). * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t -test).

Article Snippet: The primary antibodies used in this study were sourced from commercial suppliers as follows: Rabbit polyclonal antibodies against RBP4 (1 : 2,000, 11774–1-AP), CD36 (1 : 2,000, 18836–1-AP), STRA6 (1 : 3,000, 22001–1-AP), p65 (1 : 1,000, 10745–1-AP) and β-actin (1 : 20,000, 66009–1-lg) were purchased from Proteintech Group Inc. Rabbit polyclonal antibodies against CD36 (1 : 1,000, CY5796) and FLAG (1 : 10,000, AB0030) were purchased from Abways.

Techniques: Expressing, Plaque Assay, Virus, Derivative Assay, Infection, Western Blot, Immunofluorescence, Fluorescence

Journal: PLOS Pathogens

Article Title: Retinol binding protein 4 enhances cellular cholesterol uptake to facilitate influenza A virus infection

doi: 10.1371/journal.ppat.1013623

Figure Lengend Snippet: (A) Schematic illustration of IAV attachment and internalization, along with qPCR analysis of viral RNA (vRNA) levels during these stages in various cell types. WT and RBP4-deficient cells (as indicated) were infected with IAV WSN strain (MOI = 5). Data are pooled from three independent experiments (mean ± SD). (B) Immunofluorescence staining of viral NP proteins (green) in WT and RBP4-deficient PK-15 cells infected with IAV as described in (A). Cell membranes were stained with Dil dye (red), and nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (C, D) Confocal microscopy images (C and D) of α-2,3-linked SA (stained with MAL) or α-2,6-linked SA (stained with SNA) in WT and RBP4-deficient HEK293T cells (C) or in WT and RBP4-deficient BMDMs (D). Scale bars, 100 μm. (E, F) Flow cytometry analysis (E and F) of α-2,3-linked SA or α-2,6-linked SA in the same cell types as in (C and D). Mean fluorescence intensity of α-2,3-linked or α-2,6-linked SA was quantified using Image J software (for confocal microscopy) or FlowJo software (for flow cytometry). Data are representative (B, C and D, left, E and F, upper) or pooled from at least three independent experiments (C and D, right, E and F, lower). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t -test).

Article Snippet: The primary antibodies used in this study were sourced from commercial suppliers as follows: Rabbit polyclonal antibodies against RBP4 (1 : 2,000, 11774–1-AP), CD36 (1 : 2,000, 18836–1-AP), STRA6 (1 : 3,000, 22001–1-AP), p65 (1 : 1,000, 10745–1-AP) and β-actin (1 : 20,000, 66009–1-lg) were purchased from Proteintech Group Inc. Rabbit polyclonal antibodies against CD36 (1 : 1,000, CY5796) and FLAG (1 : 10,000, AB0030) were purchased from Abways.

Techniques: Infection, Immunofluorescence, Staining, Confocal Microscopy, Flow Cytometry, Fluorescence, Software

Journal: PLOS Pathogens

Article Title: Retinol binding protein 4 enhances cellular cholesterol uptake to facilitate influenza A virus infection

doi: 10.1371/journal.ppat.1013623

Figure Lengend Snippet: (A, B) Quantification of total cholesterol levels in multiple WT and RBP4-deficient cell types (A) and in serum from WT and RBP4-deficient mice (B). (C) Heatmap representation of RNA sequencing analysis showing differentially expressed genes related to cholesterol metabolism in WT and RBP4-deficient BMDMs. (D, E) qPCR analysis of CD36 mRNA expression in WT and RBP4-deficient BMDMs (D, left) and HEK293T cells (D, right), or in lung tissues from WT and RBP4-deficient mice (E). (F, G) Immunoblotting analysis of CD36 protein levels in multiple WT and RBP4-decficent cells as indicated (F) or in lung tissues from WT and RBP4-deficient mice (G). (H to J) Immunoblotting analysis of protein levels of CD36, total JNK, phosphorylated (p)-JNK, STAT1, and p-STAT1 as indicated in PK-15 or 3D4/21 cells pretreated with SP600125 (SP, 10 μM) or Resatorvid (TAK, 1 μM) for 2 hours, followed by treatment with rpRBP4 for 24 hours. (K) Immunoblotting analysis of protein expression of CD36, NP and M1 in PK-15 cells treated as described in (H), followed by infection with IAV WSN strain (MOI = 0.1) for 24 hours. (L) Immunoblotting analysis of CD36, p-STAT1, total STAT1 and RBP4 protein levels in HEK293T cells transfected with RBP4 expression plasmid or control empty vector, followed by treatment with Ruxolitinib (Ruxo, 5 μM) for 2 hours. (M) Immunoblotting analysis of CD36, p-STAT1, STAT1, STRA6 and RBP4 protein levels in HEK293T cells co-transfected with RBP4 expression plasmid and siRNA for STRA6 or negative control for 36 hours. (N) Immunoblotting analysis of CD36, p-JAK2, JAK2, p-STAT1, STAT1 and RBP4 protein levels in HEK293T cells transfected with RBP4 expression plasmid or control empty vector, followed by treatment with Ruxo (5 μM) for 2 hours or transfected with siRNA for STRA6 or negative control for 36 hours. Data are pooled from three independent experiments (A, B, D, E, mean ± SD) or representative of two independent experiments (F-N). * p < 0.05, ** p < 0.01 (Student’s t -tes t ).

Article Snippet: The primary antibodies used in this study were sourced from commercial suppliers as follows: Rabbit polyclonal antibodies against RBP4 (1 : 2,000, 11774–1-AP), CD36 (1 : 2,000, 18836–1-AP), STRA6 (1 : 3,000, 22001–1-AP), p65 (1 : 1,000, 10745–1-AP) and β-actin (1 : 20,000, 66009–1-lg) were purchased from Proteintech Group Inc. Rabbit polyclonal antibodies against CD36 (1 : 1,000, CY5796) and FLAG (1 : 10,000, AB0030) were purchased from Abways.

Techniques: RNA Sequencing, Expressing, Western Blot, Infection, Transfection, Plasmid Preparation, Control, Negative Control

Journal: PLOS Pathogens

Article Title: Retinol binding protein 4 enhances cellular cholesterol uptake to facilitate influenza A virus infection

doi: 10.1371/journal.ppat.1013623

Figure Lengend Snippet: (A to D) Confocal microscopy (A and B) or flow cytometry (C and D) analysis of cholesterol uptake by Dil-OxLDL in WT and RBP4-deficient HEK293T cells or BMDMs. Scale bars, 100 μm. Statistics of mean fluorescence intensity was calculated by Image J software (A and B, right) or Flow Jo software (C and D, right). (E, F) Flow cytometry analysis of cholesterol uptake by Dil-OxLDL in WT, RBP4-deficient and CD36 overexpressing RBP4-deficient HEK293T cells (E) and BMDMs (F). Mean fluorescence intensities were quantified with Flow Jo software. (G, H) Flow cytometry analysis of SA expression in HEK293T cells (G, upper) transfected with hCD36 plasmid or in stable CD36-overexpressing iBMDMs (H, upper). Cells were left untreated or incubated with cholesterol for 1 hour at 37°C, followed by staining with SNA or MAL. Mean fluorescence intensities were quantified by Flow Jo software (lower panels). (I) Confocal microscopy analysis of SA expression and distribution in WT and CD36-overexpressing iBMDMs. Cells were left untreated or incubated with cholesterol for 1 hour at 37°C, followed by staining with SNA or MAL (green). Cell membranes were stained with Dil dye (red), and nuclei were counterstained with DAPI (blue). Data are representative (A-F, left, G, H, upper and I) or pooled from at least three independent experiments (A-F, right, G and H, lower, mean ± SD). * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t -test).

Article Snippet: The primary antibodies used in this study were sourced from commercial suppliers as follows: Rabbit polyclonal antibodies against RBP4 (1 : 2,000, 11774–1-AP), CD36 (1 : 2,000, 18836–1-AP), STRA6 (1 : 3,000, 22001–1-AP), p65 (1 : 1,000, 10745–1-AP) and β-actin (1 : 20,000, 66009–1-lg) were purchased from Proteintech Group Inc. Rabbit polyclonal antibodies against CD36 (1 : 1,000, CY5796) and FLAG (1 : 10,000, AB0030) were purchased from Abways.

Techniques: Confocal Microscopy, Flow Cytometry, Fluorescence, Software, Expressing, Transfection, Plasmid Preparation, Incubation, Staining

Journal: PLOS Pathogens

Article Title: Retinol binding protein 4 enhances cellular cholesterol uptake to facilitate influenza A virus infection

doi: 10.1371/journal.ppat.1013623

Figure Lengend Snippet: (A, B) Flow cytometry analysis of α-2,3-linked SA or α-2,6-linked SA in WT, RBP4-deficient and CD36 overexpressing RBP4-deficient HEK293T cells, as wells as murine BMDMs transduced with CD36 retrovirus. Mean fluorescence intensities were quantified with FlowJo software. (C, D) qPCR analysis of vRNA levels of the NP gene or NP and M1 mRNA expression in HEK293T cells (C) or BMDMs (D) infected with IAV WSN strain (MOI = 5) during the viral attachment stage. (E) Flow cytometry analysis of α-2,3-linked SA or α-2,6-linked SA in WT and stable CD36-overexpressing iBMDMs cultured in serum-free medium. Cells were cultured with serum-free medium for 6 hours and subsequently stained with Lectins. Mean fluorescence intensities were quantified with FlowJo software. Data are pooled from three independent experiments (A-E, mean ± SD). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t -test).

Article Snippet: The primary antibodies used in this study were sourced from commercial suppliers as follows: Rabbit polyclonal antibodies against RBP4 (1 : 2,000, 11774–1-AP), CD36 (1 : 2,000, 18836–1-AP), STRA6 (1 : 3,000, 22001–1-AP), p65 (1 : 1,000, 10745–1-AP) and β-actin (1 : 20,000, 66009–1-lg) were purchased from Proteintech Group Inc. Rabbit polyclonal antibodies against CD36 (1 : 1,000, CY5796) and FLAG (1 : 10,000, AB0030) were purchased from Abways.

Techniques: Flow Cytometry, Transduction, Fluorescence, Software, Expressing, Infection, Cell Culture, Staining

Journal: PLOS Pathogens

Article Title: Retinol binding protein 4 enhances cellular cholesterol uptake to facilitate influenza A virus infection

doi: 10.1371/journal.ppat.1013623

Figure Lengend Snippet: (A) Experimental scheme for lentiviral transduction and influenza virus infection in mice. WT and RBP4-deficient mice were intravenously injected with lentivirus (10 9 PFU in 200 μL per mouse) followed by infection with 10 4 PFU of IAV (WSN strain) 5 days later. Lungs were collected 3 days post-IAV infection. (B) qPCR analysis of CD36 mRNA levels in lung tissues from WT, RBP4-deficient, and CD36-overexpressing RBP4-deficient mice as treated in (A). (C) Body weight changes of indicated mouse genotypes at day 3 post-IAV infection. (D) Plaque assay analysis of virus titer in lung tissues from mice described in (A). (E, F) Immunofluorescence images (E, left) and quantitative analysis of NP protein intensity (E, right) or immunoblotting analysis of M1, NP and CD36 protein expression (F) in lung tissues from the mice described in (A). Mean fluorescence intensity was determined by Image J software (E, right). Scale bars, 100 μm. (G, H) Representative H&E-stained lung sections (G) and corresponding histopathological scores (H) from indicated groups. Each dot in (B-D, H) represents an individual mouse. (I) Proposed model of RBP4-mediated promotion of influenza virus infection. Created in BioRender. Huang, L. (2025) https://BioRender.com/rll2ll1 . Data are representative of two independent experiments (E-G). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t -test).

Article Snippet: The primary antibodies used in this study were sourced from commercial suppliers as follows: Rabbit polyclonal antibodies against RBP4 (1 : 2,000, 11774–1-AP), CD36 (1 : 2,000, 18836–1-AP), STRA6 (1 : 3,000, 22001–1-AP), p65 (1 : 1,000, 10745–1-AP) and β-actin (1 : 20,000, 66009–1-lg) were purchased from Proteintech Group Inc. Rabbit polyclonal antibodies against CD36 (1 : 1,000, CY5796) and FLAG (1 : 10,000, AB0030) were purchased from Abways.

Techniques: Transduction, Virus, Infection, Injection, Plaque Assay, Immunofluorescence, Western Blot, Expressing, Fluorescence, Software, Staining