Fig 1: UGRP1 deficiency protects against S. pneumoniae‐induced pneumonia. (A) WT and UGRP1 KO mice were infected intratracheally with S. pneumoniae (4 × 105 CFU/mouse) and the survival time was assessed. (B–E) WT and UGRP1 KO mice were infected intratracheally with S. pneumoniae (105 CFU/mouse). IL‐6 in BAL (bronchoalveolar lavage) fluid was detected by ELISA at 1 day after inoculation (B, n = 5). Lungs were also harvested at 1 day post infection and Il6 (C, n = 6), Tlr2 and Nod2 (E, n = 4) mRNA levels were checked by qRT‐PCR. Haematoxylin and eosin (H&E) staining of lung tissue was performed as well (D, scale bar: 20 μm). (F) WT mice pre‐treated with control IgG or anti‐PDPN (B‐11) antibody (1 μg/mouse) were infected intratracheally with S. pneumoniae (4 × 105 CFU/mouse) to examine the survival time. (G) WT mice pre‐treated with control IgG or anti‐PDPN (B‐11) antibody (1 μg/mouse) were infected intratracheally with S. pneumoniae (105 CFU/mouse). IL‐6 in BAL fluid was detected by ELISA at 1 day after inoculation (n = 5). (H) WT mice treated with BSA or UGRP1 (1 μg/mouse) for 1 day, following by infected intratracheally with S. pneumoniae (105 CFU/mouse). IL‐6 in BAL fluid was detected by ELISA at 1 day after inoculation (n = 5). (I and J) WT mice pre‐treated with control IgG or anti‐PDPN (B‐11) antibody (1 μg/mouse) were infected intratracheally with S. pneumoniae (105 CFU/mouse). Lungs were harvested at 1 day post infection and Il6 mRNA levels were checked by qRT‐PCR (I, n = 4). H&E staining of lung tissue was performed as well (J, scale bar: 20 μm). (K) AMs from IgG or anti‐PDPN (B‐11) (1 μg/mouse) treated mice were stimulated with Pam3CSK4 for 6 h to check Il6 mRNA by qRT‐PCR (n = 3). (L) WT and UGRP1 KO mice pre‐treated with DMSO or RhoA activator (Narciclasine, 5 μg/mouse) were infected intratracheally with S. pneumoniae (4 × 105 CFU/mouse) to examine the survival time. (M and N) WT and UGRP1 KO mice pre‐treated with DMSO or RhoA activator (Narciclasine, 5 μg/mouse) were infected intratracheally with S. pneumoniae (105 CFU/mouse). IL‐6 in BAL fluid was detected at 1 day after inoculation by ELISA (K, n = 5). Lungs were also harvested at 1 day post infection and Il6 mRNA levels were checked by qRT‐PCR (L, n = 4). *p < .05, **p < .01 and ***p < .001, using a two‐tailed, unpaired Student's t‐test (C, E and I), one‐way analysis of variance (ANOVA) with Holm–Sidak's multiple comparisons test (M and N), two‐way ANOVA with Holm–Sidak's multiple comparisons test (B, G, H and K) or log‐rank (Mantel–Cox) test (A, F and L). Data from at least three independent experiments (mean ± SD) or representative data (D and J). Please also see Figure S6
Fig 2: UGRP1 enhances expression of TLR2, NOD2, MyD88 and NLRP3 by activation of NF‐κB. (A) PEMs were treated with the protein synthesis inhibitor CHX (10 μM) for 1 h followed by Pam3CSK4 stimulation with BSA or UGRP1 (0.5 μg/ml) for 6 h to measure Il1b mRNA by qRT‐PCR (n = 3). (B) The relative mRNA levels of gene from TLRs and NLRs pathway in PEMs after UGRP1 (0.5 μg/ml) treatment for 3 h (n = 3). (C) PEMs were treated with UGRP1 (0.5 μg/ml) for 3 or 6 h to check Tlr2, MyD88, Nod2 and Nlrp3 mRNA by qRT‐PCR (n = 3). (D) PEMs were treated with UGRP1 (0.5 μg/ml) for the indicated periods to check NLRP3, TLR2, MyD88 and NOD2 protein levels by immunoblot analysis. (E) PEMs were treated with the NF‐κB inhibitor PDTC (100 μM) for 1 h followed by BSA or UGRP1 (0.5 μg/ml) treatment for 6 h to measure Tlr2 and Nod2 mRNA by qRT‐PCR (n = 3). (F) PEMs were treated with UGRP1 (0.5 μg/ml) for 1 h and the relative amount of Tlr2 and Nod2 DNA binding to p65 was analysed by ChIP assay. (G) MyD88 KO PEMs were treated with UGRP1 (0.5 μg/ml) for 6 h to check Tlr2 and Nod2 mRNA by qRT‐PCR (n = 3). (H) PEMs were treated with IRAK1/4 inhibitor IRAK‐1‐4 Inhibitor I (10 μM), TAK1 inhibitor Takinib (10 μM), IKKβ inhibitor LY2409881 trihydrochloride (5 μM) or p‐IκBα inhibitor BAY 11–7082 (20 μM) for 1 h followed by UGRP1 (0.5 μg/ml) treatment for 6 h to measure Tlr2 and Nod2 mRNA by qRT‐PCR (n = 3). *p < .05, **p < .01 and ***p < .001, using a two‐tailed, unpaired Student's t‐test (G), one‐way analysis of variance (ANOVA) with Holm–Sidak's multiple comparisons test (C and H) or two‐way ANOVA with Holm–Sidak's multiple comparisons test (A and E). Data from at least three independent experiments (mean ± SD) or representative data (D). Please also see Figure S3
Fig 3: UGRP1 activates RhoA to enhance the interaction of IKKγ and IKKβ. (A and B) PEMs (A) and Cas9‐control or Cas9‐Pdpn‐deficient RAW264.7 cells (B) were treated with UGRP1 for indicated periods and the cell lysates were subjected to GST‐Rhotekin‐RBD pull‐down assay to precipitate the activated RhoA. (C) Myc‐RhoA, Myc‐RhoA (V14, constitutively active mutant) or Myc‐RhoA (N19, constitutively negative mutant) were transfected into 293T cells with FLAG‐IKKγ and HA‐IKKβ. Cell lysates were subjected to immunoprecipitation with anti‐FLAG. (D) Cas9‐control or Cas9‐Pdpn‐deficient RAW264.7 cells were treated with UGRP1 (0.5 μg/ml) for indicated periods and the cell lysates were subjected to immunoprecipitation with anti‐IKKγ. (E) RAW264.7 cells were treated with DMSO control or RhoA inhibitor (CCG‐1423, 20 μM) for 1 h followed by UGRP1 (0.5 μg/ml) stimulation for indicated periods and the cell lysates were subjected to immunoprecipitation with anti‐IKKγ. (F) Cas9‐control or Cas9‐Pdpn‐deficient RAW264.7 cells were treated with UGRP1 (0.5 μg/ml) for indicated periods and the cell lysates were subjected to detect phosphorylated (p‐) p65. (G) RAW264.7 cells were treated with DMSO control or RhoA inhibitor (CCG‐1423, 20 μM) for 1 h followed by UGRP1 (0.5 μg/ml) stimulation for indicated periods and the cell lysates were subjected to detect phosphorylated (p‐) p65. (H) RAW264.7 cells stably overexpressing WT RhoA or RhoA N19 mutant were stimulated by Pam3CSK4 (0.5 μg/ml) with BSA or UGRP1 (0.5 μg/ml) for 9 h. IL‐6 protein levels were measured by ELISA (n = 3, right pane) and overexpression of RhoA was checked by immunoblot (left panel). *p < .05, **p < .01 and ***p < .001, using two‐way analysis of variance (ANOVA) with Holm–Sidak's multiple comparisons test (H left panel). Data from at least three independent experiments (mean ± SD) or representative data (A–G, H right panel). Please also see Figure S5
Fig 4: UGRP1 enhances TLR2‐induced NF‐κB activation. (A–C) Immunoblot analysis of phosphorylated (p‐) p65 (A), p‐IKKα/β, degradation of IκBα (B) and p65 in the whole cell lysate (C, left) and nucleus (C, right) in BSA or UGRP1‐treated PEMs for 6 h following Pam3CSK4 stimulation for the indicated periods. β‐Actin and tubulin were used as the whole lysate protein control, Lamin‐B served as the nucleic protein control. (D) Immunoblot analysis of p‐IKKα/β, p‐p65 and degradation of IκBα in PEMs with treatment of UGRP1 for the indicated periods. (E) Alveolar macrophages (AMs) from WT or UGRP1 KO mice were stimulated with Pam3CSK4 or S. pneumoniae for 6 h to check Il6, Il1b and Tnfa mRNA by qRT‐PCR (n = 3). (F) PEMs were treated with the NF‐κB inhibitor PDTC (100μM) for 1 h followed by BSA or UGRP1 (0.5 μg/ml) treatment and Pam3CSK4 stimulation for 6 h to measure Il6 mRNA by qRT‐PCR (n = 3). (G) PEMs were treated with the NF‐κB inhibitor PDTC (100 μM) for 1h followed by BSA or UGRP1 (0.5 μg/ml) treatment for 6 h, then stimulated by Pam3CSK4 for 0.5 h, p65 in the whole cell lysate (left) and nucleus (right) was analysed by immunoblot. Tubulin were used as the whole lysate protein control, Lamin‐B served as the nucleic protein control. *p < .05, **p < .01 and ***p < .001, using two‐way analysis of variance (ANOVA) with Holm–Sidak's multiple comparisons test (E and F). Data from at least three independent experiments (mean ± SD) or representative data (A–D, G). Please also see Figure S2
Fig 5: UGRP1 was identified to positively regulate inflammation in S. pneumoniae stimulated macrophages. (A) Immunoblot analysis of UGRP1 protein expression in murine lung, spleen, liver, kidney, PBMCs, BMDMs and PEMs. (B) PEMs were treated with BSA or UGRP1 (0.5 μg/ml) for 1 h followed by S. pneumoniae (104 CFU/well) infection for 6 h to check Il6, Il1b and Tnfa mRNA by qRT‐PCR (n = 3). (C) RAW264.7 cells were treated with BSA or UGRP1 (0.5 μg/ml) for 1 h followed by Pam3CSK4 (0.5 μg/ml) stimulation for 6 or 9 h to check Il6, Il1b and Tnfa mRNA by qRT‐PCR (n = 3). (D) PEMs were treated with BSA or UGRP1 (0.5 μg/ml) for 1 h followed by MDP (1 μg/ml) or LTA (0.5 μg/ml) stimulation for 6h to check Il6, Il1b and Tnfa mRNA by qRT‐PCR (n = 3). (E and F) PEMs (E, n = 3) or RAW264.7 cells (F, n = 3) were treated with BSA or UGRP1 (0.5 μg/ml) for 1 h followed by Pam3CSK4 (0.5 μg/ml), LTA (0.5 μg/ml), MDP (1 μg/ml) stimulation or S. pneumoniae (104 CFU/well) infection for 12 h to check IL‐6 and TNFα concentrations by ELISA. *p < .05, **p < .01 and ***p < .001, using two‐way analysis of variance (ANOVA) with Holm–Sidak's multiple comparisons test (B–F). Data from at least three independent experiments (mean ± SD) or representative data (A). Please also see Figure S1
Supplier Page from Abcam for Anti-SCGB3A2 antibody [EPR11463]