Knockdown of Hif-1α inhibited induction of activation markers upon hypoxia treatment in HSC-T6 cells.

<p>HSC-T6 cells in 6-well plates were transfected with either 100 nM Hif-1α siRNA or nonspecific (NS) siRNA for 24h and then cultured in room air or in 1% oxygen for 48h. (A) Cells were collected and cell lysates were subjected to detect Hif-1α, vimentin, α-SMA and β-actin with western blot; Densitometric analysis was performed using pooled data from three such experiments. Data are mean ± SD. (B) Hif-1α; (C) vimentin; (D) α-SMA. * : <i>P</i><0.05, ** : <i>P</i><0.01 (RA: room air, KD: knock down)..</p

1% O₂ induced apparent accumulation of Hif-1α and reorganization of F-actin in HSC-T6 cells.

<p>HSC-T6 cells, rat hepatic stellate cell line, were cultured in room air or in 1% oxygen. (A) Cells were collected at indicated time and cell lysates were subjected to detect Hif-1α and β-actin with western blot. (B) F-actin was stained using Fluorescein Isothiocyanate (FITC)-labeled phalloidin to capture F-actin of cells grown on the coverslips.</p

1% O₂ induced increased expression of vimentin and α-SMA in HSC-T6 cells.

<p>HSC-T6 cells were cultured in room air or in 1% oxygen. (A) Vimentin expression in HSC-T6 cells grown on the coverslips at 48 hours post-seeding were co-stained with anti-vimentin (red) and DAPI (blue) by immunocytochemistry. (B) Cells were collected at indicated time and cell lysates were subjected to detect vimentin and α-SMA. Densitometric analysis was performed using pooled data from three such experiments. Data are mean ± SD. (C) vimentin; (D) α-SMA. * : <i>P</i><0.05.</p

Expression of Hif-1α apparently increased in liver tissues of <i>Schistosoma</i><i>japonicum</i> infected mice.

<p>BALB/c female mice, 6–8 weeks old, were percutaneously infected with 25 cercariae of <i>Schistosoma</i><i>japonicum</i> through the shaved abdomen, sacrificed at 6 weeks post-infection and samples of liver were collected. The expression of Hif-1α and α-SMA in <i>Schistosoma</i><i>japonicum</i> infected (n=3) and non-infected (n=3) mice liver was detected with western blot and immunohistochemistry. (A) 1: protein extract from the liver of non-infected Balb/C mice, 2: protein extract from livers of <i>Schistosoma</i><i>japonicum</i> cercariae infected Balb/C mice. (B) Densitometric analysis (n=3). Data are mean ± SD. * : <i>P</i><0.05, ** : <i>P</i><0.01. (C) Acute inflammatory cell infiltration and granulomatous inflammation, surrounding eggs of <i>Schistosoma</i><i>japonicum</i>. (D) Expression of Hif-1α in non-infected mice liver. (E) Expression of Hif-1α in infected liver.</p

Rictor positively regulates B cell receptor signaling by modulating actin reorganization via ezrin

<div><p>As the central hub of the metabolism machinery, the mammalian target of rapamycin complex 2 (mTORC2) has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. Rictor is involved in B cell development, especially the peripheral development. However, the role of Rictor on B cell receptor (BCR) signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell–specfic Rictor knockout (KO) mice to investigate how Rictor regulates BCR signaling. We found that the key positive and negative BCR signaling molecules, phosphorylated Brutons tyrosine kinase (pBtk) and phosphorylated SH2-containing inositol phosphatase (pSHIP), are reduced and enhanced, respectively, in Rictor KO B cells. This suggests that Rictor positively regulates the early events of BCR signaling. We found that the cellular filamentous actin (F-actin) is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration is associated with a decreased humoral immune response in Rictor KO mice. The inhibition of actin polymerization with latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, in which Rictor regulates BCR signaling via actin reorganization.</p></div

B cell receptor (BCR) mobility is reduced in Rictor knockout (KO) B cells after antigenic stimulation.

<p>The primary B cells labeled with low concentration Alexa Fluor 647–Fab anti-immunoglobulin M (IgM) (Fc fragment antibody [Fc] specific) and high concentration Alexa Fluor 647–Fab anti-IgM (Fc specific) was placed on planar lipid bilayers containing anti-major histocompatibility complex (MHC) or anti-IgM, and single BCR molecule total internal reflection fluorescent (TIRF) images were acquired. Individual BCR molecules in TIRF images from 1 typical primary B cell indicate the instant diffusion coefficient (D0) by pseudocolored spots from 5 min to 15 min after stimulation. The display range of the pseudocolor is based on the D0 value of 10–4–0 μm²/s, a range that included most of the D0 values of the tracked BCRs. The accumulated trajectory footprints of individual BCR molecules in the entire time course (A–D). All of the D0 values were displayed as mean-square displacement (MSD) plots (E and F), single BCR molecules of the indicated numbers (G and H), and cumulative distribution probability (CDP) plots (I and J) from 3 independent experiments. Mann–Whitney <i>U</i> test was used to do the statistics, *<i>p</i> < 0.01. The numerical data (for A, B, C, D, E, F, G, H, I, and J) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001750#pbio.2001750.s006" target="_blank">S1 Data</a>.</p

Rictor deficiency reduces the humoral immune response.

<p>Flow cytometry analysis of folicular (FO) and marginal zone (MZ) B cells in spleen of wild-type (WT) and Rictor knockout (KO) mice (<i>n</i> = 8) immunized with 4-hydroxy-3-nitrophenylacetyl–keyhole limpet hemocyanin (NP-KLH) (A). The quantification of percentage (B) and number (C) of FO and MZ B cells in the spleen of WT and Rictor KO mice. Flow cytometry analysis of germinal center (GC) B cells in the spleen of WT and Rictor KO mice immunized with NP-KLH (D). The quantification of percentage and number of GC B cells in the spleen of WT and Rictor KO mice (E). Flow cytometry analysis of memory B (MBC), plasmablast (PBC), and plasma cell (PC) cells in the spleen of WT and Rictor KO mice immunized with NP-KLH (F and H). The quantification of percentage and number of MBC, PBC, and PC cells in the spleen of WT and Rictor KO mice (G, I, and J). Mean results from ELISA detecting NP-specific immunoglobulin M (IgM) and Immunoglobulin G1 (IgG1) from immunized WT and Rictor KO mice (<i>n</i> = 6) (K). T-test was used to do the statistics, **<i>p</i> < 0.001. The numerical data (for C, E, G, I, J, K, and L) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001750#pbio.2001750.s006" target="_blank">S1 Data</a>.</p

B cell receptor (BCR) cluster formation, B cell spreading, and BCR signalsome are reduced and actin polymerization is enhanced in Rictor knockout (KO) B cells.

<p>Splenic B cells were incubated with Alexa Fluor (AF) 546– monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, cells were stained for Alexa Fluor 488 (AF488)-phallodin. Cells were analyzed using confocal microscopy (CFm) and flow cytometry. Shown are representative images at indicated times (A and B) and the average values (±SD) of mean fluorescence intensity (MFI) of phallodin in the cytoplasm and on the plasma membrane at 10 min (C). About 50 cells from 3 independent experiments using NIS-Elements AR 3.2 software and the MFI of phallodin using flow cytometry (D). Splenic B cells from wild-type (WT) and Rictor KO mice were incubated with AF546-mB-Fab′–anti-Ig tethered to lipid bilayers at 37°C for indicated times. Cells were fixed, permeabilized, and stained. Shown are representative images (E and F) and the average values (± SD) of the MFI of filamentous actin (F-actin) (G), the MFI of the BCR (H), B cell contact area (I), and the MFI of phosphorylated Brutons tyrosine kinase pBtk (J) in the contact zone. Total internal reflection fluorescent microscopy (TIRFm) analysis of the spatial relationship of BCR with phosphotyrosine (pY) and pBtk in the contact zone of splenic B cells incubated with membrane-tethered Fab′–anti-immunoglobulin (Ig). The colocalization coefficients between BCR and pY and pBtk staining were determined using NIS-Elements AR 3.2 software (K). The data were generated using 20–90 cells from 3 independent experiments. Scale bars, 2.5 μm. Mann–Whitney <i>U</i> test was used to do the statistics, *<i>p</i> < 0.01. The numerical data (for C, D, G, H, I, J, and K) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001750#pbio.2001750.s006" target="_blank">S1 Data</a>.</p

B cell receptor (BCR)-induced serine/threonine kinase (Akt) activation is inhibited in Rictor knockout (KO) B cells.

<p>To mimic soluble antigen (sAg), splenic wild-type (WT) or Rictor KO B cells were incubated with Alexa Fluor (AF) 546–monobiotinylated (mB)-Fab′–anti-immunoglobulin (Ig) for 10 min at 4°C to label the BCR. Then, the cells were either incubated with streptavidin or with the medium alone (0 min) as a control at 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for phosphorylated Rictor (pRictor) (A) or phosphorylated Akt (pAkt) (D and E) and analyzed using confocal microscopy (CFm). The mean fluorescence intensity (MFI) of pRictor (C) or pAkt (F) and the correlation coefficients (B and H) between the BCR and pRictor or pAkt were quantified using NIS-Elements AR 3.2 software. Flow cytometry analysis of MFI of pAkt after stimulation with sAg (G). Shown are representative images and mean values (±SD) from 3 independent experiments, in which over 50 cells were individually analyzed using NIS-Elements AR 3.2 software. Scale bars, 2.5 μm. Mann–Whitney <i>U</i> test was used to do the statistics, * <i>p</i> < 0.01. The numerical data (for B, C, E, F, G, and H) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001750#pbio.2001750.s006" target="_blank">S1 Data</a>.</p

The recruitment of phosphorylated SH2-containing inositol phosphatase (pSHIP) to B cell receptor (BCR) clusters in B cells stimulated by soluble antigen (sAg) is increased in Rictor knockout (KO) B cells.

<p>Splenic B cells were incubated with Alexa Fluor (AF)546–monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for pSHIP and analyzed using confocal microscopy (CFm) or flow cytometry (A, B and D). The mean fluorescence intensity (MFI) of pSHIP was generated using NIS-Elements AR 3.2 software (C). Flow cytometry analysis of the MFI of pSHIP after stimulation with sAgs (D). The Pearson’s correlation coefficients between BCR and pSHIP staining in sAg-stimulated cells were determined using NIS-Elements AR 3.2 software (E). Shown are representative images at indicated times and the average values (±SD) of about 50 cells from 3 independent experiments. Scale bars, 2.5 μm. Mann–Whitney <i>U</i> test was used to do the statistics, *<i>p</i> < 0.01. The numerical data (for C, D, and E) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001750#pbio.2001750.s006" target="_blank">S1 Data</a>.</p