recombinant mouse fstl1 proteins  (R&D Systems)

Journal: Diagnostics

Article Title: FSTL1 Suppresses Triple-Negative Breast Cancer Lung Metastasis by Inhibiting M2-like Tumor-Associated Macrophage Recruitment toward the Lungs

doi: 10.3390/diagnostics13101724

Figure Lengend Snippet: Lower FSTL1 expression in breast cancer tissues compared to normal breast tissues, and high expression of FSTL1 meant prolonged survival. ( A – D ) FSTL1 mRNA expression decreased in primary breast cancer. ( E , F ) FSTL1 had no significant effect on the survival rate of patients with breast invasive carcinoma (BRCA) and TNBC. ( G , H ) High FSTL1 expression could increase the survival rate of patients with breast cancer and those with positive nodal metastasis. *** p < 0.001.

Article Snippet: Recombinant mouse IL-4 (Catalog: #214-14; PeproTech, Cranbury, NJ, USA) and recombinant mouse FSTL1 proteins (Catalog: #1738-FN-050; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing

Journal: Diagnostics

Article Title: FSTL1 Suppresses Triple-Negative Breast Cancer Lung Metastasis by Inhibiting M2-like Tumor-Associated Macrophage Recruitment toward the Lungs

doi: 10.3390/diagnostics13101724

Figure Lengend Snippet: FSTL1 had no effect on the proliferation and EMT markers of 4T1 cells. ( A ) The proliferation ability of 4T1 TNBC cells showed no change after 600 ng/mL FSTL1 treatment for 1–4 d. ( B ) Under different FSTL1 concentration treatments, expression of 4T1 EMT markers, including E-cadherin and N-cadherin, showed no change after 12 h treatment.

Article Snippet: Recombinant mouse IL-4 (Catalog: #214-14; PeproTech, Cranbury, NJ, USA) and recombinant mouse FSTL1 proteins (Catalog: #1738-FN-050; R&D Systems, Minneapolis, MN, USA).

Techniques: Concentration Assay, Expressing

Journal: Diagnostics

Article Title: FSTL1 Suppresses Triple-Negative Breast Cancer Lung Metastasis by Inhibiting M2-like Tumor-Associated Macrophage Recruitment toward the Lungs

doi: 10.3390/diagnostics13101724

Figure Lengend Snippet: Total and M2 macrophage ratios increased in breast cancer lung metastasis in Fstl1 +/− mice. ( A , B ) Total (F4/80+) and M2 (F4/80+CD11c-CD206+) macrophage ratios in WT and Fstl1 +/− mice on the 0th and 28th day. ( C – E ) Expression of several M2-related macrophage markers (IL4, IL13, Arg-1, and IL10) in WT and Fstl1 +/− mice on the 0th and 28th day. ( F , G ) Western blot of metastasis-related markers (TGFβ and MMP9) of WT and Fstl1 +/− mice on the 28th day. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant mouse IL-4 (Catalog: #214-14; PeproTech, Cranbury, NJ, USA) and recombinant mouse FSTL1 proteins (Catalog: #1738-FN-050; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Western Blot

Journal: Diagnostics

Article Title: FSTL1 Suppresses Triple-Negative Breast Cancer Lung Metastasis by Inhibiting M2-like Tumor-Associated Macrophage Recruitment toward the Lungs

doi: 10.3390/diagnostics13101724

Figure Lengend Snippet: FSTL1 inhibited macrophage migration toward 4T1 breast cancer cells. ( A , B , D , E ) RAW264.7/Ana-1 (the upper transwell) and 4T1 cells (the lower transwell), stimulator: FSTL1 (1 μg/mL) ± IL4 (20 ng/mL), transmembrane macrophage morphology was observed, and numbers (5 fields per slide) were calculated after 12 h co-culture. ( C , F ) Migrated RAW264.7/Ana-1 macrophages toward 4T1 breast cancer cells decreased in FSTL1 (1 μg/mL) treatment group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant mouse IL-4 (Catalog: #214-14; PeproTech, Cranbury, NJ, USA) and recombinant mouse FSTL1 proteins (Catalog: #1738-FN-050; R&D Systems, Minneapolis, MN, USA).

Techniques: Migration, Co-Culture Assay

Journal: Diagnostics

Article Title: FSTL1 Suppresses Triple-Negative Breast Cancer Lung Metastasis by Inhibiting M2-like Tumor-Associated Macrophage Recruitment toward the Lungs

doi: 10.3390/diagnostics13101724

Figure Lengend Snippet: Recombinant mouse FSTL1 protein inhibited several cytokines produced by 4T1 TNBC cells. ( A ) FSTL1 (1 μg/mL)-treated 4T1 cells with or without IL4 (20 ng/mL) in vitro. Cytokine secretions: ( B ) CCL2, ( C ) CCL5, ( D ) CSF1, ( E ) VEGF-α, and ( F ) TGF-β. ( G ) Schematic representation of the effect of FSTL1 on breast cancer metastatic tumor growth. The model depicts the suppression of CSF1, VEGF-α, and TGF-β secretion in 4T1 breast cancer cells by FSTL1, which decreases macrophage recruitment toward TNBC cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant mouse IL-4 (Catalog: #214-14; PeproTech, Cranbury, NJ, USA) and recombinant mouse FSTL1 proteins (Catalog: #1738-FN-050; R&D Systems, Minneapolis, MN, USA).

Techniques: Recombinant, Produced, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/β-catenin signaling in obstructed kidneys in vivo

doi: 10.1016/j.jbc.2022.102010

Figure Lengend Snippet: Expression of FSTL1 in fibrogenic kidneys. A , Fstl1 expression in sham-operated and UUO kidneys. The data were extracted from the bulk RNA-Seq dataset . The y -axis shows fragments per kilobase per million mapped fragments (FPKM) (n = 4 per group). B – D , mRNA and protein levels of FSTL1 in the kidneys of mice subjected to UUO. Eight-week-old male mice were subjected to UUO on the left ureters. Mice were sacrificed 1, 3, and 7 days after UUO surgery. Right kidneys (Ctrl) and left kidneys (UUO) were collected and analyzed for Fstl1 mRNA levels ( B ) or for FSTL1 protein levels ( C ). Quantitative analysis of FSTL1 protein levels was performed by densitometry ( D ). E and F , violin plots of Fstl1 gene expression in different cell populations of UUO kidneys and in the mesenchymal/stromal cell cluster marked by Pdgfrb. The data were extracted from the single-cell RNA-Seq dataset on the renal cortex of UUO kidneys . The y -axis shows the log-scale normalized read count. G and H , cellular localization of FSTL1 in mouse UUO kidneys and human kidneys with IgA nephropathy (IgAN). Frozen sections of kidneys collected 3 days (D3) after UUO surgery ( G ) or IgAN kidneys at grade III ( H ) were subjected to immunofluorescent staining for FSTL1 ( red ) and PDGFR-β ( green ). Red asterisks indicate nonspecific bands, and black asterisks indicate FSTL1-specific bands. GAPDH was used as the loading control for Western blotting, and Rpl19 was used as the internal control for real-time PCR. n = 4 for B . ∗ p < 0.05; ∗∗ p < 0.01; and ∗∗∗ p < 0.001. FSTL1, follistatin-like 1; IgA, immunoglobulin A; PDGFR-β, platelet-derived growth factor receptor beta; UUO, unilateral ureteral obstruction.

Article Snippet: To determine whether there are direct interactions between FSTL1 and WNT3a, or between FZD4 and FSTL1, recombinant WNT3a (catalog no.: 315-20; PeproTech), FSTL1 (catalog no.:1738-FN; R&D Systems), and FZD4-Fc (catalog no.: 194-FZ; R&D Systems) proteins were added to Tris-buffered saline/0.1% Triton X-100 at indicated concentrations and incubated overnight at 4 °C.

Techniques: Expressing, RNA Sequencing, Gene Expression, Staining, Control, Western Blot, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/β-catenin signaling in obstructed kidneys in vivo

doi: 10.1016/j.jbc.2022.102010

Figure Lengend Snippet: FSTL1 enhances canonical Wnt signaling. A , both mFSTL1 and hFSTL1 proteins augmented mWnt3a mediated 8× TOPflash luciferase reporter activity in HEK293T cells. Cells were transfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids. Cells then were treated with and without mWnt3a protein (20 ng/ml) in the absence or the presence of mFSTL1 or hFSTL1 (200 ng/ml) before luciferase assay was performed. B , both mFSTL1 and hFSTL1 proteins promoted mWnt3a-mediated 8× TOPflash luciferase reporter activity in NRK-49F cells. Cells were transfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids. Cells then were treated with and without mWnt3a protein (20 ng/ml) in the presence of increasing amounts of mFSTL1 protein ( left panel ) or treated with and without mWnt3a protein (10 ng/ml) in the absence or the presence of hFSTL1 protein ( right panel ), before luciferase assay was performed. C , transfected FSTL1 promoted mWnt3a-induced 8× TOPflash luciferase reporter activity in a dose-dependent manner. NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids and increasing doses of FSTL1-HA plasmid. Cells then were treated with or without mWnt3a protein (15 ng/ml) before luciferase assay was performed ( right panel ). The left panel shows FSTL1 expression in cell culture medium. Albumin was used as loading control. D , transfected FSTL1 potentiated transfected Wnt3a-induced 8× TOPflash luciferase reporter activity. NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids, either alone or with FSTL1-HA plasmid, in the absence or the presence of Wnt3a plasmid. About 24 h after transfection, cells were serum starved for 16 h before luciferase assay was performed. E and F , siRNA-mediated inhibition of FSTL1 attenuated Wnt3a-induced 8× TOPflash luciferase reporter activity. NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids in the presence of scramble (Ctrl) or two Fstl1 siRNA sequences (siFstl1, 110 nM) either separately ( E ) or in combination ( F ). About 24 h after transfection, cells were incubated with or without Wnt3a protein (30 ng/ml) in serum-free medium for 16 h before cells were harvested for luciferase assay. G , the effects of siRNA-mediated inhibition of FSTL1 on Wnt3a signaling were rescued by FSTL1 overexpression. NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids in the presence of scramble (Ctrl) or Fstl1 siRNAs and with or without FSTL1-HA plasmid. About 24 h after transfection, cells were incubated with or without Wnt3a protein in serum-free medium for 16 h before cells were harvested for luciferase assay. n = 3 to 4. ∗∗∗ p < 0.001. FSTL1, follistatin-like 1; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; hFSTL1, human FSTL1; mFSTL1, mouse FSTL1; NRK-49F, normal rat kidney 49 fibroblast cell.

Article Snippet: To determine whether there are direct interactions between FSTL1 and WNT3a, or between FZD4 and FSTL1, recombinant WNT3a (catalog no.: 315-20; PeproTech), FSTL1 (catalog no.:1738-FN; R&D Systems), and FZD4-Fc (catalog no.: 194-FZ; R&D Systems) proteins were added to Tris-buffered saline/0.1% Triton X-100 at indicated concentrations and incubated overnight at 4 °C.

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Cell Culture, Control, Inhibition, Incubation, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/β-catenin signaling in obstructed kidneys in vivo

doi: 10.1016/j.jbc.2022.102010

Figure Lengend Snippet: FSTL1 interacts with canonical Wnt ligands. A , FSTL1 interacts with Wnt1. HEK293T cells were transfected with Wnt1 plasmid in the absence or the presence of FSTL1-HA plasmid. Cell lysates were immunoprecipitated with anti-FSTL1 to pull down Wnt1 ( left panel ). Reciprocal IP was performed ( right panel ). Western blotting was performed on whole lysates and precipitates as indicated. B , FSTL1 interacts with Wnt2 and Wnt3a. HEK293T cells were transfected with FATL1-HA in the absence or the presence of Wnt2-V5 or Wnt3a-V5 plasmid. Cell lysates were immunoprecipitated with IgG or anti-V5 antibody to pull down FSTL1 ( left panel ). Reciprocal IP was also performed ( right panel s ). C , FSTL1 interacts with Wnt8a, Wnt8b, Wnt9a, and Wnt10b. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of V5-tagged Wnt8a, Wnt8b, Wnt9a, and Wnt10b plasmids. Cell lysates were immunoprecipitated with anti-V5, followed by Western blotting with anti-FSTL1 or anti-V5 antibody on the precipitates and lysates as indicated. D , interaction of FSTL1 and Wnt2 occurs in the extracellular space. HEK293T cells were transfected with Wnt2-V5 in the absence or the presence of FSTL1-HA plasmid. Cell culture medium was immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and medium as indicated. E and F , interaction of FSTL1 and Wnt3a ( E ) or Wnt10b ( F ) occurs in the extracellular space. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of Wnt3a-V5 ( E ) or Wnt10b-V5 ( F ) plasmid. Cell culture medium was immunoprecipitated with anti-V5 antibody, followed by Western blotting with anti-V5 or anti-FSTL1 on the precipitates and medium as indicated. G , ALK3 does not interact with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 in the absence or the presence of FSTL1-HA or ALK3-HA plasmid. Cell lysates were immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and lysates as indicated. H , FSTL1 interacts with Wnt1 and Wnt3a in the kidney. Lysates from mouse kidneys with UUO for 7 days were immunoprecipitated with a combination of anti-Wnt1 and anti-Wnt3a antibodies or normal IgG, and the precipitates and lysates were subjected to Western blotting as indicated. I , FSTL1 directly interacts with Wnt3a. Recombinant Wnt3a protein (1.5 μg) was incubated with and without recombinant FSTL1 protein (3 μg) in 300 μl Tris-buffered saline/0.1% Triton X-100 overnight at 4 °C. Input samples were collected, and remaining mixtures were incubated with anti-FSTL1 antibodies followed by Protein G agarose. Eluted proteins and inputs were analyzed by Western blotting as indicated. J , domain organization of full-length (FL) and mutant FSTL1 proteins. K , interactions of EC–VWC and EC domains of FSTL1 with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of FL, EC–VWC, or EC plasmid. Cell lysates were immunoprecipitated with anti-FSTL1. Western blotting was performed on whole lysates and precipitates as indicated. L , VWC but not FS domain of FSTL1 interacts with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of 3× FLAG-FS or 3× FLAG-VWC plasmid. Cell lysates were immunoprecipitated with anti-FLAG. Western blotting was performed on whole lysates and precipitates as indicated. EC, the extracellular calcium–binding domain; FS, the follistatin-like domain; FSTL1, follistatin-like 1; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; IgG, immunoglobulin G; IP, immunoprecipitation; UUO, unilateral ureteral obstruction; VWC, a domain with homology to von Willebrand factor type C domain.

Article Snippet: To determine whether there are direct interactions between FSTL1 and WNT3a, or between FZD4 and FSTL1, recombinant WNT3a (catalog no.: 315-20; PeproTech), FSTL1 (catalog no.:1738-FN; R&D Systems), and FZD4-Fc (catalog no.: 194-FZ; R&D Systems) proteins were added to Tris-buffered saline/0.1% Triton X-100 at indicated concentrations and incubated overnight at 4 °C.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Cell Culture, Recombinant, Incubation, Saline, Mutagenesis, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/β-catenin signaling in obstructed kidneys in vivo

doi: 10.1016/j.jbc.2022.102010

Figure Lengend Snippet: Impacts of overexpression and neutralization of FSTL1 on renal fibrosis and on the canonical Wnt/β-catenin pathways in UUO kidneys. A – C , effects of FSTL1 overexpression on renal fibrosis and on the Wnt/β-catenin pathway. Male mice at 8 weeks of age were subjected to UUO on left ureters. FSTL1-HA or pcDNA3.1 plasmid was injected via the tail vein on day 2 after UUO surgery. Mice were sacrificed the next day, and right (Ctrl) and left (UUO) kidneys were collected for analysis. A , kidney lysates were analyzed by Western blotting for FSTL1 expression and the fibrosis markers α-SMA, fibronectin-1 (Fn-1), and collagen 1 (Col IA1) ( left panel ). Levels of α-SMA, Fn-1, and Col IA1 relative to GAPDH are presented ( right panels ). B , immunofluorescence for α-SMA and Fn-1 and Masson’s trichrome staining (MTS) on sections from the UUO kidneys of mice injected with pcDNA3.1 or FSTL1-HA plasmids. Frozen sections were used for immunofluorescent staining for α-SMA ( green ) and Fn-1 ( red ). Paraffin sections were used for MTS. The fibrotic blue area was quantified. C , kidney lysates were analyzed by Western blotting for the Wnt/β-catenin pathway (p-LRP6, p-GSK3β, active β-catenin, and β-catenin) ( left panel ). Levels of p-LRP6 relative to LRP6; p-GSK3β relative to GSK3β; and active β-catenin and β-catenin relative to GAPDH are presented ( right panels ). D , effects of neutralization of FSTL1 on the Wnt/β-catenin pathway. Male mice at 8 weeks of age were subjected to UUO on left ureters. Normal goat IgG or goat anti-FSTL1 antibody was injected (i.p.) at 5 μg/g body weight 7 days after UUO surgery. About 6 h later, kidney samples were harvested: kidney lysates were analyzed by Western blotting for p-LRP6, active β-catenin, and β-catenin ( left panel ). Levels of p-LRP6 relative to LRP6 and active β-catenin relative to β-catenin and GAPDH are presented ( right panels ). ∗∗ p < 0.01; ∗∗∗ p < 0.001. FSTL1, follistatin-like 1; HA, hemagglutinin; IgG, immunoglobulin G; LRP6, low-density lipoprotein receptor–related protein 6; α-SMA, alpha-smooth muscle actin; TGF-β, transforming growth factor beta; UUO, unilateral ureteral obstruction.

Article Snippet: To determine whether there are direct interactions between FSTL1 and WNT3a, or between FZD4 and FSTL1, recombinant WNT3a (catalog no.: 315-20; PeproTech), FSTL1 (catalog no.:1738-FN; R&D Systems), and FZD4-Fc (catalog no.: 194-FZ; R&D Systems) proteins were added to Tris-buffered saline/0.1% Triton X-100 at indicated concentrations and incubated overnight at 4 °C.

Techniques: Over Expression, Neutralization, Plasmid Preparation, Injection, Western Blot, Expressing, Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/β-catenin signaling in obstructed kidneys in vivo

doi: 10.1016/j.jbc.2022.102010

Figure Lengend Snippet: FSTL1 activates β-catenin and fibroblasts. A , FSTL1 protein elevated active β-catenin levels induced by Wnt3a proteins in NRK-49F cells. FSTL1 protein (500 ng/ml) and Wnt3a (15 ng/ml) were preincubated overnight before they were added to serum-starved NRK-49F cells for 3 h. Cell lysates were used for Western blotting for active β-catenin levels ( upper panel ). Levels of active β-catenin relative to GAPDH are presented ( lower panel ). B – D , transfected FSTL1-HA promoted β-catenin levels and fibroblast activation induced by transfected Wnt3a in NRK-49F cells. Cells were transfected with Wnt3a plasmid (50 ng/ml) in the presence or the absence of FSTL1-HA plasmid (200 ng/ml). Cells were serum starved for 24 h before cells were harvested for Western blotting for p-LRP6, LRP6, active β-catenin, β-catenin, fibronectin (Fn-1), collagens (Col 1A1 and Col 1A2), FSTL1, and Wnt3a ( B ). Levels of p-LRP6 relative to LRP6, active β-catenin relative to β-actin, and β-catenin relative to β-actin were presented ( C ). Levels of Fn-1, Col 1A1, and Col 1A2 relative to β-actin were also presented ( D ). FSTL1, follistatin-like 1; HA, hemagglutinin; NRK-49F, normal rat kidney 49 fibroblast cell.

Article Snippet: To determine whether there are direct interactions between FSTL1 and WNT3a, or between FZD4 and FSTL1, recombinant WNT3a (catalog no.: 315-20; PeproTech), FSTL1 (catalog no.:1738-FN; R&D Systems), and FZD4-Fc (catalog no.: 194-FZ; R&D Systems) proteins were added to Tris-buffered saline/0.1% Triton X-100 at indicated concentrations and incubated overnight at 4 °C.

Techniques: Western Blot, Transfection, Activation Assay, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/β-catenin signaling in obstructed kidneys in vivo

doi: 10.1016/j.jbc.2022.102010

Figure Lengend Snippet: FSTL1 interacts with FZD4 but not LRP6 and increases the association of Wnt3a with FZD4. A , FSTL1 does not interact with LRP6. HEK293T cells were transfected with LRP6 plasmid in the absence or the presence of FSTL1-HA plasmid. Cell lysates were immunoprecipitated with anti-FSTL1, and Western blotting was performed on whole lysates and precipitates as indicated ( left panel ). In reciprocal immunoprecipitation, HEK293T cells were transfected with FSTL1-HA plasmid in the absence or the presence of LRP-6 plasmid. Cell lysates were immunoprecipitated with anti-LRP6, and Western blotting was performed on whole lysates and precipitates as indicated ( right panel ). Short exposure (SE) did not show any FSTL1 band in the two lanes, and long exposure (LE) showed similar FSTL1 bands in density between the two lanes. B , FSTL1 interacts with FZD4. HEK293T cells were transfected with FZD4 in the absence or the presence of FSTL1-HA plasmid. Cell lysates were immunoprecipitated with anti-FSTL1 antibody to pull down FZD4. Western blotting was performed on whole lysates and precipitates as indicated. C , interaction of FZD4 and FSTL1 in the presence of LRP6. HEK293T cells were transfected with FSTL1-HA plasmid in the absence or the presence of FZD4 or LRP6 plasmids. Cell lysates were immunoprecipitated with anti-FZD4 antibody. Western blotting was performed on whole lysates and precipitates as indicated. D , FSTL1 directly interacts with FZD4. Recombinant FSTL1 protein (1 μg) was incubated with and without recombinant FZD4-Fc protein (3 μg) in 300 μl TBS/0.1% Triton X-100 overnight at 4 °C. Input samples were collected, and remaining mixtures were incubated with Protein A agarose. Eluted proteins and inputs were analyzed by Western blotting as indicated. E , interactions of EC–VWC and EC domains of FSTL1 with FZD4. HEK293T cells were transfected with SNAP-FZD4 plasmid in the absence or the presence of full-length (FL), EC–VWC, or EC plasmid. Cell lysates were immunoprecipitated with anti-FSTL1. Western blotting was performed on whole lysates and precipitates as indicated. F , neither VWC nor FS domain of FSTL1 interacts with Wnt3a. HEK293T cells were transfected with SNAP-FZD4 plasmid in the absence or the presence of FLAG-FS or FLAG-VWC plasmid. Cell lysates were immunoprecipitated with anti-FLAG. G , FSTL1, FZD4, and LRP6 form a complex. HEK293T cells were transfected with FSTL1-HA plasmid in the absence or the presence of FZD4 or LRP6 plasmid. Cell lysates were immunoprecipitated with anti-LRP6 antibody. Western blotting was performed on whole lysates and precipitates as indicated. H , interaction of FZD4 and LRP6 in the presence of FSTL1. HEK293T cells were transfected with LRP6 plasmid in the absence or the presence of FZD4 or FSTL1-HA plasmid. Cell lysates were immunoprecipitated with anti-FZD4 antibody. Western blotting was performed on whole lysates and precipitates as indicated. I , summary of the interactions between various FSTL1 domains with WNT3a and FZD4. FSTL1, follistatin-like 1; FZD, Frizzled; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; LE, long exposure; LRP, low-density lipoprotein receptor–related protein; SE, short exposure; SNAP-FZD4, SNAP-tagged FZD4; TBS, Tris-buffered saline.

Article Snippet: To determine whether there are direct interactions between FSTL1 and WNT3a, or between FZD4 and FSTL1, recombinant WNT3a (catalog no.: 315-20; PeproTech), FSTL1 (catalog no.:1738-FN; R&D Systems), and FZD4-Fc (catalog no.: 194-FZ; R&D Systems) proteins were added to Tris-buffered saline/0.1% Triton X-100 at indicated concentrations and incubated overnight at 4 °C.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Recombinant, Incubation, Saline

Journal: The Journal of Biological Chemistry

Article Title: Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/β-catenin signaling in obstructed kidneys in vivo

doi: 10.1016/j.jbc.2022.102010

Figure Lengend Snippet: FSTL1 increases the association of Wnt3a with FZD4. A , FSTL1 increases the level of Wnt3a pulled down by FZD4-Fc in a solution. Wnt3a protein (750 ng) was incubated overnight at 4 °C with and without FZD4-Fc protein (1 μg), and in the absence or the presence of FSTL1 protein (2 μg) in TBS buffer containing 0.1% Triton X-100 (500 μl). Input samples were collected, and remaining mixtures were incubated with protein A beads, which had been blocked with 0.1% BSA in TBS. Eluted proteins and inputs were analyzed by Western blotting as indicated. B , FSTL1 increases the binding of Wnt3a to FZD4 on cell surface. HEK293 cells were transfected with plasmids for SNAP-FZD4 (100 ng/ml) and the chaperone MESD (50 ng/ml). About 24 h after transfection, cells were serum starved overnight. Cells then were incubated for 3 h with Wnt3a conditioned medium (CM) in the absence or the presence of FSTL1-HA CM. Cells were incubated with 1 μM cell-impermeable biotinylated SNAP substrate (BG-PEG12-Biotin) for 15 min at room temperature. After washing with PBS, lysates were incubated with streptavidin–agarose, and the precipitates were used for Western blotting as indicated. C , working model showing how FSTL1 enhances renal fibrosis. In CKD, FSTL1 expression increases, and FSTL1 increases the presentation of Wnt ligands to FZD receptors, thus enhancing Wnt/β-catenin signaling and renal fibrosis. D – F , the EC domain enhances Wnt3a signaling. In E and F , HEK293T ( D ) or NRK-49F cells ( E ) were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids, either alone or with Wnt3a plasmid, in the absence or the presence of the plasmid for EC domain. In F , NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids, and increasing doses of EC plasmid before the cells were incubated with Wnt3a CM. ∗∗∗ p < 0.001. BSA, bovine serum albumin; CKD, chronic kidney disease; EC, extracellular calcium–binding domain; FSTL1, follistatin-like 1; FZD, Frizzled; HA, hemagglutinin; HEK293 human embryonic kidney 293 cell line; MESD, mesoderm development LRP chaperone; NRK-49F, normal rat kidney 49 fibroblast cell; SNAP-FZD4, SNAP-tagged FZD4; TBS, Tris-buffered saline.

Article Snippet: To determine whether there are direct interactions between FSTL1 and WNT3a, or between FZD4 and FSTL1, recombinant WNT3a (catalog no.: 315-20; PeproTech), FSTL1 (catalog no.:1738-FN; R&D Systems), and FZD4-Fc (catalog no.: 194-FZ; R&D Systems) proteins were added to Tris-buffered saline/0.1% Triton X-100 at indicated concentrations and incubated overnight at 4 °C.

Techniques: Incubation, Western Blot, Binding Assay, Transfection, Expressing, Luciferase, Plasmid Preparation, Saline

Journal: Molecular Metabolism

Article Title: Ups and downs: The PPARγ/p-PPARγ seesaw of follistatin-like 1 and integrin receptor signaling in adipogenesis

doi: 10.1016/j.molmet.2021.101400

Figure Lengend Snippet: The expression of FSTL1 in preadipocytes differentiation and adipose tissues of mice. (A) The protein levels of FSTL1 in inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), retroperitoneal white adipose tissue (rWAT) and brown adipose tissue (BAT) of C57BL/6J mice. 50 kDa, glycosylated; 37 kDa, hypoglycosylated. (B) mRNA levels of Fstl1 in iWAT, eWAT, rWAT, and BAT were detected by RT-PCR. (C) Time course of FSTL1 protein levels during MEFs differentiation detected by western blot. (D) Time course of Fstl1, Pparγ, and C/ebpα gene expression during MEF differentiation by RT-PCR. (E, F) Time course of FSTL1 protein levels during SVF and 3T3L1 differentiation.

Article Snippet: Recombinant mouse FSTL1 protein was incubated with cells for 24 h (1738-FN, R&D Systems, 100 ng/ml).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Gene Expression

Journal: Molecular Metabolism

Article Title: Ups and downs: The PPARγ/p-PPARγ seesaw of follistatin-like 1 and integrin receptor signaling in adipogenesis

doi: 10.1016/j.molmet.2021.101400

Figure Lengend Snippet: Fstl1 deletion inhibited the differentiation of MEFs and SVF by promoting PPARγ phosphorylation. (A) MEFs extracted from E13.5 wild-type (WT) and Fstl1−/− (KO) mouse embryos were induced to differentiate into adipocytes for 10 days and stained with Oil Red O. The oil red O stain was extracted and quantitated by measuring absorbance at 492 nm (n = 3), Scale bar, 200 μm 100 μm, 50 μm. (B) Western blot analysis of protein levels of PPARγ and p-PPARγ during MEF differentiation. PPARγ and p-PPARγ band intensities were normalized relative to the GAPDH bands, and the PPARγ/p-PPARγ ratio was calculated and analyzed. (C) Relative mRNA levels of adipogenic genes (Pparγ, Fabp4, C/ebpα, Adipoq) of MEFs (as in A) detected by qPCR. Two-way ANOVA followed by Bonferroni post-tests, n = 3. (D) SVF extracted from iWAT of E18.5 wild-type (WT) and Fstl1−/− (KO) mouse embryos were induced to differentiate into adipocytes and stained with Oil Red O. The oil red O stain was extracted and quantitated by measuring absorbance at 492 nm (n = 3), Scale bar, 200 μm 100 μm, 50 μm. (E) Western blot analysis of protein levels of PPARγ and p-PPARγ during SVF differentiation. PPARγ and p-PPARγ band intensities were normalized relative to the GAPDH bands, and the PPARγ/p-PPARγ ratio was calculated and analyzed. (F) Relative mRNA levels of adipogenic genes (Pparγ, Fabp4, C/ebpα, Adipoq) of SVF (as in D) detected by qPCR. Two-way ANOVA followed by Bonferroni post-tests, n = 3. Data are expressed as mean ± SEM, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005.

Article Snippet: Recombinant mouse FSTL1 protein was incubated with cells for 24 h (1738-FN, R&D Systems, 100 ng/ml).

Techniques: Phospho-proteomics, Staining, Western Blot

Journal: Molecular Metabolism

Article Title: Ups and downs: The PPARγ/p-PPARγ seesaw of follistatin-like 1 and integrin receptor signaling in adipogenesis

doi: 10.1016/j.molmet.2021.101400

Figure Lengend Snippet: Differentiation of 3T3L1 cells was inhibited or promoted by Fstl1 knockdown or overexpression. (A) 3T3L1 fibroblasts expressing shFstl1 or vector control were differentiated into adipocytes and stained with Oil Red O at day 8. The oil red O stain was extracted and quantitated by measuring absorbance at 492 nm, Student's t test, n = 3. Scale bar, 100 μm. (B) The protein levels of PPARγ and p-PPARγ during differentiation of the 3T3L1-NC and 3T3L1-shFstl1 groups were detected by western blot. PPARγ and p-PPARγ band intensities were normalized relative to the GAPDH bands, and the PPARγ/p-PPARγ ratio was calculated and analyzed. (C) mRNA levels of adipogenic genes (Fabp4, C/ebpα, Adipoq) in 3T3L1 cells (as in A) were detected by qPCR. Two-way ANOVA followed by Bonferroni post-tests, n = 3. (D) 3T3L1 fibroblasts with Fstl1 overexpression or control were induced for differentiation using cocktail with no rosiglitazone, and stained with Oil Red O at day 8. The oil red O stain was extracted and quantitated by measuring absorbance at 492 nm, n = 3. Scale bar, 100 μm. (E) The protein levels of PPARγ and p-PPARγ during differentiation of 3T3L1 cells with Fstl1 OE and control groups were detected by western blot. The PPARγ/p-PPARγ ratio was analyzed as mentioned above. (F) mRNA levels of adipogenic genes in 3T3L1 cells (as in D) were detected by qPCR. Two-way ANOVA followed by Bonferroni post-tests, n = 3. Data are expressed as mean ± SEM, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005.

Article Snippet: Recombinant mouse FSTL1 protein was incubated with cells for 24 h (1738-FN, R&D Systems, 100 ng/ml).

Techniques: Knockdown, Over Expression, Expressing, Plasmid Preparation, Control, Staining, Western Blot

Journal: Molecular Metabolism

Article Title: Ups and downs: The PPARγ/p-PPARγ seesaw of follistatin-like 1 and integrin receptor signaling in adipogenesis

doi: 10.1016/j.molmet.2021.101400

Figure Lengend Snippet: A single site mutation of N142 glycosylation was sufficient for the function of FSTL1. (A) Western blot analysis of FSTL1 protein extracted from 3T3L1 cells and supernatant (upper panel). Western blot analysis of FSTL1 protein extracted from supernatant or supernatant reacted with deglycosylation enzymes. (B) Western blot analysis of FSTL1 protein both in cell lysate and media of 3T3L1 with N-glycosylation mutants (N142, N173, N178). (C) Glycosylated modification sites (N142, N173, N178) were mutated separately or totally in 3T3L1 fibroblasts. Oil Red O staining was measured after inducing differentiation for 8 days. The oil red O stain was extracted and quantitated by measuring absorbance at 492 nm, n = 3. Scale bar, 100 μm. (D) mRNA levels of adipogenic genes (Fabp4, C/ebpα, Adipoq) of 3T3L1 cells with N142 site mutation after inducing differentiation for 8 days. Two-way ANOVA followed by Bonferroni post-tests, n = 3. (E) Relative protein levels of PPARγ and p-PPARγ during differentiation of 3T3L1 cells with N142 site mutation by western blot. PPARγ and p-PPARγ band intensities were normalized relative to the GAPDH bands, and the PPARγ/p-PPARγ ratio was calculated and analyzed. Data are expressed as mean ± SEM, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005.

Article Snippet: Recombinant mouse FSTL1 protein was incubated with cells for 24 h (1738-FN, R&D Systems, 100 ng/ml).

Techniques: Mutagenesis, Glycoproteomics, Western Blot, Modification, Staining

Journal: Molecular Metabolism

Article Title: Ups and downs: The PPARγ/p-PPARγ seesaw of follistatin-like 1 and integrin receptor signaling in adipogenesis

doi: 10.1016/j.molmet.2021.101400

Figure Lengend Snippet: The conversion of PPARγ to p-PPARγ was contributed by ERK1/2 phosphorylation when Fstl1 was deficient. (A) The protein levels of p-ERK1/2 and T-ERK1/2 in 3T3L1 cells treated with a p-ERK1/2 inhibitor (U0126) were determined by western blot. (B) Oil red O staining of the lipid droplets in three groups (Fstl1 knockdown, Fstl1 KD + U0126, control) of 3T3L1 cells after inducing differentiation for 8 days. Oil red O stain was quantified as mentioned above, One-way ANOVA followed by Dunnett's multiple comparison test. Scale bar, 100 μm. (C) mRNA levels of adipogenic (Fabp4, C/ebpα, Adipoq) and lipogenic genes (Acc, Fas) in 3T3L1 cells as in B. Two-way ANOVA followed by Bonferroni post-tests, n = 3. (D) Western blot analysis of protein levels of p-ERK1/2, T-ERK1/2, p-PPARγ, and PPARγ during differentiation of 3T3L1 cells as in B. p-ERK1/2, T-ERK1/2, p-PPARγ, and PPARγ band intensities were normalized relative to the GAPDH bands. p-ERK/T-ERK and PPARγ/p-PPARγ ratios were analyzed. Data are expressed as mean ± SEM, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005.

Article Snippet: Recombinant mouse FSTL1 protein was incubated with cells for 24 h (1738-FN, R&D Systems, 100 ng/ml).

Techniques: Phospho-proteomics, Western Blot, Staining, Knockdown, Control, Comparison

Journal: Molecular Metabolism

Article Title: Ups and downs: The PPARγ/p-PPARγ seesaw of follistatin-like 1 and integrin receptor signaling in adipogenesis

doi: 10.1016/j.molmet.2021.101400

Figure Lengend Snippet: Fstl1 deficiency promoted ERK1/2 phosphorylation and then the conversion of PPARγ to p-PPARγ. (A) Western blot analysis of protein levels of PPARγ, p-PPARγ, p-ERK1/2 and T-ERK1/2 in 3T3L1 cells treated with rosiglitazone at indicated concentrations. (B) Oil red O staining of lipid droplets in 3T3L1 cells of Fstl1 knockdown, Fstl1 KD + RG and control group after inducing differentiation for 8 days. The oil red O stain was extracted and quantitated by measuring absorbance at 492 nm, One-way ANOVA followed by Dunnett's multiple comparison test. Scale bar, 100 μm. (C) mRNA levels of adipogenic (Fabp4, C/ebpα, Adipoq) and lipogenic genes (Acc, Fas) in cells as mentioned in B. Two-way ANOVA followed by Bonferroni post-tests, n = 3. (D) Western blot analysis of protein levels of p-PPARγ, PPARγ, p-ERK1/2, and T-ERK1/2 during differentiation of 3T3L1 cells as in B. p-ERK1/2, T-ERK1/2, p-PPARγ, and PPARγ band intensities were normalized relative to the GAPDH bands, PPARγ/p-PPARγ and p-ERK/T-ERK ratios were analyzed. Data are expressed as mean ± SEM, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005.

Article Snippet: Recombinant mouse FSTL1 protein was incubated with cells for 24 h (1738-FN, R&D Systems, 100 ng/ml).

Techniques: Phospho-proteomics, Western Blot, Staining, Knockdown, Control, Comparison

Journal: Molecular Metabolism

Article Title: Ups and downs: The PPARγ/p-PPARγ seesaw of follistatin-like 1 and integrin receptor signaling in adipogenesis

doi: 10.1016/j.molmet.2021.101400

Figure Lengend Snippet: FSTL1 exerted biological effects by inhibiting the integrin β1-mediated activation of the focal adhesion kinase pathway. (A, B) Heatmap of selected differential genes enrichment based on the RNA sequencing of the 3T3L1-NC and 3T3L1-shFstl1 groups after inducing for differentiation. (C) Gene Ontology analysis of differential genes in RNA-seq. (D) Western blot analysis of ITGB1 protein levels in the 3T3L1-NC and 3T3L1-shFstl1 groups after inducing for differentiation. GAPDH was used for loading control, ITGB1 band intensity was normalized relative to the GAPDH band. Student's t -test, ∗∗∗P < 0.005. (E, F) The culturing dishes were coated or not coated with Collagen Type I, and the protein levels of p-FAK (s397, s576/577, s925), FAK, p-ERK1/2 and T-ERK1/2 in 3T3L1 cells with Fstl1 knockdown (overexpression in F) and control group were analyzed by western blot. GAPDH was used for the loading control. (G) 293T cells were transfected with plasmids expressing Flag-tagged Fstl1 and HA-tagged Itgb1 . Total cell lysate were subjected to IP with Flag or HA antibodies and immunoblotted for FSTL1 and ITGB1. Unspecific IgG antibodies were used as a control. (H) FSTL1 and ITGB1 protein complex was pulled down using glutathione-Sepharose beads and then subjected to western blot analyses with anti-FSTL1 antibody to confirm the presence of FSTL1 (upper). The presence of ITGB1 was detected with anti-ITGB1 antibody (lower). (I) 293T cells were transfected with HA-tagged Itgb1 . The culturing dishes were coated or not coated with collagen Type I. ITGB1-HA was immunoprecipitated with HA-agarose and incubated with 100 ng FSTL1 protein. COL1 and ITGB1 were detected with anti-COL1 and anti-HA antibodies, respectively.

Article Snippet: Recombinant mouse FSTL1 protein was incubated with cells for 24 h (1738-FN, R&D Systems, 100 ng/ml).

Techniques: Activation Assay, RNA Sequencing, Western Blot, Control, Knockdown, Over Expression, Transfection, Expressing, Immunoprecipitation, Incubation

Journal: Molecular Metabolism

Article Title: Ups and downs: The PPARγ/p-PPARγ seesaw of follistatin-like 1 and integrin receptor signaling in adipogenesis

doi: 10.1016/j.molmet.2021.101400

Figure Lengend Snippet: FSTL1 was required for obesity development in adipose knockout mice. (A) Adipocyte-specific Fstl1 mutant (f/f-aP2) and control (f/f) mice were fed high fat diet (HFD) for 14 weeks. Total body weight was determined weekly. Student's t-test, n = 9 for controls and n = 11 for KOs. (B) Weights of individual fat pads from f/f-aP2 and control mice following 14 weeks on HFD. n = 10 per group. (C) Representative images of wet fat pads of iWAT and eWAT from f/f-aP2 and f/f mice following 14 weeks on HFD. (D) Intraperitoneal glucose tolerance test (1.5 mg/kg) in male, HFD feeding f/f-aP2 and f/f mice. Two-way ANOVA followed by Bonferroni post-tests, n = 6 per group. (E, F) Cell size of iWAT and eWAT from male f/f-aP2 and f/f mice following 14 weeks on HFD. Representative images from hematoxylin and eosin (H&E) stained sections. Scale bar, 100 μm. Mean adipocyte area of the representative section from iWAT and eWAT was analyzed. Two-way ANOVA followed by Bonferroni post-tests, n = 3 per group. (G) The protein levels of FSTL1, p-PPARγ, PPARγ, p-ERK1/2, T-ERK1/2, p-FAK (s397), and FAK in iWAT of mice were analyzed by western blot. GAPDH was used for loading control. Data are expressed as mean ± SEM, ∗P < 0.05, ∗∗P < 0.01.

Article Snippet: Recombinant mouse FSTL1 protein was incubated with cells for 24 h (1738-FN, R&D Systems, 100 ng/ml).

Techniques: Knock-Out, Mutagenesis, Control, Staining, Western Blot

Journal: Scientific Reports

Article Title: BBS4 regulates the expression and secretion of FSTL1, a protein that participates in ciliogenesis and the differentiation of 3T3-L1

doi: 10.1038/s41598-017-10330-0

Figure Lengend Snippet: Cilia, BBS4 and BBS2 knockdown result in a reduction of intracellular and secreted FSTL1. (A) hTERT-RPE1 cells were transfected with control siRNA (Cneg) or siRNA BBS4 to produce BBS4 knock-down (BBS4). Secreted FSTL1 and intracellular FSTL1 levels were analyzed in cell culture media (SN) and whole cell lysates (WCL) respectively by Western blot using anti-FSTL1. Full-length blots are shown in Figure . (B) hTERT-RPE1 cells were transfected with siRNA to target FSTL1 and IFT88 or siRNA Cneg. Secreted FSTL1 and intracellular FSTL1 were analyzed in SN and WCL respectively by Western blot using anti-FSTL1. In both A and B Ponceau S staining of an unspecific blotted protein and anti-α tubulin were used as loading controls to normalize FSTL1 abundance. Full-length gels are shown in Figure . (C) Quantitative representation of densitometry readings of 3 combined experiments shown in (A) and (B). BBS4-KD cells show reduced levels of both intra- and extracellular FSTL1. (D) hTERT-RPE1 cells were transfected with siRNA BBS2 or siRNA Cneg and secreted FSTL1 was analyzed by Western blot anti-FSTL1 and Ponceau S staining was used to normalize. The Cneg and BBS2 KD set of lanes were cropped from the same blot. Full-length blots are shown in Figure . (E) Densitometry readings of Western blot shown in (D). The results shown are representative of two independent experiments. (F) SN of hTERT-RPE1 cells transfected with siRNA BBS4 were analyzed by Western blot with anti-Laminin and Ponceau S staining to normalize (upper panel). Densitometry readings of the western blot showed that BBS4-KD does not affect Laminin secretion (lower panel). The full-length blot is shown in Figure . (G) qRT-PCR was performed to analyze FSTL1 gene expression in hTERT-RPE1 cells transfected with the following siRNAs: Cneg, BBS4 , FSTL1 , IFT88 and BBS2 . FSTL1 mRNA levels are represented as % expression relative to control siRNA (Cneg)-transfected cells. FSTL1 expression is reduced by BBS2 and BBS4 KD cells as well as by IFT88/cilia knockdown. Data from at least two independent experiments, designed with biological duplicates, and three technical replicates were normalized relative to Cneg siRNA transfected cells and combined for the analysis. In all cases, error bars represent standard deviation. **: P = 0,001–0,01; ***: P = 0,0001–0,001 and ****: P < 0,0001, ANOVA or t-test.

Article Snippet: Primary antibodies: mouse anti-α tubulin clone B-5-1-2, mouse anti-acetylated tubulin clone 6-11-B-1, rabbit anti-γ tubulin and rabbit anti-laminin from Sigma; goat anti-human FSTL1, goat anti-mouse Fstl1, and mouse Fstl1 recombinant protein from R&D Systems, rabbit anti-BBS4 from ProteinTech, rabbit anti-LC3B from Cell Signaling Technology, mouse anti-human golgin-97 clone CDF4 from Invitrogen, and rabbit anti-calnexin, mouse anti-LAMP2 clone H4B4 and rat anti-BrdU from Abcam.

Techniques: Transfection, Cell Culture, Western Blot, Staining, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: Scientific Reports

Article Title: BBS4 regulates the expression and secretion of FSTL1, a protein that participates in ciliogenesis and the differentiation of 3T3-L1

doi: 10.1038/s41598-017-10330-0

Figure Lengend Snippet: Lysosome inhibition rescues the FSTL1 degradation induced by BBS4 knockdown. (A) hTERT-RPE1 cells were transfected with siRNA Cneg, siRNA IFT88 or siRNA BBS4 for 48 hours and incubated for 6 hours with MG132. Cell lysates were analyzed by Western blot anti-FSTL1, anti α tubulin and anti-Ubiquitin. Full-length blots are shown in Figure . (B) hTERT-RPE1 cells were transfected with siRNA Cneg, siRNA BBS4 (top panel) or siRNA IFT88 (bottom panel; for each condition, lanes cropped from the same gel are shown) during 24 hours and incubated with chloroquine for an additional 24 hours. Full-length blots are shown in Figure . (C-F) Quantification of experiment shown in ( B ). Densitometry readings were used to calculate ratios of FSTL1/Tubulin to quantitate intracellular FSTL1 and FSTL1/Ponceau S to measure secreted FSTL1. Chloroquine treatment rescued intracellular levels of FSTL1 without affecting its extracellular abundance. Results shown are representative of three independent experiments performed with biological triplicates or duplicates as shown. Error bars represent standard deviation and ns: P > 0.05; *: P = 0,01–0,05; **: P = 0,001–0,01; ***: P = 0,0001–0,001, ANOVA test.

Article Snippet: Primary antibodies: mouse anti-α tubulin clone B-5-1-2, mouse anti-acetylated tubulin clone 6-11-B-1, rabbit anti-γ tubulin and rabbit anti-laminin from Sigma; goat anti-human FSTL1, goat anti-mouse Fstl1, and mouse Fstl1 recombinant protein from R&D Systems, rabbit anti-BBS4 from ProteinTech, rabbit anti-LC3B from Cell Signaling Technology, mouse anti-human golgin-97 clone CDF4 from Invitrogen, and rabbit anti-calnexin, mouse anti-LAMP2 clone H4B4 and rat anti-BrdU from Abcam.

Techniques: Inhibition, Transfection, Incubation, Western Blot, Standard Deviation

Journal: Scientific Reports

Article Title: BBS4 regulates the expression and secretion of FSTL1, a protein that participates in ciliogenesis and the differentiation of 3T3-L1

doi: 10.1038/s41598-017-10330-0

Figure Lengend Snippet: FSTL1 accumulates in lysosomes in the absence of BBS4. (A ) Control hTERT-RPE1 were co-immunostained for Golgi (top row, Golgin 97 in cyan ) or ER (bottom row, Calnexin, cyan ) together with FSTL1 ( magenta ). (B) Analysis of colocalization of FSTL1 with each organelle marker represented as Manders coefficients showing that FSTL1 colocalizes with both organelles. (C) hTERT-RPE1 control and BBS4-KD cells were co-immunostained to study colocalization of FSTL1 ( red ) and lysosomes (LAMP2 in green ). Colocalization is evidenced by colocalized pixels in white . (D) Manders coefficients were determined showing that in BBS4 KD cells, FSTL1 is accumulated in lysosomes. In all cases, scale bars represent 10 µm and ****: P < 0,0001, t-test.

Article Snippet: Primary antibodies: mouse anti-α tubulin clone B-5-1-2, mouse anti-acetylated tubulin clone 6-11-B-1, rabbit anti-γ tubulin and rabbit anti-laminin from Sigma; goat anti-human FSTL1, goat anti-mouse Fstl1, and mouse Fstl1 recombinant protein from R&D Systems, rabbit anti-BBS4 from ProteinTech, rabbit anti-LC3B from Cell Signaling Technology, mouse anti-human golgin-97 clone CDF4 from Invitrogen, and rabbit anti-calnexin, mouse anti-LAMP2 clone H4B4 and rat anti-BrdU from Abcam.

Techniques: Marker

Journal: Scientific Reports

Article Title: BBS4 regulates the expression and secretion of FSTL1, a protein that participates in ciliogenesis and the differentiation of 3T3-L1

doi: 10.1038/s41598-017-10330-0

Figure Lengend Snippet: FSTL1 regulates ciliogenesis. ( A ) Upper panels: primary cilia length was measured in hTERT-RPE1 cells. FSTL1 KD cells present shorter cilia than control cells and comparable to IFT88 KD cells. Scatter plots with a line at the mean are shown and error bars represent 95% confidence intervals. Lower panels: micrographs showing basal bodies (γ-tubulin) in cyan , cilia (acetylated tubulin) in red and nuclei in blue (DAPI). The results shown are representative of four independent experiments. (B) Upper panel: hTERT-RPE1 FSTL1 KD cells were incubated with conditioned media starting at 24 hours after silencing of FSTL1 and primary cilia length was measured after 24 additional hours. In cells incubated with conditioned media, cilia length was comparable to that of control cells. Scatter plots with a line at the mean are shown and error bars represent 95% confidence intervals. The results shown are representative of two independent experiments. Lower panels: micrographs showing basal bodies (γ-tubulin) in cyan , cilia (acetylated tubulin) are in red and nuclei are in blue (DAPI). Statistical significance is shown as compared to siRNA Cneg except when noted. In all cases, scale bars represent 10 µm. ns: P > 0.05; and ****: P < 0,0001, ANOVA test.

Article Snippet: Primary antibodies: mouse anti-α tubulin clone B-5-1-2, mouse anti-acetylated tubulin clone 6-11-B-1, rabbit anti-γ tubulin and rabbit anti-laminin from Sigma; goat anti-human FSTL1, goat anti-mouse Fstl1, and mouse Fstl1 recombinant protein from R&D Systems, rabbit anti-BBS4 from ProteinTech, rabbit anti-LC3B from Cell Signaling Technology, mouse anti-human golgin-97 clone CDF4 from Invitrogen, and rabbit anti-calnexin, mouse anti-LAMP2 clone H4B4 and rat anti-BrdU from Abcam.

Techniques: Incubation

Journal: Scientific Reports

Article Title: BBS4 regulates the expression and secretion of FSTL1, a protein that participates in ciliogenesis and the differentiation of 3T3-L1

doi: 10.1038/s41598-017-10330-0

Figure Lengend Snippet: Cilia, BBS4 and FSTL1 are coordinated during 3T3-L1 differentiation. (A) 3T3-L1 cells were transfected with control siRNA or siRNA Bbs4 and secreted Fstl1 was detected in the supernatant by Western blot anti-Fstl1 (upper panel). Densitometry analysis of western blot normalized with Ponceau-S staining of an unspecific blotted protein as a loading control (lower panel). Knocking-down Bbs4 also reduces Fstl1 secretion in 3T3-L1 cells. Full-length blot and gel are shown in Figure . (B) qRT-PCR analysis of Fstl1 and Bbs4 genes expression in 3T3-L1 cells during differentiation from proliferative cells to day 11 after induction to differentiate. Error bars represent standard deviation. Three biological replicates were analyzed. (C) The percentage of ciliated cells was analyzed in 3T3-L1 cells at different time points during differentiation: day 0, day 4 and day 10. Mean values are shown and error bars represent 95% confidence intervals for the mean. (D) Cilia length was analyzed in 3T3-L1 cells at the same time points. Data is shown as scatter plots with a line at the mean and error bars representing 95% confidence intervals. (E) mRNA levels of the adipocyte differentiation marker Pparγ , Fstl1 and Bbs4 were analyzed in 3T3-L1 cells at the same time points during differentiation. The results shown are representative of multiple independent experiments (see also controls in Figs and ). Statistical significance is shown as compared to D0 except when noted and ns: P > 0.05; *: P = 0,01-0,05; **: P = 0,001–0,01; ***: P = 0,0001–0,001 and ****: P < 0,0001, hypothesis test for proportions, ANOVA or Kruskal Wallis test.

Article Snippet: Primary antibodies: mouse anti-α tubulin clone B-5-1-2, mouse anti-acetylated tubulin clone 6-11-B-1, rabbit anti-γ tubulin and rabbit anti-laminin from Sigma; goat anti-human FSTL1, goat anti-mouse Fstl1, and mouse Fstl1 recombinant protein from R&D Systems, rabbit anti-BBS4 from ProteinTech, rabbit anti-LC3B from Cell Signaling Technology, mouse anti-human golgin-97 clone CDF4 from Invitrogen, and rabbit anti-calnexin, mouse anti-LAMP2 clone H4B4 and rat anti-BrdU from Abcam.

Techniques: Transfection, Western Blot, Staining, Quantitative RT-PCR, Expressing, Standard Deviation, Marker

Journal: Scientific Reports

Article Title: BBS4 regulates the expression and secretion of FSTL1, a protein that participates in ciliogenesis and the differentiation of 3T3-L1

doi: 10.1038/s41598-017-10330-0

Figure Lengend Snippet: Blocking Fstl1-mediated ciliogenesis inhibits 3T3-L1 preadipocyte differentiation. (A ) Western blot of Fstl1 showing upregulation of the protein upon induction to differentiate and the levels of the protein in Fstl1 KD 3T3-L1 cells. The data is representative of two independent experiments. Full-length blots are shown in Figure . ( B ) Clonal expansion in 3T3-L1 cells upon induction to differentiate was measured by a pulse of BrdU and immunofluorescence. The difference in the percentage of BrdU positive cells between induced an uninduced cultures is shown. Fstl1 KD does not inhibit clonal expansion. Result of two independent experiments with three biological replicates each. Approximately 500 cells were counted per condition, per experiment. (C and G) The percentage of ciliated cells was analyzed in 3T3-L1 cells at day 4 (C) and day 10 (G) of differentiation. Data are shown as a line at the mean and error bars represent 95% confidence intervals. (D and H) Cilia length was measured in 3T3-L1 cells at day 4 (D) and day 10 (H). Approximately 100 cilia were measured per condition, per experiment. Scatter plots with a line at the mean are shown and error bars represent 95% confidence intervals. (E and I ) Micrographs showing examples of cilia used in the analysis at D4 and D10 respectively. Basal bodies (γ-tubulin) are in cyan , cilia (acetylated tubulin) are in red and nuclei (DAPI) are in blue . Scale bars represent 5μm. (F and J ) qRT-PCR analysis of the adipocyte differentiation markers Pparγ , Cebpα , Fabp4 , Scd1 and Atgl in 3T3-L1 cells at day 4 (F) and day 10 (J) of differentiation. Bars represent the fold change upon induction of each gene relative to Gapdh comparing induced with uninduced cells. Error bars represent standard deviation. ( K ) Oil red staining comparing lipid accumulation between control and Fstl1 KD cells. The data shown are representative of two independent experiments. ns: P > 0.05; ** P = 0,001-0,01; *** P = 0,0001–0,001 and **** P < 0,0001, hypothesis test for proportions, Kruskal Wallis or t-test.

Article Snippet: Primary antibodies: mouse anti-α tubulin clone B-5-1-2, mouse anti-acetylated tubulin clone 6-11-B-1, rabbit anti-γ tubulin and rabbit anti-laminin from Sigma; goat anti-human FSTL1, goat anti-mouse Fstl1, and mouse Fstl1 recombinant protein from R&D Systems, rabbit anti-BBS4 from ProteinTech, rabbit anti-LC3B from Cell Signaling Technology, mouse anti-human golgin-97 clone CDF4 from Invitrogen, and rabbit anti-calnexin, mouse anti-LAMP2 clone H4B4 and rat anti-BrdU from Abcam.

Techniques: Blocking Assay, Western Blot, Immunofluorescence, Quantitative RT-PCR, Standard Deviation, Staining

Journal: Scientific Reports

Article Title: BBS4 regulates the expression and secretion of FSTL1, a protein that participates in ciliogenesis and the differentiation of 3T3-L1

doi: 10.1038/s41598-017-10330-0

Figure Lengend Snippet: Addition of recombinant Fstl1 interferes with 3T3-L1 preadipocyte differentiation. ( A ) Western blot showing comparable levels of Fstl1 in supernatant from control cells and recombinant Fstl1 diluted in fresh culture media at 200 ng/ml. (B) Cilia length was measured in control and Fstl1 KD 3T3-L1 cells supplemented with 0, 100 ng/ml or 200 ng/ml of recombinant Fstl1. Cilia length was rescued in a dose dependent manner by the addition of Fstl1. Data are shown as a line at the mean and error bars represent 95% confidence intervals. At least 120 cilia were measured per condition and results shown are representative of three independent experiments. (C) Cilia length at D4 was measured in uninduced and induced to differentiate control cells and cells cultured with 200 ng/ml of recombinant Fstl1. Scatter plots with a line at the mean are shown and error bars represent 95% confidence intervals. ( D , E ) qRT-PCR analysis of gene expression of differentiation markers Pparγ , Cebpα , Fabp4 , Scd1 and Atgl in 3T3-L1 cells at day 6 ( D ) and day 10 ( E ) of differentiation. Bars represent the fold change upon induction of each gene relative to Gapdh comparing induced cells with uninduced cells. Error bars represent standard deviation. Two biological and three technical replicates were analyzed. ( F ) Oil red staining showing lipid accumulation in induced control cells in comparison with cells supplemented with recombinant Fstl1 protein. ns: P > 0.05; *: P = 0,01-0,05; **: P = 0,001-0,01; ***: P = 0,0001–0,001 and ****: P < 0,0001, ANOVA, or t-test.

Article Snippet: Primary antibodies: mouse anti-α tubulin clone B-5-1-2, mouse anti-acetylated tubulin clone 6-11-B-1, rabbit anti-γ tubulin and rabbit anti-laminin from Sigma; goat anti-human FSTL1, goat anti-mouse Fstl1, and mouse Fstl1 recombinant protein from R&D Systems, rabbit anti-BBS4 from ProteinTech, rabbit anti-LC3B from Cell Signaling Technology, mouse anti-human golgin-97 clone CDF4 from Invitrogen, and rabbit anti-calnexin, mouse anti-LAMP2 clone H4B4 and rat anti-BrdU from Abcam.

Techniques: Recombinant, Western Blot, Cell Culture, Quantitative RT-PCR, Expressing, Standard Deviation, Staining, Comparison