Journal: Nature medicine

Article Title: Interactions among HCLS1, HAX1 and LEF-1 proteins are essential for G-CSF-triggered granulopoiesis

doi: 10.1038/nm.2958

Figure Lengend Snippet: HCLS1 interacts with LEF-1. (a) In silico analysis of LEF-1 using ScanSite software43,44; the putative HCLS1-binding site in LEF-1 surrounds Pro191 (bold). LEF-1 contains a β-catenin binding domain (β-catenin BD); context-dependent domain (CDD) and high-mobility group protein domain (HMG D). (b) Representative micrograph of a bone marrow section from a healthy individual, showing staining for HCLS1 by immunohistochemistry (brown) and counterstaining with hematoxylin (blue). Inset shows a segmented granulocyte expressing high levels of HCLS1 protein. Scale bar, 5 μm. (c) HEK 293T cells were transfected with the indicated hemagglutinnin-tagged LEF-1 constructs and with an HCLS1 construct and lysates were used for immunoprecipitation (IP) of LEF-1 and HCLS1 followed by western blotting of the immunoprecipitates using the indicated antibodies. The isotype control used for IP was negative (not shown). HA, hemagglutinin. (d) Representative western blot of LEF-1 immunoprecipitates from Jurkat cells using the indicated antibodies. (e) The interaction between native LEF-1 and HCLS1 was analyzed by isolating complexes on BN-PAGE (first dimension) followed by identification of proteins within the complexes by SDS-PAGE (second dimension) and western blotting for HCLS1, LEF-1, dnLEF-1 and HAX1. Shown are representative images of BN-PAGE with marker and Jurkat cell lysates as a positive control (left) and of western blot of complexes isolated from slices 3–7 of BN-PAGE (right). (f) Following pull-down assays of Jurkat cell lysates using human recombinant HCLS1-GST-tagged protein or GST protein, representative western blots are shown using antibodies to LEF-1 and HCLS1. (g) CD34+ bone marrow cells from two healthy individuals were treated with 10 ng ml−1 G-CSF. At the indicated time points, total and phospho-HCLS1 proteins were quantified using FACS analysis. Shown is the percentage of phospho-HCLS1+ cells in the HCLS1+ cell population. Data are mean ± s.d. and are derived from three independent experiments, each in duplicate, *P < 0.05. (h) Endogenous HCLS1 from primary bone marrow CD34+ cells treated or untreated with 10 ng ml−1 G-CSF for the indicated lengths of time was immunoprecipitated with antibody to HCLS1. Coimmunoprecipitates of LEF-1 and HCLS1 were detected by western blotting. Shown are representative western blot images of immunoprecipitates (top) and of the inputs used for immunoprecipitation (bottom); the isotype control for IP was negative (not shown). Lysate of untreated CD34+ cells was used as a positive control.

Article Snippet: Billadeau), rabbit polyclonal antibody to HCLS1 (1:400, Cell Signaling Technology, 4503S), mouse monoclonal antibody to HCLS1 (1:500, BD Bioscience, 610541), rabbit polyclonal antibody to phospho-HCLS1 (Tyr397; 1:100, Cell Signaling Technology, 4507S), rabbit monoclonal antibody to LEF-1 (1:500, Cell Signaling Technology, 22305), mouse monoclonal antibody to LEF-1 (1:1,000, CalBiochem, NA64), mouse monoclonal antibody to Lyn (1:200, Santa Cruz Biotechnology, SC-7274), mouse monoclonal antibody to Syk (1:400, Biolegend, 626201), rabbit polyclonal antibody to HAX1 (Santa Cruz Biotechnology, SC-28268), rabbit polyclonal antibody to HA tag (1:1,000, Santa Cruz Biotechnology, SC-2365), mouse monoclonal antibody to β-actin (1:1,000, Santa Cruz Biotechnology, SC-47778) and secondary bovine anti-mouse or goat anti-rabbit HRP-conjugated antibody (1:5,000, Santa Cruz Biotechnology, SC-2371 and SC-2004, respectively).

Techniques: In Silico, Binding Assay, Staining, Immunohistochemistry, Expressing, Transfection, Construct, Immunoprecipitation, Western Blot, Control, SDS Page, Marker, Positive Control, Isolation, Recombinant, Derivative Assay

Journal: Nature medicine

Article Title: Interactions among HCLS1, HAX1 and LEF-1 proteins are essential for G-CSF-triggered granulopoiesis

doi: 10.1038/nm.2958

Figure Lengend Snippet: HCLS1 is essential for G-CSF-triggered granulopoiesis in vivo and in vitro. (a) HCLS1 mRNA expression in bone marrow CD33+ cells isolated from healthy individuals (Ctrl) and from patients with severe congenital neutropenia (CN), idiopathic neutropenia (IN) or neutropenia due to metabolic defects (MN) treated (+) or untreated (−) with G-CSF. HCLS1 mRNA expression was measured by quantitative RT-PCR (qRT-PCR) and normalized to β-actin. AU, arbitrary units; data are mean ± s.d. of triplicates, *P < 0.05. (b) CD33+ bone marrow cells from two healthy individuals (Ctrl) and two patients with congenital neutropenia (CN) were treated with 10 ng ml−1 G-CSF; at the indicated time points total and phospho-HCLS1 protein were quantified using FACS analysis. Shown is the percentage of phospho-HCLS1+ cells in HCLS1+ cell population. Data are mean ± s.d. and are derived from three independent experiments, each in duplicate, *P < 0.05. (c–e) CD34+ cells from healthy individuals (n = 3) were transduced with the indicated constructs and RFP+ cells were sorted on day 4 of culture. Two different shRNA constructs, HCLS1b and HCLS1f, targeting two different regions of HCLS1 mRNA, were used. Mock indicates untransduced cells cultured in the same conditions as transduced cells. (c) Expression of the indicated mRNAs was assessed using qRT-PCR. Data are mean ± s.d. and are derived from three independent experiments, each in triplicate, *P < 0.05, **P < 0.01. (d) Western blot analysis of HCLS1 and LEF-1. β-actin was used as a loading control. (e) The indicated CFU assays were performed. Data are mean ± s.d. and are derived from three independent experiments, each in duplicate, **P < 0.01.

Article Snippet: Billadeau), rabbit polyclonal antibody to HCLS1 (1:400, Cell Signaling Technology, 4503S), mouse monoclonal antibody to HCLS1 (1:500, BD Bioscience, 610541), rabbit polyclonal antibody to phospho-HCLS1 (Tyr397; 1:100, Cell Signaling Technology, 4507S), rabbit monoclonal antibody to LEF-1 (1:500, Cell Signaling Technology, 22305), mouse monoclonal antibody to LEF-1 (1:1,000, CalBiochem, NA64), mouse monoclonal antibody to Lyn (1:200, Santa Cruz Biotechnology, SC-7274), mouse monoclonal antibody to Syk (1:400, Biolegend, 626201), rabbit polyclonal antibody to HAX1 (Santa Cruz Biotechnology, SC-28268), rabbit polyclonal antibody to HA tag (1:1,000, Santa Cruz Biotechnology, SC-2365), mouse monoclonal antibody to β-actin (1:1,000, Santa Cruz Biotechnology, SC-47778) and secondary bovine anti-mouse or goat anti-rabbit HRP-conjugated antibody (1:5,000, Santa Cruz Biotechnology, SC-2371 and SC-2004, respectively).

Techniques: In Vivo, In Vitro, Expressing, Isolation, Quantitative RT-PCR, Derivative Assay, Transduction, Construct, shRNA, Cell Culture, Western Blot, Control

Journal: Nature medicine

Article Title: Interactions among HCLS1, HAX1 and LEF-1 proteins are essential for G-CSF-triggered granulopoiesis

doi: 10.1038/nm.2958

Figure Lengend Snippet: HCLS1 and HAX1 are involved in nuclear transport, activation and autoregulation of LEF-1. (a) Western blot analysis of cytoplasmic (cyto) and nuclear (nucl) lysates of CD34+ cells transduced with lentiviral constructs expressing WT LEF-1 cDNA or LEF-1 Ala16 cDNA and treated with G-CSF for 15 min after starvation for 12–14 h. (b–d) Intracellular localization of LEF-1 (b), LEF-1–HCLS1 complexes (c) and LEF-1–phospho-HCLS1 complexes (d) in CD34+ cells using the Duolink in situ PLA assay (red dots; Online Methods). CD34+ cells were transduced with the indicated constructs, starved for 14 h and either left unstimulated (left) or stimulated with 10 ng ml−1 of G-CSF for 15 min (right). Cy3 (red) dots, PLA signals of single proteins or protein–protein complexes; DAPI (blue), nuclei. Representative images are shown. On average, 100 cells were investigated in at least three experiments and evaluated using automated image analysis. Scale bars, 1 μm. (e) CD34+ cells of healthy individuals (n = 3) were transfected with the indicated constructs and treated or not with G-CSF, and expression of a LEF-1 reporter gene (diagrammed) was measured. Data are mean ± s.d. derived from three independent experiments, each in triplicate, *P < 0.05 compared to cells not treated with G-CSF. RLU, relative light units. (f) Fold induction after G-CSF stimulation of LEF-1 and C/EBPα mRNA expression in CD34+ cells transduced with indicated constructs. Data are mean ± s.d. compared to WT HCLS1 and are derived from three independent experiments, each in triplicate, *P < 0.05 compared to WT HCLS1. (g) CFU assays of CD34+ cells from healthy individuals (n = 3) transduced with the indicated constructs. Data are mean ± s.d. derived from three independent experiments, each in duplicate; *P < 0.05, **P < 0.01 compared to HCLS1 WT. (h) ChIP-re-ChIP assays of nuclear extracts of Jurkat cells to assess HCLS1 binding to LEF-1 at the LEF1, CEBPA and CCND1 gene promoters. Antibody to LEF-1 was used in the first ChIP step and antibody to LEF-1 or to HCLS1 for the second ChIP step (Online Methods). Shown is ΔΔCt of target regions to isotype control (fold enrichment). Data are mean ± s.d. (derived from two independent experiments).

Article Snippet: Billadeau), rabbit polyclonal antibody to HCLS1 (1:400, Cell Signaling Technology, 4503S), mouse monoclonal antibody to HCLS1 (1:500, BD Bioscience, 610541), rabbit polyclonal antibody to phospho-HCLS1 (Tyr397; 1:100, Cell Signaling Technology, 4507S), rabbit monoclonal antibody to LEF-1 (1:500, Cell Signaling Technology, 22305), mouse monoclonal antibody to LEF-1 (1:1,000, CalBiochem, NA64), mouse monoclonal antibody to Lyn (1:200, Santa Cruz Biotechnology, SC-7274), mouse monoclonal antibody to Syk (1:400, Biolegend, 626201), rabbit polyclonal antibody to HAX1 (Santa Cruz Biotechnology, SC-28268), rabbit polyclonal antibody to HA tag (1:1,000, Santa Cruz Biotechnology, SC-2365), mouse monoclonal antibody to β-actin (1:1,000, Santa Cruz Biotechnology, SC-47778) and secondary bovine anti-mouse or goat anti-rabbit HRP-conjugated antibody (1:5,000, Santa Cruz Biotechnology, SC-2371 and SC-2004, respectively).

Techniques: Activation Assay, Western Blot, Transduction, Construct, Expressing, In Situ, Transfection, Derivative Assay, Binding Assay, Control

Journal: Nature medicine

Article Title: Interactions among HCLS1, HAX1 and LEF-1 proteins are essential for G-CSF-triggered granulopoiesis

doi: 10.1038/nm.2958

Figure Lengend Snippet: HCLS1 and HAX1 are required for G-CSFR-triggered phosphorylation of PI3K p85 and Akt and for F-actin rearrangement. (a–d) CD34+ cells of healthy individuals (n = 3) were transduced with the indicated constructs, and RFP+ or GFP+ cells were sorted and treated with 10 ng ml−1 of G-CSF or left untreated. At the indicated time points, the cells were harvested, fixed and permeabilized cells and amounts of total PI3K p85 and phospho-PI3K p85 (Tyr458) (a, c) and total Akt and phospho-Akt (Ser473) (b, d) were quantified using FACS analysis. Shown is the percentage of phospho- to total protein-positive cells. Data are mean ± s.d. (derived from two independent experiments, each in duplicate), *P < 0.05. (e–g) CD34+ cells were transduced with the indicated constructs, treated with 10 ng ml−1 of G-CSF for the indicated lengths of time or left untreated, and F-actin staining was assessed by FACS. Shown are the percentage of RFP+ cells that are F-actin+ (e) and the percentage increase of F-actin+ cells after stimulation with G-CSF (f, g). Data are mean ± s.d. (derived from three independent experiments each in duplicate); *P < 0.05, **P < 0.01. (h, i) Representative images showing the distribution of F-actin in HEK293T cells transduced with G-CSFR cDNA (293T-GCSFR) and subsequently treated with 10 ng ml−1 of G-CSF for 3 min or left untreated was assessed by confocal microscopy. In i cells were co-transduced with control or HAX1 shRNA. Insets show higher magnification views.

Article Snippet: Billadeau), rabbit polyclonal antibody to HCLS1 (1:400, Cell Signaling Technology, 4503S), mouse monoclonal antibody to HCLS1 (1:500, BD Bioscience, 610541), rabbit polyclonal antibody to phospho-HCLS1 (Tyr397; 1:100, Cell Signaling Technology, 4507S), rabbit monoclonal antibody to LEF-1 (1:500, Cell Signaling Technology, 22305), mouse monoclonal antibody to LEF-1 (1:1,000, CalBiochem, NA64), mouse monoclonal antibody to Lyn (1:200, Santa Cruz Biotechnology, SC-7274), mouse monoclonal antibody to Syk (1:400, Biolegend, 626201), rabbit polyclonal antibody to HAX1 (Santa Cruz Biotechnology, SC-28268), rabbit polyclonal antibody to HA tag (1:1,000, Santa Cruz Biotechnology, SC-2365), mouse monoclonal antibody to β-actin (1:1,000, Santa Cruz Biotechnology, SC-47778) and secondary bovine anti-mouse or goat anti-rabbit HRP-conjugated antibody (1:5,000, Santa Cruz Biotechnology, SC-2371 and SC-2004, respectively).

Techniques: Phospho-proteomics, Transduction, Construct, Derivative Assay, Staining, Confocal Microscopy, Control, shRNA

Journal: Nature medicine

Article Title: Interactions among HCLS1, HAX1 and LEF-1 proteins are essential for G-CSF-triggered granulopoiesis

doi: 10.1038/nm.2958

Figure Lengend Snippet: Defective granulopoiesis in Hcls1−/− mice. (a) Percentage and number of peripheral blood PMNs in Hcls1−/− (n = 12) and WT (n = 10) mice. Data are mean ± s.d.; *P < 0.05, **P < 0.01. (b) Percentage of Gr1+Mac1+ cells in bone marrow cells of Hcls1−/− and WT mice. Data are mean ± s.d., *P < 0.05. (c) mRNA expression of the indicated proteins in bone marrow CD33+ myeloid cells of HCLS1−/− (n = 3) and WT (n = 3) mice measured by qRT-PCR. Data are mean ± s.d. (derived from three independent experiments, each in triplicate), *P < 0.05. (d) LEF-1 expression in bone marrow CD33+ myeloid progenitor cells from HCLS1−/− (n = 3) and WT (n = 3) mice measured by western blotting (representative data). Tubulin was used as a loading control. (e) CFU assays using bone marrow cells of Hcls1−/− (n = 4) and WT mice (n = 4). Data are mean ± s.d. (derived from three independent experiments, each in duplicate); *P < 0.05, **P < 0.01. (f,g) Bone marrow mononuclear cells from Hcls1−/− and WT mice were transduced with the indicated GFP-tagged lentiviral constructs and granulocyte differentiation was assessed. Shown are the percentage of GFP+Gr-1hiCD11bhi mature granulocytes as measured by FACS (f) and LEF-1 and C/EBPα mRNA expression in GFP+ cells as measured by qRT-PCR (g). Data are mean ± s.d., *P < 0.05.

Article Snippet: Billadeau), rabbit polyclonal antibody to HCLS1 (1:400, Cell Signaling Technology, 4503S), mouse monoclonal antibody to HCLS1 (1:500, BD Bioscience, 610541), rabbit polyclonal antibody to phospho-HCLS1 (Tyr397; 1:100, Cell Signaling Technology, 4507S), rabbit monoclonal antibody to LEF-1 (1:500, Cell Signaling Technology, 22305), mouse monoclonal antibody to LEF-1 (1:1,000, CalBiochem, NA64), mouse monoclonal antibody to Lyn (1:200, Santa Cruz Biotechnology, SC-7274), mouse monoclonal antibody to Syk (1:400, Biolegend, 626201), rabbit polyclonal antibody to HAX1 (Santa Cruz Biotechnology, SC-28268), rabbit polyclonal antibody to HA tag (1:1,000, Santa Cruz Biotechnology, SC-2365), mouse monoclonal antibody to β-actin (1:1,000, Santa Cruz Biotechnology, SC-47778) and secondary bovine anti-mouse or goat anti-rabbit HRP-conjugated antibody (1:5,000, Santa Cruz Biotechnology, SC-2371 and SC-2004, respectively).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Western Blot, Control, Transduction, Construct

Journal: Nature medicine

Article Title: Interactions among HCLS1, HAX1 and LEF-1 proteins are essential for G-CSF-triggered granulopoiesis

doi: 10.1038/nm.2958

Figure Lengend Snippet: HCLS1 is hyperactivated in AML. (a) Representative micrographs of immunohistochemistry of HCLS1 (brown) counterstained with hematoxylin (blue) of bone marrow sections of one healthy individual (control) and three patients with AML with different AML types (AML M2, M4 and M5). (b) Primary blasts of three patients with AML were transduced with control-RFP shRNA or either of two HCLS1-RFP shRNAs. RFP+ cells were sorted and used for further experiments. Top, representative western blot showing HCLS1 expression; β-actin was used as a loading control. Bottom, on day 7 cell proliferation was assessed by counting of viable RFP+ cells and apoptosis was assessed by annexin V staining of RFP+ cells. Data are mean ± s.d. (derived from three independent experiments, each in triplicate), *P < 0.05. (c) Representative images of intracellular staining of G-CSF in CD34+ cells of healthy individuals (n = 3) and in blasts of three patients with AML were measured using the Duolink in situ PLA assay with antibody to G-CSF. Nuclear DAPI staining (blue) and G-CSF staining (red). Inset, G-CSFR mRNA expression in AML blasts (red bar, n = 3) and in CD34+ cells of healthy individuals (blue bar, n = 3). Data are mean ± s.d. (derived from three independent experiments, each in triplicate). (d) Representative images of Sanger DNA sequencing of the HLCS1 gene from one healthy individual with WT HCLS1 and one patient with AML with the 12-bp insertion (MUT HCLS1). (e) Model of the interplay among HCLS1, HAX1 and LEF-1. Activation of G-CSF receptor leads to phosphorylation and activation of HCLS1 via Lyn and Syk. HCLS1 together with HAX1 binds LEF-1, transporting LEF-1 into the nucleus, where LEF-1 activates target genes (for example, LEF1, CEBPA and HCLS1) and promotes granulocytic differentiation.

Article Snippet: Billadeau), rabbit polyclonal antibody to HCLS1 (1:400, Cell Signaling Technology, 4503S), mouse monoclonal antibody to HCLS1 (1:500, BD Bioscience, 610541), rabbit polyclonal antibody to phospho-HCLS1 (Tyr397; 1:100, Cell Signaling Technology, 4507S), rabbit monoclonal antibody to LEF-1 (1:500, Cell Signaling Technology, 22305), mouse monoclonal antibody to LEF-1 (1:1,000, CalBiochem, NA64), mouse monoclonal antibody to Lyn (1:200, Santa Cruz Biotechnology, SC-7274), mouse monoclonal antibody to Syk (1:400, Biolegend, 626201), rabbit polyclonal antibody to HAX1 (Santa Cruz Biotechnology, SC-28268), rabbit polyclonal antibody to HA tag (1:1,000, Santa Cruz Biotechnology, SC-2365), mouse monoclonal antibody to β-actin (1:1,000, Santa Cruz Biotechnology, SC-47778) and secondary bovine anti-mouse or goat anti-rabbit HRP-conjugated antibody (1:5,000, Santa Cruz Biotechnology, SC-2371 and SC-2004, respectively).

Techniques: Immunohistochemistry, Control, Transduction, shRNA, Western Blot, Expressing, Staining, Derivative Assay, In Situ, DNA Sequencing, Activation Assay, Phospho-proteomics

Journal: Clinical, Cosmetic and Investigational Dermatology

Article Title: Identification of a Gene Expression Signature to Predict the Risk of Abdominal Aortic Aneurysm in Psoriasis Patients

doi: 10.2147/CCID.S495890

Figure Lengend Snippet: Establishment of gene signature by LASSO regression. ( A and B ) Heatmap of 20 hub genes in GSE226244 ( A ) and GSE57691 ( B ). Gene expression is scaled by the Z-score. The bar plot beside the heatmap reveals the log2FC of genes. ( C ) The PPI network constructed by STRING reveals the interaction between these 20 genes. ( D ) Friend analysis discovers the rank of 20 genes. The x-axis indicates the gene similarity. The y-axis represents genes. ( E ) The log (lambda) sequence was used to construct a coefficient profile diagram. ( F ) LASSO coefficient profiles of the 20 genes in psoriasis. ( G and H ) ROC curve of CCR7, CD3D, GBP5, HCLS1, IL7R, ITGAL, and the gene signature. ( I ) The visible nomogram for diagnosing psoriasis.

Article Snippet: We performed immunohistochemical (IHC) analysis to assess the differential expressions of CCR7 (1:100 dilution, Proteintech,55425-1-AP, Wuhan, China); CD3D (1:200 dilution, Proteintech,16669-1-lg, Wuhan, China); GBP5 (1:200 dilution, Proteintech,13220-1-AP, Wuhan, China); HCLS1 (1:200 dilution Proteintech,25003-1-lg, Wuhan, China); IL7R (1:100 dilution, Abcam, ab259806, Cambridge, UK), and ITGAL (1:100 dilution, Proteintech,15574-1-AP, Wuhan, China) in different groups.

Techniques: Gene Expression, Construct, Sequencing

Journal: Nature communications

Article Title: HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels.

doi: 10.1038/ncomms13889

Figure Lengend Snippet: Figure 2 | HS1BP3 regulates autophagy in zebrafish. (a) Representative confocal images of GFP-LC3 puncta (autophagosomes) in the trunk area of GFP-LC3 transgenic zebrafish embryos injected with control morpholino (C), Hs1bp3 translational-blocking morpholino (K), and the human Hs1bp3 mRNA coinjected with the morpholino (R) and imaged at 2 dpf with or without pre-treatment with chloroquine (10 mM) for 6 h. Scale bars, 10 mm. (b) GFP-LC3 puncta were counted in the trunk region (marked in d) of the transgenic zebrafish embryos at 2 dpf (mean±s.e.m., n ¼ 3). Total of 7–13 embryos were used for each condition per experiment. *Po0.05, **Po0.01, ***Po0.001, by Student’s t-test. (c) Representative immunoblotting of Hs1bp3 and Tubulin in whole lysates of zebrafish embryos at 2 dpf, treated with or without chloroquine for 6 h before harvest. (d) Representative light fluorescent microscopy images of whole embryos at 2 dpf. Scale bars, 300 mm.

Article Snippet: For immunoprecipitation from lysates, GFP, GFP-HS1BP3 or GFP-PLD1 were immunoprecipitated by GFP trap (Chromotek) following the manufacturer’s protocol.

Techniques: Transgenic Assay, Injection, Control, Blocking Assay, Western Blot, Microscopy

Journal: Nature communications

Article Title: HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels.

doi: 10.1038/ncomms13889

Figure Lengend Snippet: Figure 3 | HS1BP3 localizes to ATG16L1- and ATG9-positive vesicles. HEK293 and U2OS cells expressing the indicated proteins were starved for 2 h before fixation and immunostaining with the indicated antibodies. Confocal micrographs show: (a) HEK cells expressing mCherry-HS1BP3 stained for endogenous ATG16L1 and WIPI2. Yellow arrows mark HS1BP3- and ATG16L1-positive structures. White arrow marks HS1BP3-, ATG16L1- and WIPI2-positive structure. (b) Co-localization of GFP-HS1BP3 with endogenous ATG9 and TfR (white arrows show triple co-localization) in U2OS cells. (c) Co-localization of endogenous HS1BP3 with endogenous ATG9 in HEK cells. (d) Control or HS1BP3-depleted U2OS cells expressing GFP-ATG16L1 stained for endogenous HS1BP3. Yellow arrows indicate ATG16L1-positive structures that are positive for HS1PB3, while white arrow heads indicate ATG16L1-positive structures that are not positive for HS1BP3. Note that in addition to the specific staining (co-localization with ATG16L1), the HS1BP3 antibody also recognizes other proteins non-specifically both on immunofluorescence and western blotting (Fig. 1d). (e) HEK cells expressing GFP-HS1BP3 stained for endogenous LC3. Scale bars, 10 mm.

Article Snippet: For immunoprecipitation from lysates, GFP, GFP-HS1BP3 or GFP-PLD1 were immunoprecipitated by GFP trap (Chromotek) following the manufacturer’s protocol.

Techniques: Expressing, Immunostaining, Staining, Control, Western Blot

Journal: Nature communications

Article Title: HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels.

doi: 10.1038/ncomms13889

Figure Lengend Snippet: Figure 6 | PLD1 co-localization with ATG16L1 is affected by HS1BP3. (a) HEK cells were transfected with GFP-tagged PLD1 or PLD2. After starvation and fixation the cells were immunostained for ATG16L1 and analysed by confocal microscopy. (b) HEK cells were transfected with GFP-PLD1, starved, fixed and immunostained for ATG16L1 and TfR. (c) HEK cells were first treated with non-targeting or HS1BP3 siRNA, then transfected to express GFP-PLD1, starved, fixed and immunostained for ATG16L1. Yellow arrows indicate ATG16L1 vesicles positive for PLD1 and white arrow heads indicate ATG16L1 vesicles negative for PLD1. Co-localization of GFP-PLD1 to ATG16L1 vesicles was quantified in transfected cells using the ImageJ plugin Squassh, using 10 pictures of each condition from three independent experiments (mean±s.e.m., n ¼ 3). (d) HEK cells were transfected with HA-PLD1 together with GFP, GFP-HS1BP3 full-length, -PX or DPX constructs, starved and stained for endogenous ATG16L1. Arrows indicate co-localization between ATG16L1 and HA-PLD1. (e) Co-localization of HA-PLD1 with endogenous ATG16L1 vesicles was quantified in transfected cells in d with the Zen software (Zeiss) using 10 pictures of each condition from three independent experiments (mean±s.e.m., n ¼ 3). Scale bars, 10 mm. *Po0.05, by Student’s t-test.

Article Snippet: For immunoprecipitation from lysates, GFP, GFP-HS1BP3 or GFP-PLD1 were immunoprecipitated by GFP trap (Chromotek) following the manufacturer’s protocol.

Techniques: Transfection, Confocal Microscopy, Construct, Staining, Software

Journal: Nature communications

Article Title: HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels.

doi: 10.1038/ncomms13889

Figure Lengend Snippet: Figure 7 | HS1BP3 regulates autophagy through PLD1. (a) HEK cells were first treated with the indicated siRNA and then transfected with the indicated GFP-tagged construct. Cells were starved and fixed before immunostaining for endogenous LC3. LC3 spots were counted only in transfected cells, minimum 200 transfected cells per condition in three independent experiments (mean±s.e.m., n ¼ 3). *Po0.05, by Student’s t-test. (b) Model for the role of HS1BP3 in autophagy. PLD1 generates PA on ATG16L1-positive autophagosome precursor membranes. HS1BP3 is recruited to these membranes by the generated PA, inhibiting PLD1 activity and displacing it from the ATG16L1 vesicles. HS1BP3 thus provides a negative feedback on PA generation on these vesicles. If HS1BP3 is depleted from the cells, this negative feedback is lost, causing the PA concentrations of these membranes to increase and thereby drive increased autophagosome formation.

Article Snippet: For immunoprecipitation from lysates, GFP, GFP-HS1BP3 or GFP-PLD1 were immunoprecipitated by GFP trap (Chromotek) following the manufacturer’s protocol.

Techniques: Transfection, Construct, Immunostaining, Generated, Activity Assay