Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: A P4HA2 interacts with KIF7. HEK293T cells were transfected with Flag-tagged P4HA2. Cell lysates were prepared and subjected to immunoprecipitation (IP) with M2 anti-Flag beads. The immunoprecipitates were analyzed by Western blotting (WB) with anti-Flag and KIF7 antibodies. B KIF7 interacts with P4HA2. HEK293T cells were transfected with Flag-tagged KIF7. Cell lysates were prepared and subjected to Flag IP. The immunoprecipitants were analyzed by WB with anti-Flag and P4HA2 antibodies. C Schematic representation of the SHH signaling pathway. SAG is a chlorobenzothiophene-containing Hh pathway agonist and binds to the SMO heptahelical bundle in a manner that antagonizes cyclopamine action. SAG activates SMO, leading to the release of GLI1 by SUFU, and the Hh signaling pathway is then activated. D – I Knockout of P4HA2 causes suppression of Hedgehog signal transduction. NIH/3T3 ( D – F ) and OP9 ( G – I ) P4HA2 knockout (KO) or vehicle cells were treated with (+) or without (−) 200 nM SAG. The Hh targeted genes, Gli1 ( E , H ) and Ptch1 ( F , I ), were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Data are shown as the mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All experiments were repeated three times independently.

Article Snippet: P4ha2 knockout mice were purchased from Cyagen Biosciences Inc.

Techniques: Transfection, Immunoprecipitation, Western Blot, Knock-Out, Transduction, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: A Schematic of full-length KIF7 proteins. The KIF7 protein encodes a 1343 aa protein with a kinesin motor domain, GLI-binding domain (BD), Nephoronophthisis-1-interacting domain (NPHP1-ID) and a Structural Maintenance of Chromosomes (SMC) domain representing the ATPase activity. Various truncated mutation constructs of KIF7-Flag are shown schematically. B Mapping the interaction domain of KIF7 with P4HA2. HEK293T cells were transfected with constructs shown as ( A ). Cell lysates were prepared and subjected to immunoprecipitation (IP) with M2 anti-Flag beads. C Mapping the minimum interaction domain of KIF7 with P4HA2. HEK293T cells were transfected with constructs “a”, “f” and “g” mutants. Cell lysates were prepared and subjected to Flag IP. D , E Endogenous co-immunoprecipitated P4HA2 and KIF7 interact with each other separately in HEK293T cells. Endogenous KIF7 and P4HA2 were precipitated from a HEK293T cells extract and analyzed by immunoblotting. F P4HA2 interacts with SUFU and GLI2. HEK293T cells were transfected with Flag-tagged P4HA2. Cell lysates were subjected to Flag IP. The immunoprecipitants were analyzed by WB with anti-Flag, KIF7, SUFU and GLI2 antibodies. G P4HA2 interacts with GLI1. HEK293T cells were transfected with Flag-tagged P4HA2 and V5-tagged GLI1. Cell lysates were subjected to Flag IP. The immunoprecipitants were analyzed by WB with anti-Flag, KIF7 and V5 antibodies. H P4HA2 directly interacts with KIF7 in vitro. His-tagged KIF7, GLI1, SUFU, and Flag-tagged P4HA2 proteins were purified and incubated. After His pulldown, the complex was analyzed by WB. I KIF7 knockdown decreases the interaction between P4HA2 with SUFU. IgG-P4HA2 was overexpressed in shKIF7-1 and shKIF7-2 HEK293T cells. Cell lysates were subjected to IgG IP. Co-immunoprecipitated SUFU was detected by WB. J KIF7 knockdown decreases the interaction between P4HA2 and GLI1. IgG-P4HA2 and V5-GLI1 were co-overexpressed in KIF7 stable knockdown HEK293T cells (shKIF7-1 and shKIF7-2). Cell lysates were subjected to IgG IP. Co-immunoprecipitated V5-GLI1 was detected by WB.

Article Snippet: P4ha2 knockout mice were purchased from Cyagen Biosciences Inc.

Techniques: Binding Assay, Activity Assay, Mutagenesis, Construct, Transfection, Immunoprecipitation, Western Blot, In Vitro, Purification, Incubation, Knockdown

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: A The trafficking of the P4HA2-KIF7 complex from the cytoplasm to the cilium responds to Hedgehog signaling. NIH/3T3 cells were co-infected with KIF7-BFP and P4HA2-EGFP lentivirus. White arrow indicates the co-localization of cilia, KIF7-BFP and P4HA2-EGFP. Yellow arrow indicates the co-localization of cilia and P4HA2-EGFP, which may bind with endogenous KIF7. Cells were treated with 200 nM SAG (+) or not (−). Cells were fixed and stained with antibodies for ARL13B to mark primary cilia. Scale bars: 5 μm. B Statistical analysis of the relative colocalization rate in ( A ) indicated cells. Data are shown as the mean ± SEM; SAG (−), n = 11; SAG (+), n = 13. ** P < 0.01. C The regulation of the Hedgehog signaling by P4ha2 is dependent on KIF7. KIF7 knockout (KO) and the control cells were knocked down with P4HA2 (shP4HA2). Cells were treated with (+) or without (−) 200 nM SAG. qRT-PCR analysis of GLI1 transcripts was performed using RNA isolated from the indicated cell lines. Data are shown as the mean ± SEM ( n = 3). *** P < 0.001. All experiments were repeated three times independently.

Article Snippet: P4ha2 knockout mice were purchased from Cyagen Biosciences Inc.

Techniques: Infection, Staining, Knock-Out, Control, Quantitative RT-PCR, Isolation

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: A , B The regulation of Hh signaling by P4HA2 is dependent on P4HA2 hydroxylase activity. HEK293T P4HA2 KO cells were expressed with wild-type P4HA2 (P4HA2) or hydroxylase dominant negative mutant (P4HA2-HDH). Cells were treated with (+) or without (−) 200 nM SAG. qRT-PCR analysis of GLI1 ( A ) and GLI2 ( B ) transcripts was performed using RNA isolated from the indicated cell lines. Data are shown as the mean ± SEM ( n = 3). * P < 0.05, *** P < 0.001. All experiments were repeated three times independently. C – E The hydroxylation levels of GLI1 and SUFU decrease in P4HA2 knockout cells. P4HA2 KO and the vehicle cells were transfected with Flag-tagged KIF7, GLI1, and SUFU. Cell lysates were subjected to Flag IP and then analyzed by WB. Hydroxylated proteins were recognized by the Pan-hydroxylation antibody (OH). The relative quantification of Flag-KIF7 ( C ), GLI1 ( D ), and SUFU ( E ) hydroxylation level was analyzed and normalized. The values shown are the means of three independent experiments. F – H LC-MS/MS spectra of tryptic peptides. 3 tryptic peptides Pro 46 ( F ), Pro 134 ( G ), and Pro 249 ( H ) from SUFU. The b ion and y ion are fragment ions of tryptic peptide in tandem mass spectrometry. The x and y axes represent m/z and relative ion intensity, respectively. I Validation of the proline 46, proline 134, and proline 249 hydroxylation sites of SUFU. P46A, P134A, P249A, and P46/134/249A proline to alanine mutants of SUFU were constructed and transfected into HEK293T cells. Cell lysates were subjected to Flag IP. The relative quantification of SUFU WT and mutant hydroxylation levels was analyzed and normalized. The values shown are the means of three independent experiments. J P4HA2 regulates the protein stability of SUFU in response to the Hh pathway activation. HEK293T P4HA2 KO cells were expressed with wild-type P4HA2 or a P4HA2 dominant negative mutant in hydroxylase activity (HDH). Cell lysates were analyzed by WB with anti-P4HA2 and GAPDH antibodies. K Hydroxylated SUFU regulate the Hh signaling. SUFU knockout (KO) NIH/3T3 and control cell lines were constructed and then infected with SUFU WT and mutant (SUFU P46/134/627 A) lentivirus. qRT-PCR analysis of GLI1 transcripts was performed using RNA isolated from the above cell lines. L SUFU KO NIH/3T3 and control cell lines were constructed and then infected with SUFU WT and mutant (SUFU P46/134/627 A) lentivirus. Utilized a dual luciferase reporter gene system, GLI1-Firefly and Renilla plasmids were overexpressed in above cell lines. Relative fold change of GLI1 luciferase activity was measured and normalized. Data are shown as the mean ± SEM ( n = 3). *** P < 0.001, **** P < 0.0001. All experiments were repeated three times independently.

Article Snippet: P4ha2 knockout mice were purchased from Cyagen Biosciences Inc.

Techniques: Activity Assay, Dominant Negative Mutation, Quantitative RT-PCR, Isolation, Knock-Out, Transfection, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Construct, Mutagenesis, Activation Assay, Control, Infection, Luciferase

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: A – D Injection of luciferased Eµ-myc arf −/ − B cells into C57BL/6 WT and P4ha2 −/ − mice. Bioluminescence imaging ( A , C ) of mice for 18 days after injection. Luminescence intensity curves ( B, D ) for mice injected with luciferased Eµ-myc arf −/ − B cells. E P4HA2 is located in stromal fibroblasts. Immunofluorescence staining of α-SMA, P4HA2 and DAPI in C57BL/6 WT mice xenograft. Scale bars: 100 μm. F Primary stromal fibroblasts were isolated from bone marrow of C57BL/6 WT and P4ha2 −/ − mice. qRT-PCR analysis of Gli2, Ptch1, Ptch2 and HH ligands transcripts was performed using RNA isolated from the primary stromal fibroblasts. G Differential genes of Hh downstream secreted factors obtained from RNA-seq analysis of primary bone marrow stromal fibroblasts. Data are shown as the mean ± SEM ( n = 3). * P < 0.1, ** P < 0.01, *** P < 0.001. H The growth curve of Eµ-myc arf −/ − B cells co-cultured with primary bone marrow stromal fibroblasts supernatant. WT and P4ha2 −/ − primary bone marrow fibroblasts were isolated and cultured. After cell counting, both primary cells were cultured at the same density, and the cultured supernatant was collected for co-culture with Eµ-myc arf −/ − B cells. The cell viability was detected every 12 hr. I qRT-PCR analysis of Gli1 and Gli2 transcripts was performed using the tissue of C57BL/6 WT and P4ha2 −/ − mice xenograft. Data are shown as the mean ± SEM ( n = 3). *** P < 0.001, **** P < 0.0001. All experiments were repeated three times independently.

Article Snippet: P4ha2 knockout mice were purchased from Cyagen Biosciences Inc.

Techniques: Injection, Imaging, Immunofluorescence, Staining, Isolation, Quantitative RT-PCR, RNA Sequencing Assay, Cell Culture, Cell Counting, Co-Culture Assay

Journal: Leukemia

Article Title: P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

doi: 10.1038/s41375-024-02313-8

Figure Lengend Snippet: In stromal cells, P4HA2 hydroxylates SUFU to regulate the Hh pathway. This leads to the activation of Hh signaling and the secretion of downstream growth factors, resulting in the malignant proliferation of tumor cells.

Article Snippet: P4ha2 knockout mice were purchased from Cyagen Biosciences Inc.

Techniques: Activation Assay