Antibodies and reagents

The following antibodies were used for western blotting (WB), immunofluorescence (IF): PAN-OH (home-made; 1:1,000 for WB); P4HA2 (13759-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC, 1:200 for IF); P4HA1 (12658-1-AP, rabbit, Proteintech; 1:1,000 for WB); KIF7 (24693-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC); SUFU (26759-1-AP, rabbit, Proteintech; 1:2,000 for WB); Flag (clone M2, F1804, mouse, Sigma-Aldrich; 1:10,000 for WB, 1:200 for IF); His (clone OGHis, D291-3, mouse, Medical & Biological Laboratories; 1:5,000 for WB); V5 (AB3792, rabbit, Merck Millipore; 1:2,000 for WB); GLI1 (clone A-7, sc-515781, mouse, Santa Cruz; 1:1,000 for WB, 1:200 for IHC); GLI2 (18989-1-AP, rabbit, Proteintech; 1:1,000 for WB, 1:100 for IHC) ; GAPDH (clone 1E6D9, 60004-1-Ig, mouse, Proteintech; 1:10,000 for WB); Arl13b (clone N295B/66, 75-287, mouse, NeuroMab; 1:200 for IF); Cy3 AffiniPure Goat Anti-Mouse IgG (H + L) (115-165-003, Jackson ImmunoResearch; 1:10,000 for IF); α-SMA (#34105, Cell Signaling Technology, 1:100 for IF) Light chain specific Mouse Anti-Rabbit IgG (211-032-171, Jackson ImmunoResearch; 1:10,000 for WB); Light chain specific Goat Anti Mouse IgG (115-035-174, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Rabbit IgG, Fc fragment specific (111-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Donkey anti-mouse IgG (H&L) (715-035-150, Jackson ImmunoResearch; 1:10,000 for WB); Goat Anti-Rabbit IgG (H + L) (111-035-003, Jackson ImmunoResearch; 1:10,000 for WB); Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific (115-035-008, Jackson ImmunoResearch; 1:10,000 for WB); Smoothened Agonist (SAG), Selleck, Cat# S7779; Fetal bovine serum, Thermo Fisher, Cat# 16140071; Penicillin/streptomycin, Thermo Fisher, Cat# 15140122; RPMI 1640, Thermo Fisher, Cat# 11875093; DMEM, Thermo Fisher, Cat# 11995065; Puromycin Dihydrochloride, Sigma, Cat# P8833; Trypsin 0.25% EDTA, Thermo Fisher, Cat# 25200072; Polyethylenimine Hydrochloride (MW 40,000), Polysciences, Cat# 24765-1; Polybrene Transfection Reagent, Sigma, Cat# TR-1003-G; SHH protein, Yun Zhao Laboratory; Anti-V5-tag mAb-Magnetic Beads, Medical & Biological Laboratories, Cat# M215-11; NTI-FLAG® M2 Affinity Gel, Sigma, Cat# F2426; Ni-NTA Agarose, QIAGEN, Cat# 30230; HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01/02; ChamQ SYBR qPCR Master Mix, Vazyme, Cat# Q311-02.

Tandem affinity purification

The P4HA2 gene was cloned into a pcDNA3.1 plasmid which coding protein A and streptavidin binding protein in its N terminal. 2 × 107 HEK293T cells were seeded in 15 cm dish. 20 μg plasmids was transfected with 60 μL PEI in DMEM with 10% FBS without PS next day. One day after transfection, the cells were harvest with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 125 mM NaCl, 0.2% NP-40, 5% glycerol, 1.5 mM MgCl2 and protease inhibitors (Sigma) and phosphatase inhibitor cocktail (Sigma) and lysate with sonicate. Then the supernatant was incubated with 30 μL IgG beads (Sigma) for 2 hr at 4 °C, IgG beads were then incubated with 1.5 μL AcTEV protease (Invitrogen, 10 U/μL) per tube at 4 °C overnight. 80 μL Streptavidin beads (Invitrogen) per tube was equilibrated with SBP buffer containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl and 0.2% NP-40. IgG beads treated with the AcTEV was spun down and the supernatant was added to the streptavidin beads and incubate at 4 °C for 4 hr. Streptavidin beads was finally spined down and boiled with 40 μL 2× SDS sample buffer and taken to the Mass spectrum analysis.

Pulldown and co-immunoprecipitation

HEK293T cells transfected with plasmids were pelleted by centrifugation, washed with 1X PBS and lysed for 15 min in RIPA buffer at 4 °C (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) or Cell Lysis Buffer (Cell Signaling) supplemented with protease inhibitors (Sigma) and phosphatase inhibitor cocktail (Sigma). Following centrifugation at 12,000 rpm at 4 °C for 15 min, supernatants were recovered. 10% volume of whole cell lysates were collected as input. Lysates were incubated with 30 μL Ni NTA agarose (QIAGEN) or Flag beads (Sigma) at 4 °C for 3 hr, and then the beads were washed four times with lysis buffer and boiled with sample buffer, then separated on 10% SDS-PAGE gel together with 0.1% input.

Cell culture

NIH/3T3(a gift from Ma Dengke at University of California, San Francisco) or HEK293T(a gift from Ma Dengke at University of California, San Francisco) cells were cultured in DMEM (Thermo Fisher) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/glutamine (Thermo Fisher) in a humidified 5% CO2 incubator at 37 °C. OP9 cells (obtained from National Collection of Authenticated Cell Cultures, China) were cultured in MEM-alpha (Thermo Fisher) with 10% fetal FBS 1% penicillin/streptomycin/glutamine (Thermo Fisher) in a humidified 5% CO2 incubator at 37 °C. Eµ-myc arf−/− B cells (a gift from Jiang Hai at Chinese Academy of Sciences [39]) were cultured in DMEM and IMDM (Thermo Fisher) supplemented with 10% fetal bovine serum (FBS), 1% 50 mM beta-ME and 1% penicillin/streptomycin/glutamine (Thermo Fisher) in a humidified 5% CO2 incubator at 37 °C.

All cell lines were free of mycoplasma contamination. Experiments assaying the Hh signaling were carried out in 0.5% FBS supplemented with Smoothened Agonist (SAG) (Sigma). Mammalian cell transfection performed using Polyethylenimine Hydrochloride (MW 40,000) Transfection Reagent (Polysciences) manufacturer protocol.

Co-culture and cell viability assays

OP9 and mice primary bone marrow cells were plated in 6-well plates as required and allowed to attach and grow for 48 hr. For co-culture, cell culture supernatant was collected and Eµ-myc arf−/− B cells were cultured in the supernatant. Cell viability assay was done using CellTiter-Glo® (Promega).

The Hedgehog signaling transduction

NIH/3T3, HEK293T or OP9 cells were cultured for over 12 hr, then 10% FBS was replaced by 0.5% FBS overnight. SAG (200 nM) or the biological ligand, Sonic Hedgehog (Human), recombinant protein (200 ng/mL) treated cells for 24 hr. The Hh pathway targeted genes were analyzed by qRT-PCR. Primers sequences for qRT-PCR are shown in Supplementary Table 2.

The Hedgehog reporter activity assay

NIH/3T3 cell line stably transfected with Gli-dependent firefly luciferase and constitutive Renilla luciferase reporters [40], which was cultured for over 12 hr, then 10% FBS DMEM was replaced by 0.5% FBS DMEM overnight. SAG (200 nM) treated cells for 24 hr. Lyse cells with 1x whole cell lysis buffer available from the Dual-Luciferase Reporter Assay System and collect aliquot of supernatant (20 μL) from cell lysis, plate them into 96-well plate. Thaw dual-luciferase reporter reagents. Flash and prime Berthold luminometer with firefly luciferase and Renilla luciferase substrate reagents. 20 μL of each substrate is added sequentially by the luminometer and light signals generated are instantly measured by the EnSpire. Read firefly and Renilla luciferase signals (firefly luciferase signal is detected at 560 nm and Renilla luciferase signal is detected at 480 nm).

Fluorescence microscopy

NIH/3T3 were co-infected with indicated viruses, which overexpressed KIF7-BFP, P4HA2-EGFP and Arl13b-mCherry for 48 hr. Puromycin was used to select the positive cells and to maintain the pressure of selection. Cells were seeded on the 35-mm glass bottom dish overnight, then cells were serum starved for 12 hr and then treated with SAG (200 nM) for 24 hr. Imaging was done with a ZEISS LSM 980 with Airyscan 2 microscope equipped with a C-plan-Apochromat 63x/1.4 objective. The excitation and emission spectra of BFP, GFP and mCherry was 405 nm 1.5%/574-720 nm, 488 nm 6.5%/420-480, 495-550 nm, 561 nm 2.6% / 300-720 nm respectively.

Cell Immunofluorescence staining

NIH/3T3 were co-infected with indicated viruses, which overexpressed KIF7-BFP, P4HA2-EGFP for 48 hr. Puromycin was used to select the positive cells and to maintain the pressure of selection. Cells were seeded on the glass coverslips in 24-well plate overnight, then cells were serum starved for 12 hr and then treated with SAG (200 nM) for 24 hr. Cells were fixed with 4% paraformaldehyde in PBS buffer, and were incubated for 15 min at room temperature. Then, samples were blocked by incubation in 5% normal horse serum in PBS. Primary cilia were detected by Arl13b (1:200, NeuroMab), and the second antibody was Cyanine 3 (1:2,000, Jackson ImmunoResearch). Samples were mounted in polyvinyl-based mounting media (Southern Biotech), and were imaged with A1R Multiphoton laser confocal microscope (Nikon).

Lentiviral transduction

Knockout or knockdown: CRISPR/Cas9 genome editing was used to generate stable knockout cell lines. Cells were co-transfected with lentiCRISPR v2-EGFP (Knockout) or lentiviral shRNA (knockdown) expression vector, the lentiviral packaging vectors psPAX2 and the vesicular stomatitis virus G glycoprotein expression vector pMD2.G using PEI reagent. The supernatant containing lentivirus was collected at the second and third day after transfection, followed by filtration (Acrodisc Syringe Filter 0.45 μm Supor Membrane, PALL Life Sciences) and concentration (Millipore 100 μM). Viral supernatant was used to infect 1 × 105 cells in a 35-mm dish with 5 μg/mL polybrene (Sigma), followed by 1500 rpm centrifugation for 2 hr at room temperature. The Knockout cell lines were determined by sequencing and Western blotting. The knockdown efficiency was determined by Western blotting. The sgRNA or shRNA sequences are shown in Supplementary Table 2.

Overexpression: coding sequences of P4HA2, KIF7 and GLI1 were fused with GFP, BFP and V5 tag and cloned into lentiviral constructs, respectively. Lentiviral constructs were packaged in HEK293T and transduced with NIH/3T3. Stably positive colonies were sorted by puromycin and expanded. The overexpression efficiency was determined by Western blotting.

In-gel digestion and LC-MS/MS analysis

The protein was analyzed by SDS-PAGE and the aim gel bands were cut. The in-gel digestion was according to the published methods [41]. Briefly, the gel bands were cut into 1 mm3 particles. The protein-cysteine residues were reduced by adding 5 mM dithiothreitol buffer at 56 °C for 1 hr. Then the cysteine residues were alkylated with 10 mM iodoacetamide in the dark at room temperature for 30 min. Proteins were digested by sequence grade trypsin enzyme at a trypsin- to-protein ratio of 1:50 (w/w). The trypsin digestion was incubated at 37 °C for 16 hr. Peptides were extracted and desalted by C18 Zip-Tips. The desalted peptides were stored in -80 °C before LC-MS/MS analysis.

The dried peptides were dissolved in buffer A (0.1% formic acid in 2% acetonitrile) and then were loaded onto the home-made capillary C18 column (75 µm ID x 20 cm length, packed with C18 resins, 3 µm particle size, 100 Å pore size (Dikma Technologies) equipping on Easy-nanoLC 1000 system (Thermo Scientific). The peptides were eluted from column by 60 min gradient at flow rate of 350 nL/min. The gradient used was as follows: 5 – 32% buffer B (0.1% formic acid in 90% acetonitrile) over 40 min, 32 – 45% buffer B over 15 min, 45 – 80% buffer B over 2 min and then kept at 80% for 3 min. The eluted peptides were ionized under high voltage (2.1 kV) and detected by Orbitrap Fusion mass spectrometer (Thermo Scientific). For the full scan, the ions were detected by Orbitrap analyzer with mass range of 350-1550 m/z at a resolution of 120,000 (m/z 200). The Automatic Gain Control (AGC) targets were set at 5 × 105 and the maximum injection time was set as 50 milliseconds (msec). The MS/MS acquisition was used by top speed mode and the cycle time was set as 3 s (sec). The precursor ions were fragmented under higher energy collision dissociation (HCD) with normalized collision energy (NCE) of 32%. The fragment ions were detected by ion trap. The AGC for the MS/MS were set to 7 × 103. The maximum injection time was 50 msec and dynamic exclusion was 30 sec. The raw data were converted into MGF format by Proteome Discoverer software (version 1.4) and then searched by Mascot search engine (version 2.3.01) against the UniProt-Homo proteome database. Search parameters were as following. Enzyme specificity was set as trypsin/P and up to two missed cleavages were allowed. Carbamidomethylation of cysteine was set as a fixed modification. The acetylation of protein N-term, oxidation of methionine and oxidation of proline were set as variable modifications. Mass tolerance of precursor ion was set as ±10 ppm, fragment mass tolerance was set as ±0.5 Da. The protein score was cut-off at 50.

Mice experiment

P4ha2 knockout mice were purchased from Cyagen Biosciences Inc. The gRNA to mouse P4ha2 gene, and Cas9 mRNA were co-injected into fertilized mouse eggs to generate targeted knockout offspring. F0 founder animals were identified by PCR followed by sequence analysis, which were bred to wildtype mice to test germline transmission and F1 animal generation.

gRNA target sequences are shown in Supplementary Table 2.

Genotyping PCR Primers are shown in Supplementary Table 2.

C57BL/6 WT and P4ha2−/− mice (6 weeks old, female) were randomly grouped and tail vein injected with 1 × 106 Luciferased Eµ-myc arf−/− B cells. Tumor volume was measured using an in vivo imaging system (IVIS). At the experimental endpoint, tumors were harvested and fixed with 4% PFA for paraffin-embedded section. Treatment with 40 mg/kg Ethyl 3,4-dihydroxybenzoate (EDHB, Sigma, E24859) or vehicle control (10% ethanol) started on day 2 after injection of Luciferased Eµ-myc arf−/− B cells. All animal care and experimentation were ethically performed according to procedures approved by the Institutional Animal Care and Use Committee at Fudan University (20190221-001, 20220228-080).

Human specimens

Symptomatic patient samples were collected as part of routine clinical examination. The study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by institutional ethics committee under number 2022-K558, 2019-Y005. All patients provided written informed consent.

RNA sequencing

RNA-seq was performed in isolated stromal fibroblasts from WT and P4ha2−/− mice aged 8 weeks. Total RNA was extracted using the Trizol reagent according to the manufacturer’s protocol. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then the libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, 150 bp paired-end reads were processed and generated, the libraries were sequenced on an Illumina HiSeq platform. Differential gene expression analysis was done using the R package DEseq2(1.42.1). P value < 0.05 and log2 fold change > 0.2 or log2 fold change < -0.2 was set as the threshold for significantly differential expression. The Differential genes table is available in Supplementary Materials.

Statistical analysis

The relative gene expression levels normalized by GAPDH were calculated by the formula 2 − ΔCt, where the ΔCt (critical threshold) = Ct of genes of interest −Ct of GAPDH. Statistical analysis was performed using the GraphPad Prism 8.0 software. Immunohistochemistry and immunoblot bands staining were measured by ImageJ. A two-tailed Student’s t-test was used to evaluate the group-level differences. Data were shown as the mean ± SEM. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.