The use of clinical samples
A panel of 40-paired HCC tumor and adjacent non-tumor samples was obtained from the Department of Pathology, the First Affiliated Hospital of Chongqing Medical University. The use of the human clinical samples in this study was approved by the Research Ethics and Regulations Committee of Chongqing Medical University, Chongqing, China.
Cell Culture And Chemicals
HEK-293 derived 293pTP and RAPA cells were previously described [38, 39]. Human hepatocyte line LO2, Human HCC lines Huh7, MHCC97H, HepG2 and Hep3B were obtained from the Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education, Chongqing Medical University. All cells were cultured in DMEM supplemented with 10% fetal bovine serum (Lonsera, Cat: S711-0015, Uruguay) containing 100 units of penicillin and 100 µg of streptomycin in a 5% CO2 incubator at 37°C. BAY-876 was purchased from Selleckchem (Cat: S8452, Houston, TX, USA). Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), Thermo Fisher Scientific (Waltham, MA, USA), or Solarbio (Beijing, China).
Construction And Amplification Of Recombinant Adenoviral Vectors Ad-b4, Ad-sib4, Ad-gfp And Ad-rfp
The AdEasy technology was used to generate recombinant adenovirus as described [40–42]. The construction and amplification of Ad-B4 that overexpresses human BMP4 was described previously [43]. The construction and amplification of Ad-siB4 that silences the human BMP4 was carried by using our recently developed methods as described [44–46]. Both Ad-GFP and Ad-RFP were used as controls [47]. Ad-B4 also expresses GFP, while Ad-siB4 co-expresses RFP. Fluorescence signals were documented under a fluorescence microscope at 48h after infection. For all adenoviral infections, polybrene (8µg/ml) was added to enhance infection efficiency as reported [19, 20].
H & E And Immunohistochemical (Ihc) Staining
The tumor tissue sections were deparaffinized, rehydrated and subjected to H & E staining and IHC staining as described [19, 20, 47]. Briefly, the tissue sections were deparaffinized, rehydrated, antigen-retrieval treated, blocked and incubated overnight with appropriate dilutions of primary antibodies against BMP4 (1:50 dilution; Bimake; Cat# A5543), SLC2A1 (1:100 dilution; Proteintech; Cat# 21829-1-AP), SMAD5 (1:50 dilution; Bimake; Cat# A5511), or p-SMAD5 (1:200 dilution; Abcam; Cat# ab92698), followed by stained with biotin-labeled goat anti-rabbit IgG and horseradish peroxidase-conjugated-labeled streptavidin. IHC scores were calculated based on the percentage of positive cells and the degree of staining. The IHC staining scores were defined into 5 levels as follow: Level 1, positive cells accounting for ≤ 20%; Level 2, positive cells accounting for 20–40%; Level 3, positive cells accounting for 40–60%; Level 4,positive cells accounting for 60–80%; and Level 5, positive cells accounting for > 80%. Samples with IHC staining scores < Level 3 were designated as low expression groups, while samples with ≥ Level 3 were designated as high expression groups. The scoring outcomes of HCC tissue and adjacent liver non-tumor tissue samples for anti-BMP4 and anti-SLC2A1 IHC are shown in Table S1. Staining results were recorded under a bright field microscope (magnification, x100 or x400). Each assay condition was done in triplicate. Representative results are shown.
Total Rna Extraction And Touchdown-quantitative Real-time Pcr (Tqpcr)
Total RNA was isolated from the cultured cells using TRIZOL Reagent (Invitrogen, China) as described [48, 49]. Briefly, Huh7 and HepG2 cells treated with various conditions were lysed in TRIZOL Reagent for total RNA isolation. Total RNA was subjected to reverse transcription with hexamer and M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA). The cDNA products were used as PCR templates. Gene-specific PCR primers were designed by using Primer 3 program. All primer sequences are shown in Table S2. TqPCR was carried out by using 2x SYBR Green qPCR Master Mix (Bimake, Shanghai, China) on the CFX-Connect unit (Bio-Rad Laboratories, Hercules, CA) as described [49]. All TqPCR reactions were done in triplicate. GAPDH was used as a reference gene. Quantification of gene expression was carried out by using the 2−ΔΔCq method as described [48].
Western Blotting Analysis
Western blotting was performed as previously described [50]. Huh7 and HepG2 cells were treated with various conditions, and lysed in RIRA Lysis Buffer containing phosphatase inhibitors and protease inhibitors. Cell lysates were separated by 10% SDS-PAGE and transferred to PVDF membranes. The proteins of interest were assessed by being incubated with the following primary antibodies at 4°C overnight: anti-BMP4 (1:1000; Bimake; Cat# A5543), anti-SLC2A1 (1:1000; Proteintech; Cat# 21829-1-AP), anti-SMAD5 (1:1000, Bimake; Cat# A5511), anti-p-SMAD5 (1:1000; Abcam; Cat# ab92698), and anti-β-Actin (1:2000, Proteintech; Cat# 60008-1-Ig). Subsequently, the membranes were incubated with HRP-conjugated goat anti-mouse or anti-rabbit secondary antibody for 1h at 37°C, respectively (1:5000; ZSGB-BIG, Cat# ZB-2306 or 2305). Lastly, the proteins were detected on the Bio-Rad ChemiDoc Imager (Hercules, CA) by using an Enhanced Chemiluminescence (ECL) kit (Millipore, USA). Each assay condition was done in triplicate. Representative results are shown. The original images of WB were shown in Supplementary Fig. S1.
Immunofluorescence (If)
The IF staining was carried out as reported [47]. Huh7 and HepG2 cells were seeded in 24-well plates onto coverslips and treated with various conditions, followed by being fixed with 4% paraformaldehyde at RT for 20 min and blocked with 5% bovine serum albumin (BSA) for 30 min at RT. The coverslips were incubated with the primary antibody anti-SLC2A1 (1:100; Proteintech; Cat# 21829-1-AP) at 4°C overnight, and incubated with Rhodamine (TRITC)–conjugated goat anti-rabbit IgG (H + L) (1:200; Proteintech; Cat# SA00007-2) and CoraLite488-conjugated goat anti-rabbit IgG(H + L) (1:200; Proteintech; Cat# SA00013-2) for 2h at RT. The nuclei were counterstained with DAPI (10µg/mL) for 5min at RT. Minus primary antibody only was used as a negative control. Fluorescent images were obtained with a laser confocal microscope (Leica TCS SP8, magnification x400). Each assay condition was done in triplicate. Representative results are shown.
Glycogen Accumulation And Quantitative Analyses
PAS staining was conducted using Glycogen Periodic Acid Schiff (PAS / Hematoxylin) Stain Kit as described [24]. The staining results were recorded under a bright field microscope (magnification, x100 or x400). Glycogen quantification assay was performed by using the glycogen assay kit (Solarbio, Glucogen Assay Kit; Cat# BC0340). Each assay condition was done in triplicate. Representative results are shown.
Wst-1 Cell Proliferation Assay
WST-1 assay was conducted as described [51]. Briefly, the Huh7 and HepG2 cells were plated in 96-well plates at a density of 2,000 cells per well and treated with different conditions. At the indicated time points, the Premixed WST-1 Reagent (Clontech, Mountain View, CA) was added, followed by incubating at 37°C for 120 min and reading absorbance at 450nm. Each assay condition was done in triplicate.
Chromatin Immunoprecipitation (Chip)
Consensus Smad1/Smad5 binding sites were previously characterized [52, 53]. Potential binding motifs for Smad1/5 at the SLC2A1 promoter were predicted by using the JASPAR program (https://jaspar.genereg.net/. Accessed 20 May 2022) [54, 55]. ChIP assay was carried out by using the ChIP Assay Kit (Beyotime, China, # P2078) as previously described [56]. Briefly, Huh7 and HepG2 cells were infected with Ad-B4 for 36h, and formaldehyde cross-linked, which was terminated by adding glycine. The cross-linked cells were collected by centrifugation at 4°C and then sonicated in an ice bath. Ultrasonic samples were centrifuged at 12,000g and then incubated with Protein A + G Agarose/Salmon Sperm DNA with occasional agitations at 4ºC for 30 minutes, followed by centrifugation to remove non-specific binding DNA/protein complexes. The cleared supernatant samples were incubated with Anti-SMAD5 (1:20 dilution; Bimake; Cat# A5511) or goat IgG to pull down the protein-DNA complex at 4°C overnight. The complexes were captured by Protein A + G Agarose/Salmon Sperm DNA, followed by being washed sequentially in different wash buffers. The pulled down precipitates (i.e., DNA-protein complexes) were dissolved in freshly prepared elution buffer (1% SDS, 0.1M NaHCO3). The mixtures were heated at 65°C for 4 h to reverse the formaldehyde cross-linking in a high salt buffer (0.2M NaCl). The pulled down genomic DNA was isolated by using the DNA Purification Kit (Beyotime# D0033) for PCR. The presence of SLC2A1 promoter fragments was detected by semi-quantitative PCR using and the primer sequences are listed in Table S3.
Xenograft tumor model of human HCC in vivo
The use and care of experimental animals was approved by the Research Ethics and Regulations Committee of Chongqing Medical University, Chongqing, China. All experimental procedures followed the approved guidelines. Athymic nude mice were obtained from and housed in the Experimental Animal Research Center of Chongqing Medical University. The cancer cell implantation experiments were carried out as previously described [56]. Briefly, Huh7 cells were infected with Ad-B4 and Ad-GFP, collected and resuspended in sterile PBS for subcutaneous injection into the flanks of athymic nude mice (5–6 week old, male, 5×106 cells/injection, 4 injections per mouse, and 3 mice per group). The mice in the Ad-B4 + BAY-876 group also were administered through gavage with BAY-876 (2.5 mg/kg) in 0.25% carboxymethyl cellulose (CMC) every 3 days, while the mice in the Ad-B4 group and Ad-GFP group were administered through gavage with equal volumes 0.25% CMC. Tumor volumes were measured every 7 days. At 28 days after injection, mice were sacrificed, and the subcutaneous tumor masses were retrieved. The tumor volume of tumor was calculated according to the formula LW2/2, where L is the length and W is the width of the tumor. The subcutaneous tumor masses were subjected to paraffin embedding and histologic and IHC staining.
Statistical analysis
All experiments performed at least three times and/or repeated in three independent batches. Data were analyzed using GraphPad Prism 8 and presented as mean ± standard deviation (SD). Statistical significance was confirmed by one-way analysis of variance and the student’s t-test for the comparisons between groups. A value of P < 0.05 was considered statistically significant.