Additional file 2 of The prognostic role of ACSL4 in postoperative adjuvant TACE-treated HCC: implications for therapeutic response and mechanistic insights
Creators: name: Feng, Ji
name: Bin, Jin-Lian
name: Liao, Xi-Wen
name: Wu, Yong
name: Tang, Yue
name: Lu, Pei-Zhi
name: Zhu, Guang-Zhi
name: Cui, Qian-Ru
name: Dan, Yock Young
name: Yang, Guo-Huan
name: Li, Li-Xin
name: Deng, Jing-Huan
name: Peng, Tao
name: Hooi, Shing Chuan
name: Zhou, Jing
name: Lu, Guo-Dong
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer

description: Supplementary Material 2. Supplementary Fig. 1. ACSLs expression comparisons in HCC patients collect from the TCGA-LIHC database. a mRNA expression of ACSLs between tumor samples (n=374) and normal samples (n=50). b ACSL4 protein was determined by western blotting in 18 representative pairs of HCC (TU) and adjacent non-tumor (NT) tissue samples, relative ACSL4 levels were normalized with α-tubulin or GAPDH. Supplementary Fig. 2. ACSL4 was associated with poorer HCC patient survival. a A Kaplan-Meier survival comparison of HCC patients with or without ACSL4 up-regulation. b With the increase of ACSL4 expression in tumor, the higher level of serum AFP observed. c-d Kaplan-Meier survival comparisons between ACSL4-present and ACSL4-absent HCC patients who had low serum AFP level (c) or who had high serum AFP level (d). e Accumulated risk to develop recurrence in HCC patients with or without ACSL4. f A Kaplan-Meier survival comparison of recurrent (within two years after curative surgery) HCC patients with or without ACSL4. g Kaplan-Meier survival comparison of recurrent HCC patients with different post-recurrent treatment methods.Supplementary Fig. 3. ACSL4 protected HCC cells from glucose starvation, rather than hypoxia or chemotherapeutic drugs. a Huh7 cells were silenced with ACSL4 siRNA or negative control siRNA, before treatment with full medium or glucose starvation for 48 hours. b ACSL4-overexpressing (OE) cells were treated with full medium or glucose starvation for 24 hours. The proteins were determined by Western blotting (up panel) while the proportion of survival cells were determined by PI assay (lower panel). c ACSL4-knockout (KO) Huh7 cells (left panel), ACSL4-OE N1S1 cells (middle panel) or ACSL4-OE HepG2 cells (right panel) were incubated in normoxia or hypoxia (Hpx) for indicated times. Then cell viability was determined by cck8 assay. d-e ACSL4-OE N1S1 cells (d) or ACSL4-OE HepG2 cells (e) were treated with cisplatin or doxorubicin for 36 hours or 24 hours respectively, with or without hypoxia treatment. f-i Comparation between NC-sg and ACSL4-KO Huh7 cells in cell proliferation (f), clone formation (g), migration (h) and invasion (i). j-m Comparation between NC and ACSL4-OE HepG2 cells in proliferation (j), clone formation (k), migration (l) and invasion (m). n Comparation between NC and ACSL4-OE N1S1 cells in cell proliferation. o Western blot analyses of ACSL4 in indicated cells incubated in full or glucose starvation medium for 48 hours. Compared with NC, #P &lt;0.05,##P &lt;0.01, ###P&lt; 0.001. ***P &lt;0.001. NS, not significant. Supplementary Fig. 4. ACSL4-OE maintenance of cellular ATP levels by promoting β-oxidation under glucose starvation. a-b ACSL4-OE N1S1 cells (a) or HepG2 cells (b) were treated with full medium or glucose starvation for 36 hours or 20 hours respectively. Then cellular ATP were detected. c ACSL4-OE cells were treated with glucose starvation for 12 hours, mitochondrial membrane potential was detected using JC-1 dye by flow cytometry. The ratios of red/green florescence were shown. d  Indicated cells treated with or without 50μM CQ were incubated in full or glucose starvation medium for 24 hours, then the cellular lipid droplets were staining with O oil red. e-f ACSL4-OE cells were treated with glucose starvation for 12 hours. The cellular lipid droplets (e) and triglycerides (f) were detected. g ACSL4-OE cells were treated with glucose starvation for 20 hours, then cellular β-hydroxybutyrate (β-HB) were detected. Supplementary Fig. 5. ACSL4 promote cell survival by mediating fatty acid activation and β-oxidation. a-bACSL4-OE N1S1 cells (a) or HepG2 cells (b) were treated with glucose starvation medium supplied with/without BSA or indicated BSA-fatty acid conjugates (AA-CoA, AA, LA, PA, OA, SA; 25 μM for each). Then the proportion of survival cells were detected. c-d ACSL4-OE N1S1 cells (upper panel) or HepG2 cells (lower panel) were treated with glucose starvation medium supplied with/without AA-CoA (25 μM), then cellular ATP (c) and β-HB (d) were detected respectively. e ACSL4-OE cells were treated with glucose starvation medium supplied with AA-CoA (25 μM) and/or etomoxir (ETO, 40 μM). The proportion of survival cells were detected. f-g Oxygen consumption rates (OCRs) in ACSL4-OE cells and respective control cells were determined by Seahorse XF analyzer (f). Basal OCR (left panel), ATP-linked OCR (middle panel) and maximal OCR (right panel) of indicated cells are summarized, respectively (g). Abbreviations: AA-CoA, arachidonoyl coenzyme A lithium salt; CON, control; BSA, bovine serum albumin; ETO, etomoxir; Oligo, oligomycin; Rot/ AA, Rotenone/ Antimycin A. Compared with NC, #P &lt;0.05, ##P &lt;0.01, ###P &lt;0.001. *P&lt;0.05, **P &lt;0.01; ***P &lt;0.001. NS, not significant. Supplementary Fig. 6. ACSL4-OE prevented cells from inhibition caused by canagliflozin mimicked glucose starvation in vitro and in vivo. a-b The 3D Spheroid tumor developed by indicated cells were treated with glucose starvation (a) or canagliflozin (Cana, 20 μM) in glucose-present (0.9 g/L) medium (b) for 60 hours. The dead cells were stained with PI (red fluorescence) while live cells were stained with calcein (green fluorescence) before observation under a fluorescent microscopy. The volume of spheroid (middle panel) and cell death (right panel) were detected. c The 3D Spheroid tumor developed by indicated cells were treated with canagliflozin (Cana, 20 μM) in glucose-present (0.9 g/L) medium in the present or absent of rosiglitazone (Rosig, 20 μM) for 48 hours. The volume of spheroid (lower-left panel) and cell death (lower-right panel) were detected. d-h The mice experiment was carried out as in Fig. 5c or S6i. The effects of Cana treatment on blood glucose level (d-e) and tumor glycogen (f) and body weights (g-h) were summarized respectively. h The mice bearing xenograft developed from ACSL4-OE or control HepG2 cells were treated with control PBS or Cana (30 mg/kg) for 15 days once every other day. The resected tumors were photographed (left panel). The tumor volumes (middle panel) and tumor weights (right panel) were measured. i-l Representative images of hematoxylin &amp; eosin staining (HE), IHC for ACSL4 and Ki67 are shown (i). The proportion of area of necrotic tissue (k) and the Ki-67 positive score (l) were summarized. Compared with NC, #P &lt;0.05, ##P &lt;0.01, ###P &lt;0.001. *P&lt;0.05, ***P &lt;0.001. NS, not significant. Supplementary Fig. 7. Correlation of CEBPA and ACSLs in HCC. a-d Correlation of mRNA expression between CEBPA and ACSLs (ACSL1, ACSL3, ACSL5, ACSL6) in HCC in TCGA-LIHC database. e-h Correlation of mRNA expression between CEBPA and ACSLs (ACSL1, ACSL3, ACSL5, ACSL6) in 6 human cell lines. i Expression of CEBPA protein in 7 cell lines. Supplementary Fig. 8. ACSL4 was regulated transcriptionally by CEBPA. a CEBPA-overexpression HepG2 (pcDNA3.1-CEBPA) and its negative control (pcDNA3.1-vector) were treated with glucose starvation for 24 hours. The proportion of survival cells is shown in the right panel.  b-c The luciferase reporter activity of ACSL4 promoter was assayed in indicated cells. Luciferase activity was normalized to Renilla activity and was plotted relative to NC (b) or pcDNA3.1-Vector (c). d Expression of CEBPA in different cancer types in TCGA database, which was analyzed using online website tool Timer 2.0 (http://timer.cistrome.org/), LIHC data was marked with the black frame. e-f Comparison of recurrence between ACSL4-High and ACSL4-Low HCC patients (e) or CEBPA-High and CEBPA-Low HCC patients (f) who treated TACE as post-recurrence treatment.