Fig 2: HRG deletion affects the development of experimental MASH by downregulating inflammatory response and fibrogenesis. IHC analysis for F4/80 (A), αSMA (B) and Sirius Red staining (C) performed on paraffin-embedded liver samples from six wild-type mice (WT) or from nine HRG knock-out mice (HRG–/–) fed with CDAA diet for 24 weeks. ImageJ software analysis was performed to evaluate the amount of positivity. The data are means ± SD of 6/9 animals per group. The boxes include the values within the 25th and 75th percentile, whereas the horizontal bars represent the medians. The extremities of the vertical bars (10th–90th percentile) comprise 80% of the values. Statistical differences were assessed by unpaired parametric t-test or Mann–Whitney non-parametric t-test. Original magnification as indicated (A–C).

Fig 3: HRG in human HCC of mixed etiology. Survival and disease-free survival (log rank Mantel–Cox test) in high- vs. low-HRG-expressing HCC patients in the TCGA-LIHC cohort accessed using GEPIA webserver (A, B). GEPIA analysis of HRG gene expression in the non-tumoral (n = 50) areas and in the tumor samples (n = 369) from the TCGA-LIHC cohort. *p-value <0.0001 (C).

Fig 4: HRG protein expression in MASLD/MASH-related HCC patients. IHC analysis of HRG performed on paraffin-embedded human liver specimens from MASLD/MASH-related HCC patients (n = 37, grades G2 to G3). Original magnification as indicated (A). HRG expression was semi-quantitatively scored blinded by a pathologist by means of histological score (H-score) (B). Kaplan–Meier curves of overall survival (C) and recurrence (D) according to HRG peritumoral H-score in MASLD/MASH-related HCC patients (C, D). Statistical analysis was performed using log-rank (Mantel–Cox) test (C, D). Plasma concentration of HRG measured in a cohort of patients (N = 78) with different stages of disease progression including 10 patients with a F0/F1 score, 25 patients with a F3/F4 score (six F3 and 19 F4) and 43 MASLD/MASH patients carrying HCC. Cirrh, cirrhotic liver; F, fibrosis score (E). Kaplan–Meier curves of overall survival according to HRG plasma levels in MASLD/MASH-related HCC patients (F). Statistical analysis was performed using log-rank (Mantel–Cox) test (F). ns, not significant.

Fig 5: HRG deletion affects apoptosis but not proliferation. WB analysis for proCASP3 and cleaved CASP3 (A), PCNA (B), c-MYC (C), and YAP (D) performed in HCCs from wild-type mice (WT) or from HRG knock-out mice (HRG–/–). BIORAD Quantity One software was used to perform the densitometric analysis (data are expressed as fold change relative to the normalized WT expression). Equal loading was evaluated by re-probing membranes for β-actin (A, B, D) or vinculin (C). Statistical differences were assessed by Student’s t-test or Mann–Whitney test for non-parametric values (A–D). qPCR analysis of Ki67 transcripts performed in HCC tumor masses from 12 WT mice or from 11 HRG–/– mice (E). The mRNA values are expressed as fold increase over control values after normalization to the TBP gene expression. The results are expressed as means ± SD. The boxes include the values within the 25th and 75th percentile, whereas the horizontal bars represent the medians. The extremities of the vertical bars (10th–90th percentile) comprise 80% of the values. Statistical differences were assessed by Student’s t-test or Mann–Whitney test for non-parametric values (E). ns, not significant.