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Page 4 of 14 Schwertheim et al. Hepatoma Res 2020;6:41 I http://dx.doi.org/10.20517/2394-5079.2020.23

incubated with non-immune immunoglobulin at the same concentration as the primary antibody instead of
[6]
the primary antibody as described before . We counted the membrane-bound intranuclear inclusions (NI)
in all 10 serial sections of each case; cases lacking evaluable material on all 10 serial sections were excluded
[6]
as described before . We defined an inclusion only as positive if it was delimited by an intact membrane and
completely closed. In a standardized area, inclusions were counted and calculated by: 10 × (3 × 1 mm tissue
2
2
cores) = 10 × (3 × 0.78 mm ) = 23.5 mm . The qualitative detection of membrane-bound nuclear inclusions
was recorded as positive (1) or negative (0). We defined cases containing at least one membrane-bound
nuclear inclusion as positive; haematoxylin counterstaining enabled the enumeration of the inclusions. We
analyzed the immunostainings within NI. Quantitative analysis of immunopositive NI was performed for
β-catenin, KDM2A, ubiquitin, p62, LC3B, cathepsin B and cathepsin D. Due to the small size of the NI and
staining surface, we did not quantify immunostaining intensity. Cases with positively stained membrane-
bound nuclear inclusions were classified as positive (1) or negative (0).

Next generation sequencing
FFPE tumor tissue of all cases was analyzed by next generation sequencing (NGS) with the Illumina MiSeq
[16]
sequencer (Illumina, San Diego, CA, USA) following the manufacturer’s instructions as described before .
Briefly, 45 ng of DNA was used to perform multiplex-PCR and Biomedical Genomics Workbench (CLC Bio,
Qiagen, USA) was used for analysis. We designed a customized HCC-panel containing regions of interest: 23
genes of the Wnt pathway including the CTNNB1 gene.

Transmission electron microscopy
Ultrastructural analysis of NI was carried out as described previously [6,16] . Briefly, for transmission
electron microscopy (TEM), fresh tissue from liver biopsy of a representative HCC patient was fixed in 2%
glutaraldehyde in 0.1M cacodylate buffer (cb), pH 7.3, for 4 h at room temperature. Afterwards it was washed
in cb, post-fixed with 1% osmium tetroxide in cb, dehydrated in a graded series of alcohol and embedded
in epoxy resin. In order to determine blocks of adequate quality, semi-thin sections were stained with basic
fuchsin and methylene blue. Ultrathin sections of selected blocks were mounted on copper grids, and then
treated with uranyl acetate (1%) and lead citrate (0.4%) for contrast enhancement. Digital TEM images were
acquired on the Zeiss EM 902A (Zeiss, Oberkochen, Germany) equipped with a Morada slow-scan CCD
camera using ITEM 5.2 software (both Olympus Soft-Imaging-Systems, Münster, Germany).

Double immunofluorescence of tissue sections
Spatial localization of β-catenin and p62 was investigated by double immunofluorescence staining.
One-µm-thick FFPE tissue sections (HCC) were cut, dewaxed, rehydrated and pretreated with Target
Retrieval Solution (Agilent Technologies, Ratingen, Germany) at pH 9.0 for 20 min at 97 °C. For double
labeling immunofluorescence, the primary antibodies anti-β-catenin (Transduction) and anti-p62 (Enzo)
were used. Anti-p62 antibody was labeled with Donkey Anti Rabbit AF555. For labeling of anti-β-catenin
antibody Goat Anti Mouse HRP and Goat Anti HRP AF488 were used. Details are provided in
Supplementary Table 2. DNA was stained with DAPI; image analysis and microscopy was performed by
using an Olympus BX43 (Olympus Deutschland, Hamburg, Germany).

Statistical analysis
Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS 24.0, Chicago,
IL, USA) program. Relationships between categorical parameters were investigated using the two-sided
Fisher’s exact test. Overall survival (OS) curves were performed using the Kaplan-Meier method, and
differences in survival curves were compared by the log-rank test. Mann-Whitney U-test was used to assess
whether positive KDM2A immunostaining in NI correlates with the number of NI; P ≤ 0.05 was defined as
statistically significant.