Reaction buffer

A total of 20 mg of tissue was used for total RNA isolation from liver samples using an RNA isolation kit (Macherey-Nagel, Allenton, PA, USA) following the manufacturer’s instructions. RNA concentration was measured using a NanoDrop spectrophotometer and stored at −80 °C.
Reverse transcription was performed with 1 µg of total RNA in a reaction buffer (Roche Diagnostics, Mannhein, Germany) containing 5 mM MgCl₂, 10 µM dNTPs (Biotools, Jupiter, FL, USA), 5 µM random hexamers (Invitrogen, Waltham, MA, USA) and 1 unit of AMV Reverse Transcriptase (Applied Biosystems, Waltham, MA, USA). Then, qPCR reaction using a Taqman Universal PCR Master Mix (Applied Biosystems) was performed as previously described [24 (link)]. Primers sequences for qPCR are shown in Supplementary Materials, Table S2.

Formaldehyde (4%)-fixed specimens were paraffin-embedded and cut at a thickness of 4 μm. Sections were dried for 1 h at 56 °C, and immunohistochemical staining was performed using a DISCOVERY XT automated staining platform (Ventana Medical Systems, Tucson, AZ, USA) with a DISCOVERY ChromoMap DAB (3,3’-diamniobenzidine) Kit as follows: Sections were deparaffinized and rehydrated using EZ Prep (Ventana) and washed with Reaction Buffer (Ventana), and antigens were retrieved by heating at 90 °C for 30 min in RiboCC citrate buffer reagent (pH 6.0; Ventana) prior to detection with an anti-Ki-67 antibody (ab15580, Abcam) or anti-NDUFB8 antibody (ab192878, Abcam) and DAB.

Formaldehyde (4%)-fixed specimens were paraffin-embedded and cut at a thickness of 4 μm. Sections were dried for 1 h at 56 °C and immunohistochemical staining was performed with the automated instrument Discovery XT (Ventana medical system, Tucson Arizona, USA) using the Chromomap DAB Detection kit as follows: sections were deparaffinized and rehydrated using EZ prep (Ventana) and washed with Reaction buffer (Ventana). Antigens were retrieved by heating at 90 °C for 30 min in Citrate buffer (pH 6.0; Ribo CC, Ventana) prior to detection with an anti-Ki-67 antibody (ab15580; Abcam, Cambridge, UK).

A431 cells (2.5 × 10⁶) was injected subcutaneously into 6-week-old Balb/c nude mice. Average tumor volume was determined as (L × W²)/2 with measurements of tumor length (L) and width (W) taken with a caliper. When the tumor reached an average volume of 30 mm³, siRNA mixtures were injected using atelogenes local use (Koken, Japan).^{49 (link)} Tumor volumes were determined twice a week after the siRNA gel injection. A431 cells (2.5 × 10⁶) expressing shEV or shREP1 were injected subcutaneously into Balb/c nude mice and tumor volumes were determined twice a week. Finally, the mice were killed and tumor tissues were either processed for immunoblotting analysis or immunohistochemistry analysis. For immunohistochemistry, tumor tissues were fixed with 10% neutral buffered formalin. Formaldehyde fixed specimens were paraffin-embedded and cut at a thickness of 4 μm. Sections were dried for 1 h at 56 °C and immunohistochemical staining was performed with the automated instrument Discovery XT (Ventana medical systems, Tucson, AZ, USA) as follows: sections were deparaffinized, rehydrated with EZ prep (Ventana medical systems), and washed with reaction buffer (Ventana medical systems). The antigens were retrieved with heat treatment in Tris–EDTA buffer (CC1, Ventana medical systems) at 90 °C for 30 min with indicated antibodies.

An immunohistochemical staining for Ki-67 was performed to evaluate the regenerative/proliferative activity of the tubular epithelial cells. The BenchMark Ultra automated staining platform (Ventana Medical System, Inc., USA) was used for this purpose. Briefly, the tissue sections from the representative paraffin blocks that were cut at 3 μm thickness onto charged slides were deparaffinized with EZ Prep. solution (Ventana medical system, cat. no: 950–102) at 75 °C. Following rehydration through alcohol series and 32 min heat-induced epitope retrieval at 100 °C with an EDTA-based buffer (Cell Conditioner 1, Ventana; cat. no: 950–124), tissue sections were incubated for 32 min with anti-Ki-67 primary antibody (Ventana; pre-diluted; monoclonal rabbit, clone SP6) at 37 °C temperature. Washing between the steps were accomplished by Reaction Buffer (Ventana medical system, cat. no: 950–300). Ultra View Detection kit (Ventana medical system, cat. no: 760–500) was used for the detection of the target protein. The reaction product was visualized with 3, 3′-diaminobenzidine chromogen and counterstaining with hematoxylin was done. Nuclear staining was considered positive. Appropriate staining of the germinal centers of normal tonsil served as a positive control. Ki-67 index was expressed as the percentage of the number of immunostained nuclei among the total number of tubular cell nuclei.

Formaldehyde (4%) fixed specimens were paraffin-embedded and cut at a thickness of 4 μm. Sections were dried for 1 h at 56 °C, and immunohistochemical staining was performed with the automated instrument Discovery XT (Ventana Medical Systems, Tucson, Arizona, USA) using the Chromomap DAB Detection kit as follows: sections were deparaffinized and rehydrated with EZ prep (Ventana, Oro Valley, AZ, USA) and washed with reaction buffer (Ventana, Oro Valley, AZ, USA). The antigens were retrieved with heat treatment in pH 6.0 citrate buffer (Ribo CC, Ventana, Oro Valley, AZ, USA) at 90 °C for 30 min for anti-Ki-67 (ab15580; Abcam, Cambridge, UK), CK-19 (ab52625, Abcam, Cambridge, UK), and α-SMA (ab5694, Abcam, Cambridge, UK).