Chemicals and antibodies

The following antibodies and chemicals were used: AAV8-RHOT1 (83070-1) and a RHOT1OE plasmid (Genechem, Shanghai, China); vector pCMV-Mito-AT1.03, Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), Tubulin-Tracker-Green, Tubulin-Tracker-Red, an Oil Red O Staining Kit, and Hoechst 33258 (Beyotime Biotechnology, Shanghai, China); and Tetramethylrhodamine (TRITC) Phalloidin, fluorescein isothiocyanate (FITC) Phalloidin, Micro Mitochondrial Respiratory Chain Complex I, III, IV, and V activity Assay Kits, an Mitochondrial extraction kit, and a Ca2+-Mg2+-ATPase activity assay Kit (Solarbio, Beijing, China). Mito-tracker-Red was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against the following proteins were used: Translocase of outer mitochondrial membrane 20 (TOMM20) (#42406), carnitine palmitoyltransferase 1 (CPT1) (#97361), acyl-CoA synthetase long chain family member 1 (ACSL1) (#9189), glycogen synthase kinase 3 beta (GSK-3β) (#12456), Tau (#46687), β-actin (#4970), cytochrome C oxidase subunit 4 (COX IV) (#4850), and sequestosome 1 (P62) (#23214) (all Cell Signaling Technology, Danvers, MA, USA). Other antibodies included those recognizing kinesin family member 5B (KIF5B) (sc-133184), mitochondrial Rho GTPase 1 (Rho-T1) (sc-398520), Dynactin1 (sc-365054), and dynein light chain LC8-type 1 (DYNLL1) (sc-136287) (Santa Cruz Biotechnology, Santa Cruz, CA, U SA).Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (Abcam) (ab6734). Other kits and reagents included, cadmium chloride (CdCl2) (Sigma-Aldrich, St. Louis, MO, USA); a Triglyceride (TG) assay kit and Total cholesterol (TC) assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); Nocodazole (Noc) and Cytochalasin D (Cytd) (MedChemExpress, Monmouth Junction, NJ, USA); GSK3B (GSK-3β), DCTN1 (Dynactin1), and SQSTM1 (P62)-specific small interfering RNAs (siRNAs; RiboBio Corporation, Guangzhou, China); and a Cell Counting Kit-8 (CCK8; Yeasen BioTechnology, Shanghai, China).

Animals and treatment

Albumin (Alb)-cre mice (used to generate conditional deletions in hepatocytes) were obtained from Cyagen Biosciences (Santa Clara, CA, USA) All mice were maintained on a C57BL/6J background. The mice were male, and were 8 weeks old. The adeno-associated virus vector AAV8-Rho-T1 was injected through the tail vein, and the mice were subsequently housed in a suitable environment (24 °C) for one month to detect RHOT1 expression levels. The 32 mice were evenly divided into four groups: (1) AAV8-Negative Control (AAV8-NC) group [double distilled water (DDW) intraperitoneally (i.p.)], (2) AAV8-NC + Cd group (1 mg/kg CdCl2, i.p.), (3) AAV8-Rho-T1 (DDW, i.p.), and (4) AAV8-Rho-T1 + Cd group (1 mg/kg CdCl2, i.p.). All mice had ad libitum access to food water and were maintained in a specific-pathogen-free facility with a 12 h:12 h light: dark cycle. Animals were randomly assigned at eight mice/group per experiment. After 2 weeks of exposure, the mice were humanely killed for tissue harvest using carbon dioxide asphyxiation. This study was approved by the Institutional Animal Care and Use Committee of Yangzhou University and carried out according to the Guide for the Care and Use of Laboratory Animals of the National Research Council (approval ID: SYXK (Su) 2022- 0044).

Cell culture and processing

The alpha mouse liver 12 (AML12) cell line (AML12, ATCC®CRL-2254™, USA)was cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 supplemented with 1% Insulin-Transferrin-Selenium-Sodium Pyruvate (ITS-A), 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin, and maintained at 37 °C with 5% CO2.

Mitochondrial tracing in living cells

pCMV-Mito-AT1.03 is a tool plasmid containing a cytomegalovirus (CMV) promoter for the expression of the AT1.03 protein (AT1.03 protein is an ATP indicator based on the ε subunit of FoF1-ATP synthetase) with a mitochondrial localization signal (MLS) in mammalian mitochondria as a mitochondrial fluorescent probe. AML12 cells were seeded in a 35 cm confocal dish and when the cell density reached 60%, pCMV-Mito-AT1.03 was transferred into the cells using Lipofectamine 3000 (Invitrogen, Cambridge, MA, USA). After 12 h, the fusion protein was observed under a fluorescence microscope. Cells expressing fusion proteins were then transferred to a live-cell workstation to observe mitochondrial dynamics (AXIO observer 7, Carl Zeiss, Germany).

Mito-tracker and CFDA-SE staining

Mito-Tracker and CFDA-SE were diluted with medium to form a staining working solution and stored in the dark for later use. AML12 cells were seeded in a petri dish and grown to 50% confluence. The cells were then immersed in the staining working solution at 37 °C for 20 min. The staining solution was aspirated off, the cells were washed three times with phosphate-buffered saline (PBS), fresh medium was added, and the cells were imaged under a fluorescence microscope.

Scanning electron microscopy and transmission electron microscopy

AML12 cells were seeded in 24-well cell glass coverslip, and when the cells grew to 70%, the medium containing different concentrations of Cd solution was replaced with the basal medium, and culture was continued for 12 h. The medium was discarded, the cells were washed with PBS twice, 2.5% glutaraldehyde was added to fix the cells at room temperature for 2 h, the glutaraldehyde was discarded, and the cells were washed with PBS thrice. The cells were then dehydrated through an ethanol gradient, with 15 min intervals between different concentrations. The sample was freeze-dried, sprayed with gold, and then observed under a scanning electron microscope (GeminiSEM 300, Carl Zeiss, Germany). For transmission electron microscopy, mouse liver tissue was cut into 1 × 1 mm blocks, the blocks were cleaned using PBS, fixed using 2.5% glutaraldehyde for 2 h at room temperature, dehydrated, embedded, and sectioned on an ultramicrotome. The sections were double-stained using 3% uranium acetate and lead citrate, and then observed on a transmission electron microscope (HT7800, HITACHI, Japan).

Establishment of a co-culture system for cells

In this study, we used Transwell assays to establish two cell culture models. 1. A group of Mito-tracker-labeled cells was seeded inside the Transwell membrane for testing. At the same time, a colony of unlabeled cells was inoculated in the internal chamber of the dish. When the cells were 80% confluent, the basal medium was replaced with a medium containing Cd. After continuing the culture for 12 h, the Transwell was removed, washed with PBS twice, and the cell in the dish were measured using fluorescence microscopy. 2. A group of Mito-tracker-labeled cells was seeded on the outside of the Transwell membrane, another group of cells labeled with CFDA-SE was seeded in inside the Transwell membrane. The two groups of cells were cultured together, and after the cells grew to 80%, the basal medium was replaced with medium containing Cd. After continuing culture for 6 h, the Transwell chamber was washed twice with PBS, the cells on the outside of the Transwell membrane were removed using a cell scraper, the Transwell chamber was washed twice with PBS, and the fluorescence changes in the cells inside the Transwell membrane were observed using fluorescence microscopy.

Flow cytometry

In this study, two groups of cells pre-stained CFDA-SE and Mito-tracker were mixed in cell culture flasks, centrifuged, collected, washed with PBS, centrifuged, collected, and observed by flow cytometry (LSRFortessa, BD Biosciences, USA). Subsequently, we established two cell models: 1. A group of CFDA-SE pre-stained cells was seeded in a 60 mm cell culture dish and grown to 70% confluence. Cd solution was added to the medium and cultured was continued for 12 h. The medium was removed and the cells were washed with PBS twice. Another group of Mito-tracker pre-stained cells was also added to a dish in a similar proportion, fresh medium was added, and culture was continued for 12 h. The cells were incubated with trypsin without EDTA for 40 s, collected by centrifugation, filtered, centrifuged again, and subjected to flow cytometry. 2. In the other model the cell labeling was the opposite, i.e., CFDA-SE-stained cells are added to the Mito-tracker pre-stained cell population to measure the co-staining rate of the cells.

Oil red O staining

Cell samples were fixed using 4% paraformaldehyde for 15 min, washed with PBS twice, added with staining wash solution to cover the cells for 20 s, after which the staining wash was discarded. Oil red O staining solution was added to the cells, incubated for 30 s, discarded, and staining wash solution was added and incubated for 30 min. The staining washing solution was then aspirated off, the cells were washed three times using PBS for each 3 min each time, and the cells were observed under the microscope. Tissue samples were fixed, dehydrated, embedded in optimal cutting temperature (OTC) resin, and sectioned to make frozen section. The staining wash solution was used to cover the sections for 20 s, the sections were immersed in the staining working solution, and stained for 20 min. The staining solution was aspirated off, the sections were covered with staining wash solution, incubated for 20 s, washed twice with PBS, and observed under the microscope.

BODIPY staining

BODIPY was made into a staining working solution. For cell samples, 4% paraformaldehyde fixation was carried out for 15 min, the cells were washed twice with PBS for 2 min each time, and then incubated with staining working solution for 20 min. The staining working solution was aspirated off, the cells were washed with PBS, and finally, Hoechst staining solution was added and incubated for 10 min. The cells were then washed with PBS three times before being imaged under the fluorescence microscope.

Hematoxylin–eosin staining

Part of the mouse liver was cut off at the same position in each mouse, trimmed, added to the fixative solution (4% paraformaldehyde), and fixed for 24 h. The fixative was discarded, and the tissue was washed, dehydrated through an ethanol concentration gradient, made transparent using a xylene solution, and impregnated with wax. The tissue was sectioned using a microtome and attached to a slide. The sections were then stained with hematoxylin for 10 min, rinsed twice under running water, stained using eosin staining solution for 1 min, rinsed twice under running water, and allowed to air dry. The dried sections were dehydrated, made transparent, and imaged under the microscope.

Western blotting analysis

Cells grown on dishes were collected using a cell scraper, centrifuged, lysed in Radioimmunoprecipitation assay (RIPA) Lysis Buffer at 4 °C for 30 min, and treated with ultrasound twice for 5 s each time. The samples were centrifuged at 12,000 × g for 10 min, the supernatant was retained, and the protein concentration in the supernatant was determined using a bicinchoninic acid (BCA) kit (Yeasen BioTechnology, Shanghai, China). The different samples were adjusted to the same protein concentration with loading buffer, and placed at 100 °C for 8 min. The proteins were then separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were subjected to blocking, washing with Tris Buffered Saline with Tween® 20 (TBST), primary antibody incubation, and secondary antibody incubation, after which the densities of the immunoreactive protein bands were measured using Image Laboratory software (Bio-Rad, Hercules, CA, USA).

Immunofluorescence

AML12 cells were seeded on 24-well cell glass coverslips, and then subjected to a series of treatments. The medium was then discarded, the cells were washed with PBS twice, fixed for 15 min in 4% paraformaldehyde, washed with PBS twice, and blocked for 2 h in blocking fluid (3% BSA solution containing 1% Triton X-100). The blocking fluid was removed and the cells were incubated with primary antibodies at 4 °C for 12 h, and then with the appropriate secondary antibodies at room temperature for 2 h, and washed with PBS three times. The cells could also be further co-stained with other staining agents (e.g., Tubulin-tracker, Phalloidin, Hoechst). After staining, anti-quencher was added, the coverslips were sealed, and then observed under a confocal microscope (TCS SP8 STED, Leica, Germany). Immunofluorescent staining of tissue samples was based on frozen sections. 0.5% TritonX-100 drops were added to the tissue sections, incubated at room temperature for 15 min, and then the TritonX-100 was discarded. The sections were blocked in PBS containing 10% normal goat serum at room temperature for 1 h. Primary antibodies were then added, incubated overnight at 4 °C, and then washed with PBS three times for 5 min each time. Then, different fluorescently labeled secondary antibodies were added, incubated at room temperature for 2 h, washed for 5 min, incubated with Hoechst in the dark for 10 min, washed with PBS three times, covered, and imaged under a fluorescence microscope.

Isolation of mitochondria

Mitochondrial isolation kits were used to isolate mitochondria within cells based on the manufacturer's requirements. Exogenous mitochondrial origin with untreated AML12 cells.Briefly, treated cells digested using pancreasin, centrifuged, and collected. The mitochondrial separation reagent was added, and then the solution was transferred to a glass homogenizer, and homogenized 30 times. The liquid in the homogenizer was transferred to a 1.5 ml centrifuge tube, centrifuged at 600 × g at 4 °C for 10 min, and the supernatant was collected. The supernatant was centrifuged at 11,000 × g at 4 °C for 10 min, and the precipitate comprised the extracted mitochondria, which were resuspended in mitochondrial storage solution, and preserved for later use.

TG and TC content determination

Tissue samples were weighed and a certain weight of tissue was added with normal saline at 1:9, homogenized, centrifuged at 2500 r/min for 10 min, and the supernatant was retained for measurement. Cell samples were made into a cell suspension using normal saline, disrupted using ultrasound, centrifuged, and the supernatant was retained for measurement. The TG and TC contents in the supernatants were determined according to the protocol of the corresponding test kit.

Micro mitochondrial respiratory chain complex I, III, IV, and V Ca2+-Mg2+-ATPase/ATP content determination

The mitochondrial respiratory chain samples were prepared as follows: 0.1 g of tissue was weighed or 5 million cells were collected, and extracted to make a tissue suspension or cell suspension. The suspension was transferred to a glass homogenizer, collect the liquid after homogenizing, and centrifuged. The supernatant was transferred into a 1.5 ml centrifuge tube, centrifuged, the pellet was resuspended in the extraction buffer, and the cells/tissue were broken ultrasonically. The resulting liquid is used for subsequent activity determination. The ATP content and Ca2+-Mg2+-ATPase activity could be measured without homogenization. The assays were performed according to the manufacturer’s protocol.

Co-immunoprecipitation

First, cells were digested and collected, added with a Cell lysis buffer for CO-IP (Beyotime Biotechnology, Shanghai, China) on ice for 30 min, and centrifuged at 4 °C and 12,000 × g for 15 min. Part of the supernatant was collected to prepare control protein samples. Then, 2 μg of the corresponding antibody was added to the remaining supernatant, and incubated overnight at 4 °C. At the same time, the magnetic beads were resuspended and cleaned with PBS (noting the damage to the beads during washing), centrifuged, placed on the magnetic stand, and the liquid was removed. The collected supernatant was added to resuspend the magnetic beads, which were incubated at room temperature for 2 h, Washed three times with the lysate, added with loading buffer, and denatured at high temperature for 10 min before electrophoresis..

Cell viability assay

The CCK8 assay kit was used to detect cell viability. First, the required CCK8 working solution (CCK8 solution and medium 1:9 configuration) was calculated according to the number of samples, and protected from light. Cells were seeded in 96-well plates, and after a series of treatments, the medium was discarded and the CCK8 working solution was added. After incubation for 1 h, the OD value of each well was determined at 450 nm.

Small interfering RNA transfection

The siRNA specific for GSK-3β, Rho-T1, Dynactin1, and P62 was synthesized by RiboBio Corporation (Guangzhou, China). AML12 cells were seeded in 6-well or 24-well plates. At 40% confluence, the cells were cultured in fresh medium without penicillin and streptomycin, together with transfection reagent (Polyplustransfection, Illkirch, France) and 20 nM GSK-3β, Rho-T1, Dynactin1,and P62 siRNA or negative control siRNA for 24 h, according to the manufacturer’s protocols. Then, the cells were subjected to other treatments as required by the experiments.

Statistical analysis

The data from at least three independent experiments were analyzed statistically and expressed as the mean ± standard deviation (SD). GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, USA) analyzed the data using one-way analysis of variance ((ANOVA) (Scheffe’s SF test). P values less than 0.05 indicated a significant difference.