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Drugs and reagents
Ruxolitinib (Ruxo, Cat: T1829), Mdivi-1 (Cat: T1907), STX-0119 (Cat: T60160), Tween 80 (Cat: T13947) and PEG300 (Cat: T7022) were procured from TargetMol (Shanghai, China) and DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride, Cat: C1002) was purchased from Beyotime Biotechnology, China. Z-VAD-FMK (Cat: A1902) was purchased from Apex Bio (Houston, TX, USA). The mitochondrial tracker deep red (Mito tracker) (Cat:8778) was purchased from Cell Signaling Technology (MA, USA). The anti-cytochrome C antibody (Cat:556432) was purchased from BD Biosciences (CA, USA). The anti-HSP60 antibody (Cat: 66041-1-Ig) was purchased from Proteintech (Wuhan, China).
Patient information and specimens
Tumor tissue specimens were procured from a collective of 4 patients diagnosed with ATC and 10 patients with PTC. Additionally, normal thyroid (NT) tissues were collected from 10 patients diagnosed with benign nodular goiter, who underwent surgery at the Zhejiang Provincial People’s Hospital between January 2016 and December 2022. The excised specimens were promptly cryopreserved in liquid nitrogen at −80 °C. In accordance with the criteria set by the World Health Organization, all specimens were reviewed by two pathologists in a retrospective manner to validate the histological diagnosis. Consent forms were duly acquired from all participating individuals as a measure of ethical responsibility. Approval number QT20222394 was granted by the Ethics Committee of Zhejiang Provincial People’s Hospital for this study.
Cell culture
The human thyroid epithelial cell line Nthy-ori 3-1 (NTHY), PTC cell lines TPC-1, BCPAP, and IHH4 were acquired from Procell Life Science & Technology (Wuhan, China). The human ATC cell lines KHM-5M, C643, CAL-62 and BHT101 were acquired from the National Collection of Authenticated Cell Cultures (Shanghai, China). Deutsche Sammlung von Mikroorganismen und Zellkulturen provided us with another ATC cell line called 8505C. All cells have STR Authentication and were cultured in HyClone RPMI-1640 with 10% fetal bovine serum (Cat: S-FBS-SA-015, SERANA, Germany), 100U/ml penicillin, and 0.1 mg/ml streptomycin. The cells were maintained at 37 °C with 5% CO2.
Cell viability
The viability of cells was estimated using a CCK-8 assay (Cat: A311-01; Vazyme Biotech Co., Ltd., China) according to the instructions provided by the manufacturer. Prior to treatment, the cells were placed in 96-well plates at a density of 5000 cells per well and incubated at 37 °C with 5% CO2 for 24 h. The vehicle solvent dimethyl sulfoxide (DMSO) was added at a concentration of 1 µL/ml or lower, and different concentrations of Ruxo (0 to 180 µM) for a duration of 24 h. Thereafter, CCK8 was introduced, and the cells were incubated at 37 °C and 5% CO2 for 2 h. The absorbance at 450 nm was then measured using Synergy LX Multi-Mode Reader (BioTek Instruments, USA).
Colony formation
8505C and KHM-5M were placed with an initial density of 2 × 104 cells containing varying concentrations of Ruxo (0, 40, and 80 µM) for one week. The plates were gently washed using PBS, fixed with paraformaldehyde for 15 min, and colored with 0.1% crystal violet for 20 min.
Transwell migration and invasion assay
The transwell cell culture chamber (Corning Costar Corp., Cambridge, MA, USA) served as the platform to assess ATC cells’ migratory and invasive capabilities. In the migration assay, cells from the 8505 C and KHM-5M lines were seeded in the upper chamber using a serum-free medium at a density of 3 × 104 cells. Meanwhile, the lower chamber was loaded with RPMI-1640 medium supplemented with 10% FBS, along with varying concentrations of Ruxo (0, 40, and 80 µM). For the invasion assay, the chambers were coated with Matrigel (BD Biosciences, diluted at a 1:4 ratio) for 1 hour, followed by the subsequent steps identical to those in the migration assay. The plates were incubated at a temperature of 37 °C with a 5% CO2 concentration. This was maintained for 24 h for migration assessment and extended to 48 h for evaluating the invasion. Subsequently, the cells were fixed using a 4% paraformaldehyde solution for 30 min, followed by staining with 0.01% crystal violet for the same duration. The migrated and infiltrated cells were scrutinized under a microscope, and their quantity was quantified using the ImageJ software.
Determination of cell survival rate
To determine the cell viability, an Annexin V-FITC and PI Kit (Liankebio, China) containing Annexin V-FITC and PI were employed according to the provided instructions. Plates were seeded with cells at a density of 1.5 × 105 and exposed to various drug concentrations for 24 h. Afterward, the cells were harvested, PBS-washed, and stained with PI and Annexin V-FITC. The NovoCyte® Quanteon® Benchtop Flow Cytometer was used to obtain fluorescence data, which were then analyzed with FlowJo V10.
Morphological examination
Morphological analyses were conducted following the methods described earlier [30]. Briefly, following the application of various substances to the cells for the specified durations, they were captured using a phase-contrast optical microscope. The observation of mitochondria involved the following steps: ATC cells were initially subjected to a specific duration of treatment with Ruxo, subsequently stained with Mito tracker for a duration of 30 min in a light-free environment, and finally examined using Confocal laser (Leica STED, Germany).
For immunofluorescence staining, the cells after treatment were subjected to a 30-minute incubation with a Mito tracker in a dark environment. Subsequently, the cells were rinsed with PBS and fixed in 4% paraformaldehyde, followed by blocking in a solution containing 3% BSA, 0.4% gelatin, and TBST. The cells were then exposed to Cyto C antibody overnight at 4 °C and washed with PBS. Following this, the cells were incubated with Alexa Fluor 488-labeled Goat Anti-Rabbit IgG(H + L) (A0423, Beyotime Biotechnology, China) for an additional hour at room temperature. Subsequently, the nuclei were subjected to staining with DAPI for a duration of 10 min, followed by examination through employment of a Confocal laser (Leica TCS SP8, Germany). The double antibody staining procedure for DRP1 and HSP60 was conducted following the above steps without Mito tracker stain. Except, Alexa Fluor 488-labeled Goat Anti-Rabbit IgG(H + L) and Alexa Fluor 555-labeled Donkey Anti-Mouse IgG(H + L) (A0460, Beyotime Biotechnology, China) were employed as secondary antibodies, and then samples were subsequently observed using a Confocal laser (Leica TCS SP8, Germany).
Transmission electron microscopy (TEM)
The cells, specifically 8505 C, and KHM-5M, underwent an 8-hour treatment with Ruxo. After this treatment period, the cells were harvested and fixed utilizing an electron microscope fixative (HaoKe Biotechnology Co. Ltd, China). Subsequently, a rinse was performed using 0.1 M phosphate buffer (PB) with a pH of 7.4. Following this, the cells were exposed to 1% OsO4 in 0.1 M PB for a duration of 2 h, followed by a process of dehydration using a gradient of alcohol and 100% acetone. The prepared samples were subjected to embedding, polymerization, ultrathin slicing, and staining procedures. The images were captured using a transmission electron microscope (HT7650, HITACHI, Tokyo, Japan).
Lactate dehydrogenase release assay (LDH)
An LDH assay (Cat: A020-1, Nanjing Jiancheng Bioengineering Institute, China) was used to determine extracellular LDH activity after various treatments according to the protocol. Briefly, after treatment, the cell culture supernatant was collected, and LDH activity was determined by following the kit procedure.
JC-1 functional assay
The mitochondrial membrane potential was determined using JC-1 (Cat 40705ES03, Yeasen Biotechnology, China) according to the instructions provided by the manufacturer. Following a 12-hour treatment, the 8505 C and KHM-5M cells were exposed to JC-1 at a concentration of 5 µg/mL for a duration of 30 min. Subsequently, they were rinsed twice and reconstituted in PBS. Stained cells were analyzed using a NovoCyte® Quanteon® Benchtop Flow Cytometer.
Intracellular ROS determination
Following an 8-hour treatment, the cells were subjected to staining using 10 µM DCFH-DA (Cat T15458, TargetMol, USA) in a dark environment for 30 min. Subsequently, the cells were rinsed twice with PBS. The cells were examined with a NovoCyte® Quanteon® Benchtop Flow Cytometer to ascertain ROS levels.
siRNA transfection and CRISPR/Cas9 knock-out cell lines
Beijing Tsingke Biotech Co., Ltd., China, synthesized the siRNA that targets STAT3 and the negative control siRNA. The siRNA sequence of STAT3 was (siRNA): 5′- GGCTGGACAATATCATTGA-3′. According to the instructions provided by the manufacturer, Lipofectamine 3000 (Cat.NO. L3000015, Invitrogen) was used to transfect the siRNAs into ATC cells. After 48 h of transfection, the cells were examined using real-time quantitative PCR (RT-qPCR) and western blotting to confirm the effectiveness of siRNA knockdown.
The CRISPR/Cas9 lentivirus plasmid was constructed by Applied Biological Materials Inc., Canada. The sgRNA sequences were as follows: caspase 3: 5′-GGATGCCGGCACTACACAAC-3′, caspase 9: 5′- GAACAGCTCGCGGCTCAGCAGGG-3′, and GSDME: 5′-GGATGCCGGCACTACACAAC-3′. A lentivirus system at an MOI of 20–50 was used to infect ATC cells, followed by selecting Lenti Guide-Puro positive cells using 2–5 μg/mL puromycin. Forty-eight hours after the infection, western blot analysis was used to validate the CRISPR/Cas9 knockdown efficiency. The remaining cells were used for subsequent experiments.
DRP1 and STAT3 overexpression
ATC cells were transfected with a DRP1 overexpression plasmid (Biocompete Inc., China) and STAT3 overexpression plasmids (Genomeditech Inc., China) using Lipofectamine 3000. After 48 h of transfection, the overexpression efficiency was validated through RT-PCR and western blotting.
Transcription factor binding site analysis and dual-luciferase reporter assay
The Jaspar database (http://jaspar.genereg.net/about/) was used to analyze the binding sites of transcription factors within the promoter region of the human DRP1 gene Applied Biological Materials Inc., Canada, synthesized both full-length and mutated human DRP1 plasmids to construct luciferase reporters. The procedure involved cloning the wild-type (WT) full-length human/mouse DRP1 promoter sequence, Mutant 1 (deletion of ttgccaggaat), DRP1 promoter, and Mutant 2 (deletion of catcctggaaa) into pLenti-Promoterless-Dual-Luc vectors. Upon transfection, ATC cells received equitably measured DNA along with a matched empty vector. Following a 48-hour incubation, the ATC cells were isolated and subjected to assessment using the Dual-Lumi™ Luciferase Reporter Gene Assay Kit (Cat: RG088S, Beyotime Biotechnology, China), following the provided guidelines by the manufacturer.
RT-qPCR
The RT-qPCR was conducted according to the previously mentioned protocol [31]. Briefly, total cellular RNA was extracted using the Trizol method, and PCR amplification and fluorescence real-time detection was performed after reverse transcription using PrimeScript™ RT Reagent Kit (Cat: RR037 Takara Biomedical Technology Co., Ltd., Japan). The PCR primer sequences for genes are shown in supplement table 1.
Western blotting and antibodies
Cells were harvested employing a western blotting and IP lysis buffer containing PMSF (Cat: P0013 and ST505, Beyotime Biotechnology, China). The protein concentration was quantified using The BCA protein assay (Thermo Fisher Scientific, USA). The protein samples were subsequently separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. For the following steps, the membranes were blocked for an hour using a TBST solution containing 5% skim milk. Following this, the membranes were exposed to primary antibodies overnight at a temperature of 4 °C. Afterward, they were incubated with suitable HRP-conjugated secondary antibodies at a temperature of 25 °C for 60 min. Chemiluminescence was observed using a ChemiDoc-MP imager (Bio-Rad, Hercules, CA, USA) coupled with an FD Bio-Dura ECL Kit (Cat FD8020, Fdbio Science, China). The band density was analyzed using the ImageJ software. The primary antibodies used are listed in Supplement Table 2. The suitable HRP-conjugated IgG secondary antibodies, namely Goat anti-rabbit (Cat: A0208) and anti-mouse (Cat: A0216), were procured from Beyotime Institute of Biotechnology, China.
Xenograft tumor model
The animal trials were carried out by the authorized procedure of the Animal Ethics Committee of Zhejiang Provincial People’s Hospital (Approval NO. 20221112233826207365).
Shanghai SLAC Animal Co. (Shanghai, China) provided female BALB/c nude mice, which were 3–4 weeks old. Subcutaneous implantation of 8505 C cells (5 × 106 cells/100 µl) was performed on the right axilla of the mice. Upon forming palpable tumors, the mice were randomly assigned (n = 6/group). Ruxo and Mdivi-1 were diluted in 5% DMSO + 30% PEG300 + 10% Tween 80 + 55% PBS, and 100 µL of the dilute drug was administered intraperitoneally daily for 12 days. The mice were weighed regularly, and the volumes of tumors were measured every alternate day. Tumor volume was determined by applying the subsequent equation: Tumor vol (mm3) = 0.5 × (short diameter)2 × (long diameter).
The mice were euthanized after a 12-day treatment period, and their tumors, blood, and organs (liver, kidneys, and heart) were gathered. After collection, the specimens were preserved with a 4% formalin solution, followed by embedding in paraffin. Subsequently, they were sliced into sections and stained with hematoxylin and eosin (HE).
Immunohistochemistry
Immunohistochemistry was performed as previously described [30]. The dilution ratios of the primary antibodies were as follows: JAK1, 1:100; p-JAK1, 1:100; p-STAT3, 1:250; DRP1, 1:100; c-PARP, 1:50, and c-caspase 3, 1:200. The scoring system comprises four levels, which are determined by the intensity of cellular staining. A score of 0 is assigned to samples with no positive staining (negative), while a score of 1 is given to samples exhibiting pale yellow staining (weakly positive). Furthermore, samples displaying tan staining are assigned a score of 2 (positive), while those exhibiting strongly positive tan staining receive a score of 3.
Statistical analysis
A minimum of three repetitions were conducted for all experiments. Results are presented as average ± standard deviation or averages ± standard error of the mean. To examine the distinctions between two groups, a t-test was employed, whereas distinctions among multiple groups were assessed using either one-way ANOVA or the two-tailed unpaired Student’s t-test, followed by Bonferroni’s test. P value < 0.05 was considered statistically significant, with all P-values being two-sided. The analysis was conducted utilizing GraphPad Prism 9 software (USA).