The new drug Tirzepatide may slow colon cancer growth by changing how cells use sugar

To our knowledge, no prior studies have reported the effective concentration range of TZP in cancer cell models.^[16^] Therefore, we conducted dose‐response experiments to determine the minimal concentration required to observe significant anti‐proliferative effects in CRC cell lines. TZP showed a dose‐dependent inhibition of the proliferation of colonic cancer MC38 cells from 5 up to 50 µM without apparent toxic effect on the mouse embryonic hepatocytes line (BNL CL.2) (Figure S1, Supporting Information). In human cell lines HCT116 and RKO, TZP exerted a concentration‐dependent inhibition of CRC cell migration and tube formation (Figure S2, Supporting Information). To assess the specificity of its antitumoral effects, a cell viability assay was used to examine its effects on four types of other cancer cell lines. TZP did not significantly inhibit the proliferation of glioma cells (U87MG), human gastric cancer cells (HGC27 and MKN45), and pancreas‐02 cells (Pan02) (Figure S3, Supporting Information), indicating that CRC cells were most sensitive to TZP.

As TZP is clinically used as an antidiabetic medication, we questioned whether its antitumor efficacy is related to glucose concentration. An in vitro model of MC38 under high glucose (HG) stress was established in the trans‐well system. TZP inhibited migration of the cancer cells across the permeable membrane in a dose‐dependent manner, and HG stimulation did not have an apparent effect (Figure 1A,B). In the wound healing assay, cell migration in TZP‐treated groups decreased at both concentrations against the control cells but was more pronounced at 50 µM (Figure 1C,D). Flow cytometry and TUNEL assays revealed that TZP significantly increased the apoptotic ratio of MC38 cells, and this effect was independent of HG stimulation (Figure 1E–H).

To explore if the anti‐tumoral effect of TZP would occur in vivo, orthotopic and subcutaneous animal models were constructed in normal mice and STZ‐induced T1DM and T2DM mice (Figures 2A and 3A). TZP was administered via intraperitoneal injection every day (50 nmol/kg/day) for 3 weeks according to the half‐life of TZP in mice (Figure S4, Supporting Information). TZP did not affect body weight and blood glucose in T1DM and control mice (Figure 2B,C). Treatment with TZP inhibited tumor growth of CRC, either diabetic or non‐diabetic, as revealed by in vivo imaging and tumor volume measurement (Figure 2D–G), indicating that the inhibitory effect of TZP on CRC in vivo is independent of blood glucose regulation. And TZP increased the survival of T1DM mice with CRC (MC38‐Luc) (Figure 2H). Next, we constructed subcutaneous models using murine colon cancer cells (MC38) in control mice and T2DM mice. We found that treatment with TZP significantly reduced blood glucose levels, body weight (Figure 3B,C), decreased fatty acid levels, and some degree of reduction of caloric intake during 7–14 days of TZP injection with T2DM mice (Figure S5A, Supporting Information). A slight reduction of plasma insulin levels was possibly due to the rescue in insulin sensitivity (Figure S5B,C, Supporting Information). The tumor volume in diabetic mice was the largest while TZP treatment significantly reduced CRC volume and weight in T2DM mice (Figure 3D–F). It might be that tumor progression and drug response require extended time periods, and short‐term studies may not capture significant weight changes in control mice. Besides, the presence of cancer might compromise body weight.

Accumulating evidence demonstrates the important roles of glucose metabolism in determining cancer cell fate.^[15, 17^] We used spatial metabolomics to further characterize whether TZP exerted its anti‐colon cancer effects by influencing metabolism. We used in situ Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI‐MS) imaging of all CRC specimens from the orthotopic mice model (Figure 2A) to visualize and quantify metabolites to identify the enriched metabolomic profiles in tumor regions (Figure 4A). Postoperative CRC tissue sections were divided into three histologic types based on the cell type and components: cancer tissue, epithelial tissue, and muscular tissue (Figure 4B). Multivariate analysis and volcanic map of metabolites showed that TZP treatment reduced the vast majority of the metabolite differences in the cancer tissue, including downregulation of 541 metabolites and upregulation of only 4 metabolites (Figure S6A,B, Supporting Information). The KEGG pathway analysis indicated that TZP treatment led to the activation of the metabolism‐related pathways and those related to cancer, cell cycle, etc. (Figure S6C,D, Supporting Information). A multitude of region‐specific glucose metabolites were screened and visualized, and the downregulation of products in the CRC tissues was apparent by TZP treatment (Figure 4C). Some key glycolytic metabolites in the tumor tissues were significantly downregulated after TZP treatment, such as D‐glucose, phosphoenolpyruvate, 2,6‐beta‐delta‐fructan, glycerophosphoric acid, (3S,4R) ‐ketose 1‐phosphate and 2′‐deoxyinosine (Figure 4D,E). These findings indicated that TZP robustly reduced glycolytic metabolism in the CRC tissues.

We performed further in vitro experiments using two murine colon cancer cell lines (MC38 and CT26) to investigate changes in the glycolytic metabolism mediated by TZP. Seahorse assay confirmed that TZP attenuated glycolytic flux (extracellular acidification rate [ECAR]) in MC38 and CT26 cancer cells (Figure 5A–D). Pyruvate and lactate production, and glucose uptake were significantly reduced in the presence of TZP in multiple CRC cell lines (Figure 5E,F; Figure S7, Supporting Information). Decreased glycolysis consumes less NAD⁺ to generate NADH. As expected, diminished aerobic glycolysis induced by TZP led to an increase NAD⁺/NADH ratio in the cytoplasm (Figure 5G,H). The majority of the genes involved in the aerobic glycolytic pathway were significantly inhibited by TZP (Figure 5I,J; Table S1, Supporting Information). TZP affected the enzyme activity of PFKFB3 and PFK‐1 which are engaged in the rate‐limiting step in glucose metabolism (Figure 5K–N). We also investigated the inhibitory effects of TZP on glycolysis levels and on glucose transporters GLUT1, GLUT3, GLUT4, and GLUT12 in HCT116. The results indicated that TZP reduced glycolysis and downregulated the expression of glucose transporters at mRNA and protein levels in HCT116 (Figure S8, Supporting Information). Interestingly, the stable isotope tracing found that TZP significantly decreased the enrichment of [U‐¹³C₆] glucose‐labeled M+3 lactate and M+2 citrate, aconitate, isocitrate, succinate, and malate, indicating the blockage of tricarboxylic acid (TCA) cycle (Figure S9, Supporting Information). These findings suggested that TZP inhibited both glycolysis and the TCA cycle, thereby impairing the energy production in the CRC cells.

We hypothesized whether TZP modulated glycolysis by targeting HIF‐1α, as it is considered a key regulator in cancer cell glucose metabolism^[18^] and controls the transcription of glycolysis‐related genes.^[19^] Immunohistochemistry results indicated that TZP reduced the expression of HIF‐1α, PFKFB3, and PFK‐1 in CRC tissues in vivo (Figure 6A,B). Immunofluorescence analysis revealed that TZP treatment effectively blocked the translocation of HIF‐1α into the nuclei of the HCT116 cells as compared with the control cells (Figure 6C,D). We then investigated whether the reduced HIF‐1α expression by TZP was due to increased ubiquitination and enhanced protein degradation. To examine the effect of TZP on HIF‐1α stability, cobalt chloride (CoCl₂) was used as HIF‐1α inducer and carbobenzoxyl‐L‐leucyl‐L‐leucyl‐L‐leucine (MG132), a proteasome inhibitor. Western blotting demonstrated that CoCl₂‐induced upregulation of HIF‐1α and its ubiquitination was inhibited by TZP (Figure 6E,F). Wound healing and tube formation assays also confirmed that TZP regulated CRC function by affecting HIF‐1α stability (Figure S10, Supporting Information). TZP treatment could markedly reduce HIF‐1α as well as the key glycolytic molecules PFKFB3 and PFK‐1 in the HCT116 cells with or without (CoCl₂) induction (Figure 6G,H). Meanwhile, TZP inhibited the enzymatic activity of PFKFB3 and PFK‐1, and this effect was counteracted by the stabilization of HIF‐1α (Figure 6I,J). Treating CRC cells with chemical inhibitors of PFKFB3 (PFK‐158, 10 µM) and HIF‐1α (LW6, 50 µM) or lentivirus to specifically knockdown PFK‐1, the results indicated that inhibition of the expression or transcription of these three key molecules significantly suppressed CRC cell migration and proliferation (Figures S11 and S12, Supporting Information). These results provided strong evidence that TZP played its anti‐CRC effect by targeting HIF‐1α degradation leading to reduced glycolysis with the inhibition of PFKFB3 and PFK‐1.

As low‐passage patient‐derived xenograft (PDX) tumor models better reflect human tumor cell heterogeneity than do conventional cell lines and xenografts and are useful for assessing in vivo response to therapeutic agents,^[20^] we surgically resected human CRC tissues and subcutaneous transplantation to NOD/ShiLtJGpt‐Prkdcem^26Cd52Il2rg^em26Cd22/Gpt (NCG mice) to conduct CRC‐PDX model to evaluate the therapeutic efficacy of TZP (Figure 7A). Due to the different genetic backgrounds of NCG and varying sensitivity to TZP, we adjusted the TZP injection duration for 9 days accordingly. The results showed that TZP injection significantly delayed the tumor growth as compared with the control group, consistently causing a significant reduction in the tumor volume and weight (Figure 7B–E). Immunohistochemistry was performed to detect the expression of HIF‐1α, PFKFB3, and PFK‐1 in the tumor tissues of both groups. Consistent with previous findings in the cell‐line derived cancer model (Figure 6A,B), TZP significantly reduced the expression of the target proteins in tumor tissues, confirming that TZP inhibited the HIF‐1α‐mediated glycolytic pathway in human tumor cells (Figure 7F,G).

Murine colon carcinoma cell line (CT26 and MC38), human glioblastoma cell line (U87), human colon cancer cell line (HCT116 and RKO), human pancreatic adenocarcinoma cell line 02 (Pan02), human gastric carcinoma cell lines HGC27 and MKN45, and mouse embryonic hepatocytes line (BNL CL.2) were acquired from American Type Culture Collection (ATCC, Manassas, Virginia, USA). All cell lines were cultured with DMEM (Gibco; USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) and antibiotic‐antimycotic (Gibco) at 37 °C in a 5% CO₂ atmosphere except HCT116 cells were routinely maintained in McCoy's 5A Modified Medium (Gibco).

Tirzepatide (LY3298176) and cobalt chloride were purchased from Selleck (Houston, TX, USA). Proteasome inhibitor (MG132), PFKFB3 chemical inhibitor (PFK‐158), and molecule inhibitor of HIF‐1α (LW6) were purchased from MedChemExpress (NJ, USA). D‐glucose (NIST917C) was bought from Sigma (MO, USA). Lentivirus to specifically knock down PFK‐1 (Target sequence: GTG GCA CTG ATA TGA CCA TTG) was conducted by GenePharma (Shanghai, China)

The proliferation of MC38, BNL, U87, Pan02, HGC27, and MKN45 cells was determined by a Cell Counting Kit‐8 assay (CCK8; Dojindo, Japan) as described.^[36^] The cells of different lines were seeded in 96‐well plates and cultured for 12, 24, 48, 72, or 96 h (h) with different doses of TZP. Absorbance at 450 nm was examined using a microplate reader (Bio‐Rad, Berkeley, USA).

Cell migration was assessed using Transwell permeable supports (Corning, Cat. No. 3421). Cells were suspended in serum‐free DMEM and seeded in the upper chamber, while DMEM containing 10% FBS was added to the lower chamber. After 48 h, migratory cells in the bottom chamber were stained with crystal violet, pictured, and counted by Adobe Photoshop 2024.

Cells were plated into wells of a 12‐well plate and cultured overnight until a confluent monolayer was formed, onto which a scratch was made with a 200 µL pipette tip. The effects of TZP (10 and 50 µM) on wound healing were measured at 0 and 48 h after wounding by imaging on the DMIL microscope system equipped with a DFC295 camera and controlled by LAS V4.0 software. (Leica, Germany). 1 µM of mitomycin (Selleck) was added to inhibit cell proliferation and eliminate its potential influence on the migration assay.

Apoptosis was detected using an Annexin V‐FITC apoptosis detection kit (Vazyme), according to the manufacturer's instructions. After treatment, cells were trypsinized, washed twice with HBSS, resuspended in 1× binding buffer, and stained with Annexin V‐FITC and PI for 10 min at room temperature (RT) in the dark. At least 1 × 10⁵ cells were analyzed by flow cytometry, using side and forward scatter to identify viable cell populations.

For the TUNEL assay, the MC38 cells were treated with TZP in two different doses (10 and 50 µM) for 48 h. The cells were fixed with a 4% paraformaldehyde solution in PBS for 30 min at −20 °C, followed by permeabilization with 0.1% Triton X‐100 in 0.1% sodium citrate solution for 2 min on ice. Subsequent procedures were performed according to the manufacturer's protocol for the TUNEL BrightGreen apoptosis detection kit (Vazyme). Fluorescent images (magnification ×100) were acquired using a DMI8 fluorescence microscope (Leica, Germany).

C57BL/6J male mice and PDX female NCG mice were brought from GemPharmatech (Nanjing, Jiangsu, China). The animals were maintained in a temperature‐controlled room (22 °C) on a 12‐h light‐dark cycle. The original PDX establishment followed the procedure approved by GemPharmatech's relevant committees and the original colon cancer samples were obtained from collaborating hospitals whose institutional committee approved the sample collection and obtained informed consent. Experimental setups and animal care were permitted by the Animal Policy and Welfare Committee of Zhejiang University (ethical approval code:2024‐NO.073).

C57BL/6J male mice were purchased from GemPharmatech (Jiangsu, China). For conducting T1DM model, mice at the age of 6 to 8 weeks were injected intraperitoneally of streptozotocin (STZ) (male mice: 100 mg kg⁻¹ body weight, 1 dose a day for 2 days). For the T2DM model, mice were fed with a high‐fat diet (Research Diets, NJ, USA) for up to 8 weeks. On a caloric basis, the high‐fat diet consisted of 60% fat from lard, 20% carbohydrate, and 20% protein (total 5.24 kcal g⁻¹), whereas the normal diet contained 11.1% fat, 67.4% carbohydrate, and 21.5% protein (total 3.41 kcal g⁻¹). After 8 weeks of feeding, the control group was treated with sterile PBS, and the T2DM group was treated by intraperitoneal injection of STZ (male mice: 30 mg kg⁻¹ body weight, 1 dose a day for 5 consecutive days). Blood glucose was measured once a week to validate diabetic hyperglycemia. Serum insulin was determined by immunoassay (Alpco Diagnostics, China), and serum‐free fatty acids were determined enzymatically (Nanjing Jiancheng Bioengineering Institute, China).

For the orthotopic mouse model, 6‐ to 8‐week C57BL/6 male mice were anesthetized with 5% isoflurane. The hairs on the abdominal region were cleared before surgery, and the cecum was exposed. A 50 µL suspension of 5 × 10⁵ MC38 cells in Matrigel VR Basement Membrane (1:1 ratio) was carefully injected into the cecal wall from the serosal side under microscopic visualization. The cecum was then returned to the abdominal cavity, and the peritoneum, muscle, and skin closures were completed.^[37^] TZP treatment started at day 35 by intraperitoneal injection daily for 21 days (50 nmol kg⁻¹). The growth of tumors was monitored using the Xenogen IVIS 200 BLI system (Caliper Life Sciences, USA). Pictures were analyzed by Living Image 2.5 software.

For the subcutaneous animal model, C57BL/6J male mice were randomly assigned to groups of control and TZP (n = 10 per group), respectively. Equal numbers of corresponding cells (5 × 10⁵ per mouse) were injected subcutaneously in the right flank to establish a CRC allograft model.^[38^] When the tumors were palpable (7 days post‐injection), TZP treatment was initiated by intraperitoneal injection daily for 21 days using the same dose as described above. Tumor volume was measured every three days using a Vernier hand caliper. Tumor volume was calculated using the formula V = 0.52(length * width²). Mice were finally sacrificed at week 4 to harvest the tumor bulks.

CRC‐PDX (patient‐derived xenograft) establishment and treatment were similar to the above model. Surgically resected tissues were minced into pieces, ≈2‐mm in size, and injected into the subcutaneous area of the flanks of 6‐week NCG mice. Daily TZP injection lasted for 9 days. The tumor volumes of the mice were checked every day for 9 days.

Spatially resolved metabolomics profiling was conducted at MetWare Biological Science and Technology Co., Ltd. (Wuhan, China). Tissue samples were sectioned at a thickness of 12 µm using a Leica CM1950 cryostat (Leica Microsystems GmbH, Wetzlar, Germany) at −20 °C. The resulting tissue sections were then arranged on electrically conductive slides coated with indium tin oxide (ITO), and the slides were dried in a vacuum desiccator for 30 min.

For matrix coating, desiccated tissue sections mounted on ITO glass slides were sprayed using an HTX TM sprayer (Bruker Daltonics, Germany) with 15 mg mL⁻¹ DHB (2,5‐dihydroxybenzoic acid), dissolved in 90% acetonitrile:10% water. The sprayer temperature was set to 60 °C, with a flow rate of 0.12 mL min⁻¹, pressure of 5 psi. Thirty passes of the matrix were applied to slides with 5 s of drying time between each pass.

MALDI timsTOF MSI experiments were performed on a prototype Bruker timsTOF flex MS system (Bruker Daltonics, Germany) equipped with a 10 kHz smartbeam 3D laser. Laser power was set to 80% and then fixed throughout the whole experiment. The mass spectra were acquired in positive mode. The mass spectra data were acquired over a mass range from m/z 50–1300 Da. The imaging spatial resolution was set to 50 µm for the tissues, and each spectrum consisted of 400 laser shots. MALDI mass spectra were normalized with the Root Mean Square, and the signal intensity in each image was shown as the normalized intensity. MS/MS fragmentations performed on the timsTOF flex MS system in the MS/MS mode were used for further detailed structural confirmation of the identified metabolites.

Total RNA of the tumors and control cells was extracted using TRIzol reagent (Invitrogen, USA), followed by cDNA synthesis using a reverse transcription kit (Yeasen, China) according to the manufacturer's instructions. Quantitative real‐time PCR was performed using a SYBR Green qPCR kit (Yeasen, China), and the primers were designed and synthesized by Tsingke Biotech Com (Shanghai, China). Primer sequences are listed in Table(Supporting Information). Relative mRNA expression was normalized to β‐actin mRNA. S1

As described in a previous publication,^[39^] MC38, CT26, and HCT116 cells were seeded onto Seahorse XF96 culture cell plates at a concentration of 2 × 10⁵ per cell and incubated with 50 µM TZP at 37 °C overnight. On the second day, the treatment media were removed, and the cells were washed once with the assay medium (XF base medium supplemented with 2 mM glutamine, pH adjusted to 7.4). The culture medium was replaced with an assay medium containing the treatments, and the plates were incubated for 1 h in a non‐CO₂ incubator at 37 °C. Following incubation, cellular analysis was performed using an XFe96 extracellular flux analyzer (Seahorse Bioscience). The following inhibitors and activators were applied: glucose (10 mM), oligomycin (1 µM), and 2‐DG (50 mM). Basel glycolysis, glycolytic capacity, and glycolytic reserve acidification were determined using the XF Wave 2.1 software.

The effect of TZP on glucose uptake in each group of CRC cells was tested using the Glucose Uptake Fluorometric Assay Kit (E‐BC‐F041, Elabscience, China) according to the manufacturer's instructions. Briefly, cells were cultured and treated. Then, the cells were starved overnight in serum‐free cell medium and the next day, they were rinsed with KRPH solution plus 10 mmol L⁻¹ 2‐DG and incubated for 30 min at 37 °C. The samples were mixed with reagent 4 working solutions and the fluorescent intensity was then measured at excitation/emission = 485/535 nm) and normalized to protein concentration measured using the Bio‐Rad protein assay (Bio‐Rad, USA).

Further, the glucose analog 2‐NBDG (MedChem Express, Monmouth Junction, NJ, USA) was also used to assess glucose uptake. Cells were treated with TZP (50 µM) for 48 h, after which the medium was replaced with PBS buffer containing 2‐NBDG (100 µM), and incubated at 37 °C for 30 min. The cells were then washed three times with PBS to eliminate residual extracellular 2‐NBDG. Subsequently, 2‐NBDG imaging was performed using a 488 nm laser and pictures were taken with an inverted fluorescence microscope (DMI8, Leica, Germany). Lactate and pyruvate production was measured using an enzymatic assay (Abcam, ab65331) and a pyruvate assay kit (Abcam, ab65342). All measurements were performed according to the manufacturer's instructions.

The glycolytic flux was assessed by measuring the rate of glucose consumption and the ratio of ¹³C incorporated into lactate, determined using liquid chromatography‐mass spectrometry (LC‐MS). In brief, cells were cultured in a medium containing either D‐glucose‐¹³C₆ or without it. After 12 h, the culture supernatants were collected, and cells were harvested using cold 80% methanol. Flux analysis was conducted on a TSQ Quantiva Triple Quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA) with positive/negative ion switching in multiple reaction monitoring (MRM) mode for data acquisition. Mobile phase A consisted of 2.376 mL tributylamine and 0.858 mL acetic acid in HPLC‐grade water, adjusted to a final volume of 1 liter. Mobile phase B was HPLC‐grade methanol. Separation of polar metabolites was achieved using a Synergy Hydro‐RP 100 A column at a column temperature of 35 °C. The mass isotopomer distributions were corrected for natural isotope abundances.

The cells were treated with cell lysis buffer for 10 min on the ice. The suspensions were then centrifuged at 120 00 g for 10 min at 4 °C, and the supernatant samples were used to measure the level of F‐2,6‐BP (mlbio, China) and F‐1,6‐BP using corresponding ELISA kits (mlbio, China) according to the manufacturer's instructions. Three replicate wells were used for each sample.^[40^]

To evaluate the levels of representative protein makers, immunohistochemical analyses were performed as described.^[41^] In brief, tissues were fixed in 10% formalin and embedded in paraffin. Tumor tissues were fixed in 1 mL 4% paraformaldehyde overnight, then dehydrated in ethanol and embedded in paraffin. IHC was performed using the antibodies against HIF‐1α, PFK‐1, and PFKFB3. Reading of IHC slides and TMA was performed using Vectra 2 (Perkin Elmer) to quantify protein expression in the samples. For TMA, A Leica APERIO AT TURBO instrument was used for scanning and analysis. Quantification of IHC staining was performed blindly.

Immunofluorescence analysis was performed briefly. The cells were seeded on glass coverslips, treated as indicated and fixed with 4% paraformaldehyde for 15 min at RT. Subsequently, the cells were washed three times with PBS and incubated with 1% BSA (Carl Roth) in PBS for 1 h at room temperature (RT). The coverslips were incubated with diluted primary HIF‐1α antibodies overnight at 4 °C, washed with PBS three times, and incubated with secondary antibodies for 1 h at RT. The cells were stained with 1× DAPI (Solarbio, China) for 5 min and washed three times with PBS. Imaging was performed with a STELLARIS 5 Cryo laser scanning confocal microscope (Leica, Germany).

Western blotting was conducted to investigate the expression and phosphorylation level of target proteins in the MC38, CT26, and HCT116 with different treatments. Protein lysates were extracted using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Haimen, China) and resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, blocked with QuickBlock Blocking Buffer (Beyotime), and incubated with primary antibodies at 4 °C overnight. After washing with TBST buffer, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies for 1 h, followed by three washes with TBST. Immunoreactive bands were detected using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Waltham, USA), and images were captured using a chemiluminescent imaging system (Amersham ImageQuant 800). Antibodies against β‐actin, HIF‐1α, PFKFB3, and GLUT1 were from Cell Signaling Technology (CST, Boston, USA) and Abcam (Cambridge, MA). Antibodies against PFK‐1 and GLUT3 were from Santa Cruz Biotechnology (Santa Cruz, California, USA), and those against GLUT4 and GLUT12, from Proteintech (Chicago, USA) and Affinity (Ohio, USA), respectively.

The impact of TZP on the stability of HIF‐1α in colon cancer cells was examined by in vivo ubiquitination assays. Briefly, the different groups of cells were treated with CoCl₂ (500 µM) under a normoxic condition for 8 h and the levels of HIF‐1α ubiquitination were determined by IP with anti‐HIF‐1α antibody, followed by western blotting using an anti‐Ub antibody (10201‐1‐AP, Proteintech, 1:1000).

Data are expressed as mean ± SEM from at least three independent experiments per condition. Significance testing was performed based on these data. Variance equality was assessed using the Brown‐Forsythe and Bartlett tests, while normality was evaluated with both normality and log‐normality functions in the software. If data met these criteria, comparisons among more than two groups were conducted using one‐way ANOVA with Tukey's multiple comparisons or two‐way ANOVA with Sidak or Tukey's multiple comparisons in GraphPad Prism (version 9.0). Two‐way repeated measures ANOVA was applied for repeated measurements from the same subjects under different conditions. Comparisons between the two groups were made using the unpaired t‐test, with a Family‐wise significance level of 0.05 and a 95% confidence interval for statistical significance.