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Miltenyi Biotec dapi
Dapi, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 97/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/dapi+staining/DAPI+Staining+Solution/pm42811304-68-17-18
Average 97 stars, based on 93 article reviews

dapi - by Bioz Stars, 2026-10

97/100 stars

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Staining:

Article Title: Repurposing the Bis-Biguanide Alexidine in Combination with Tyrosine Kinase Inhibitors to Eliminate Leukemic Stem/Progenitor Cells in Chronic Myeloid Leukemia.
Article Snippet: The IncuCyte Zoom Imaging system (Essen Bioscience, Ann Arbor, MI, USA) was used for confluence counting by capturing images of the culture dish every two hours for 24 h. The optical density of the cells was measured in 9 squares per well (in triplicate), and the IncuCyte® Zoom v2018A software (Sartorius Stedim Biotech GmbH, Goettingen, Germany) was used to analyze the images and determine the confluence index as the percentage of the dish that was covered by the cells. .. Cell viability was determined by DAPI staining prior to flow cytometry analysis in 96-well plates using MACSQuant or MACSQuant VYB flow cytometers (Miltenyi Biotec, Bergisch Gladbach, Germany). ..

Article Title: Loss of UBE3A impacts both neuronal and non-neuronal cells in human cerebral organoids.
Article Snippet: The cell pellets were resuspended in 500 μL Click-iT saponin-based permeabilization and wash reagent containing DAPI (Invitrogen). .. After DAPI staining, samples were loaded into a MACSQuant ® VYB flow cytometer (Miltenyi Biotec). .. FlowJo Software119 (v10.9.0, Becton Dickenson Life Sciences) was used to analyze flow cytometry data.

Article Title: Loss of UBE3A impacts both neuronal and non-neuronal cells in human cerebral organoids
Article Snippet: The cell pellets were resuspended in 500 μL Click-iT saponin-based permeabilization and wash reagent containing DAPI (Invitrogen). .. After DAPI staining, samples were loaded into a MACSQuantR VYB flow cytometer (Miltenyi Biotec). .. FlowJo Software (v10.9.0, Becton Dickenson Life Sciences) was used to analyze flow cytometry data.

Article Title: Repurposing the Bis-Biguanide Alexidine in Combination with Tyrosine Kinase Inhibitors to Eliminate Leukemic Stem/Progenitor Cells in Chronic Myeloid Leukemia
Article Snippet: The IncuCyte Zoom Imaging system (Essen Bioscience, Ann Arbor, MI, USA) was used for confluence counting by capturing images of the culture dish every two hours for 24 h. The optical density of the cells was measured in 9 squares per well (in triplicate), and the IncuCyte ® Zoom v2018A software (Sartorius Stedim Biotech GmbH, Goettingen, Germany) was used to analyze the images and determine the confluence index as the percentage of the dish that was covered by the cells. .. Cell viability was determined by DAPI staining prior to flow cytometry analysis in 96-well plates using MACSQuant or MACSQuant VYB flow cytometers (Miltenyi Biotec, Bergisch Gladbach, Germany). ..

Article Title: A New Method to Sort Differentiating Osteoclasts into Defined Homogeneous Subgroups.
Article Snippet: .. When DAPI staining, fixed cells were incubated with a 1 in 10 volume of DAPI staining solution (10 ug/mL, Miltenyi Biotech) for 15 min at room temperature, then stored at 4 ◦C in the dark for up to 1 week before analysis. .. Flow cytometry was performed on a BD Fortessa calibrated with calibration and tracking beads.

Article Title: hsa-miR-34a-5p enhances temozolomide anti-tumoral effects on glioblastoma: in-silico and in-vitro study
Article Snippet: .. After RNase treatment and DAPI staining, cell distribution in the cell cycle phases was studied using flow cytometry (MiltenyBiotecTM FACS Quant 10; MiltenyBiotec, Germany). .. The data were analyzed using the FlowJo software (Tree Star, CA, USA).

Article Title: Loss of UBE3A impacts both neuronal and non-neuronal cells in human cerebral organoids
Article Snippet: The cell pellets were resuspended in 500 μL Click-iT saponin-based permeabilization and wash reagent containing DAPI (Invitrogen). .. After DAPI staining, samples were loaded into a MACSQuant ® VYB flow cytometer (Miltenyi Biotec). .. FlowJo Software (v10.9.0, Becton Dickenson Life Sciences) was used to analyze flow cytometry data.

Article Title: Palbociclib impairs the proliferative capacity of activated T cells while retaining their cytotoxic efficacy
Article Snippet: .. Samples were analyzed after DAPI staining by utilizing a MACSQuant VYB flow cytometer (Miltenyi Biotec GmbH). ..

Flow Cytometry:

Article Title: Repurposing the Bis-Biguanide Alexidine in Combination with Tyrosine Kinase Inhibitors to Eliminate Leukemic Stem/Progenitor Cells in Chronic Myeloid Leukemia
Article Snippet: The IncuCyte Zoom Imaging system (Essen Bioscience, Ann Arbor, MI, USA) was used for confluence counting by capturing images of the culture dish every two hours for 24 h. The optical density of the cells was measured in 9 squares per well (in triplicate), and the IncuCyte ® Zoom v2018A software (Sartorius Stedim Biotech GmbH, Goettingen, Germany) was used to analyze the images and determine the confluence index as the percentage of the dish that was covered by the cells. .. Cell viability was determined by DAPI staining prior to flow cytometry analysis in 96-well plates using MACSQuant or MACSQuant VYB flow cytometers (Miltenyi Biotec, Bergisch Gladbach, Germany). ..

Article Title: Loss of UBE3A impacts both neuronal and non-neuronal cells in human cerebral organoids
Article Snippet: The cell pellets were resuspended in 500 μL Click-iT saponin-based permeabilization and wash reagent containing DAPI (Invitrogen). .. After DAPI staining, samples were loaded into a MACSQuant ® VYB flow cytometer (Miltenyi Biotec). .. FlowJo Software (v10.9.0, Becton Dickenson Life Sciences) was used to analyze flow cytometry data.

Incubation:

FACS:

Image Search Results

Confocal immunofluorescence images of S2-013 and HPNE cells that took up SNORA22 siRNA conjugated with FA-Dab8α and FA-Dab8γ. A S2-013 cells were incubated with Alexa Fluor 647-labeled chemically modified SNORA22 siRNA-FA-Dab8α and SNORA22 siRNA-FA-Dab8γ (green). Blue, DAPI staining. Scale bars, 10 µm. B Quantification of Alexa Fluor 647-labeled chemically modified SNORA22 siRNA-positive cells in A. Data represent the number of cells positive for the siRNA, scored across five fields per group and derived from two independent experiments. Columns , mean; bars , SD. * p <0.05 significantly different from SNORA22 siRNA-FA-Dab8γ (the Student’s t -test). C S2-013, S2-CP8, and HPNE cells were incubated with Alexa Fluor 647-labeled chemically modified SNORA22 siRNA-FA-Dab8α (green). Cells were stained with an anti-FOLR1 antibody (red). Blue, DAPI staining. Scale bars, 10 µm. D Quantification of Alexa Fluor 647-labeled chemically modified SNORA22 siRNA-positive cells in C. Data represent the number of cells positive for the siRNA, scored across five fields per group and derived from two independent experiments. Columns , mean; bars , SD. * p <0.05 significantly different from HPNE cells (the Student’s t -test)

Journal: Human Cell

Article Title: Inhibition of pancreatic cancer growth by an siRNA targeting SNORA22 and a folic acid-conjugated cationic oligopeptide

doi: 10.1007/s13577-026-01458-1

Figure Lengend Snippet: Confocal immunofluorescence images of S2-013 and HPNE cells that took up SNORA22 siRNA conjugated with FA-Dab8α and FA-Dab8γ. A S2-013 cells were incubated with Alexa Fluor 647-labeled chemically modified SNORA22 siRNA-FA-Dab8α and SNORA22 siRNA-FA-Dab8γ (green). Blue, DAPI staining. Scale bars, 10 µm. B Quantification of Alexa Fluor 647-labeled chemically modified SNORA22 siRNA-positive cells in A. Data represent the number of cells positive for the siRNA, scored across five fields per group and derived from two independent experiments. Columns , mean; bars , SD. * p <0.05 significantly different from SNORA22 siRNA-FA-Dab8γ (the Student’s t -test). C S2-013, S2-CP8, and HPNE cells were incubated with Alexa Fluor 647-labeled chemically modified SNORA22 siRNA-FA-Dab8α (green). Cells were stained with an anti-FOLR1 antibody (red). Blue, DAPI staining. Scale bars, 10 µm. D Quantification of Alexa Fluor 647-labeled chemically modified SNORA22 siRNA-positive cells in C. Data represent the number of cells positive for the siRNA, scored across five fields per group and derived from two independent experiments. Columns , mean; bars , SD. * p <0.05 significantly different from HPNE cells (the Student’s t -test)

Article Snippet: This solution was added to each well, followed by a 48-h incubation with the addition of 1 mL of fresh DMEM at 24 h. Cells were then fixed with 4% paraformaldehyde, followed by immunostaining using an anti-folate receptor 1 (FOLR1) antibody (MAB5646, R&D Systems, Minneapolis, MN, USA) and DAPI staining (HardSet Mounting Medium with DAPI, Vector Laboratories, Burlingame, CA, USA).

Techniques: Confocal, Immunofluorescence, Incubation, Labeling, Modification, Staining, Derivative Assay

Localization of SNORA22 siRNA-FA-Dab8α and SNORA22 siRNA-FA-Dab8γ within lysosomes of S2-013 cells. Confocal immunofluorescence images show S2-013 cells incubated with Alexa Fluor 647-labeled chemically modified SNORA22 siRNA-FA-Dab8α and SNORA22 siRNA-FA-Dab8γ (green) and stained for lysosomes (LysoTracker, red) and nuclei (DAPI, blue). Cells enclosed in white boxes are shown at higher magnification below each panel to clearly demonstrate the specific colocalization of the nucleic acid compounds within the lysosomal compartments. Scale bars, 10 µm

Journal: Human Cell

Article Title: Inhibition of pancreatic cancer growth by an siRNA targeting SNORA22 and a folic acid-conjugated cationic oligopeptide

doi: 10.1007/s13577-026-01458-1

Figure Lengend Snippet: Localization of SNORA22 siRNA-FA-Dab8α and SNORA22 siRNA-FA-Dab8γ within lysosomes of S2-013 cells. Confocal immunofluorescence images show S2-013 cells incubated with Alexa Fluor 647-labeled chemically modified SNORA22 siRNA-FA-Dab8α and SNORA22 siRNA-FA-Dab8γ (green) and stained for lysosomes (LysoTracker, red) and nuclei (DAPI, blue). Cells enclosed in white boxes are shown at higher magnification below each panel to clearly demonstrate the specific colocalization of the nucleic acid compounds within the lysosomal compartments. Scale bars, 10 µm

Techniques: Confocal, Immunofluorescence, Incubation, Labeling, Modification, Staining

TKTL1 drives tumor progression while potentiating anti-PD-1 immunotherapy sensitivity rather than apatinib in HCC. a Schematic of the orthotopic tumor mouse model: Hepa1-6 cells transduced with OE-TKTL1 (TKTL1 overexpression) or OE-VE (vector control) lentiviruses were subcutaneously implanted, followed by tumor transplantation to the liver. Anti-PD-1 treatment was administered, with live imaging at day 21 and sample harvest at day 42. b Tumor growth curves of orthotopic models in OE-VE, OE-TKTL1, OE-VE+Anti-PD-1, and OE-TKTL1+Anti-PD-1 groups (days 21, 28, 35). c Quantification of tumor volume and tumor weight in each treatment group. Data are mean ± SD ( n = 5, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA). d TUNEL immunofluorescence staining of orthotopic tumors to detect apoptotic cells. Scale bar: 50 µm. e Ki67 immunofluorescence staining of orthotopic tumors to assess cell proliferation. Scale bar: 50 µm. f Quantification of TUNEL-positive cells in orthotopic tumors. Data are mean ± SD ( n = 3, *** p < 0.001, **** p < 0.0001; One-way ANOVA). g Quantification of Ki67-positive cells in orthotopic tumors. Data are mean ± SD ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA). h Immunofluorescence staining and analysis of CD8 + (green) and PD-1 + (red) T cells in orthotopic tumors. Nuclei: DAPI (blue). Scale bar: 50 µm. Data are mean ± SD ( n = 3, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA). i Flow cytometry analysis of CD8 + T cell exhaustion markers (PD-1, CTLA-4, TOX) and effector cytokine (IFN-γ) in orthotopic tumor tissues. Data are mean ± SD ( n = 3, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA). j Schematic of the diethylnitrosamine (DEN)/carbon tetrachloride (CCl4)-induced orthotopic HCC model: Mice were treated with DEN+CCl4, followed by AAV-VE (vector control) or AAV-TKTL1 injection at week 20, anti-PD-1 treatment from week 24, MRI scanning, and liver tissue harvest at week 28. k Representative micro-MRI images of mouse livers pre- and post-treatment; representative photographs of excised livers. Scale bar: 1 cm. H&E staining of tumor (T) and peritumoral (P) tissues from DEN/CCl4-induced models. Scale bar: 50 µm. l TUNEL and Ki67 immunofluorescence staining of DEN/CCl4-induced tumor tissues. Scale bar: 50 µm. m Quantification of tumor size changes pre- and post-treatment in DEN/CCl4-induced models related to ( k ). Data are mean ± SD ( n = 5, ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA). n Quantification of TUNEL-positive and Ki67-positive cells in DEN/CCl4-induced models related to ( l ). Data are mean ± SD ( n = 3, ns: not significant, * p < 0.05, *** p < 0.001, **** p < 0.0001; One-way ANOVA). o Immunofluorescence staining of CD8 + and PD-1 + CD8 + T cells in DEN/CCl4-induced tumors. Scale bar: 50 µm. p Quantification of CD8 + and PD-1 + CD8 + T cell proportions in DEN/CCl4-induced tumors. Data are mean ± SD ( n = 3, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001; One-way ANOVA). q Flow cytometry analysis of CD8 + T cell exhaustion markers (PD-1, CTLA-4, TOX) and effector cytokine (IFN-γ) in DEN/CCl4-induced tumor tissues. Data are mean ± SD ( n = 3, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA). r Schematic illustrating the role of TKTL1 in promoting HCC progression and sensitizing tumors to anti-PD-1 immunotherapy via modulating CD8 + T cell function in the TME

Journal: Signal Transduction and Targeted Therapy

Article Title: Transketolase-like 1 potentiates PD-1 blockade in hepatocellular carcinoma by glycolysis to prime dendritic cell lactylation

doi: 10.1038/s41392-026-02875-2

Figure Lengend Snippet: TKTL1 drives tumor progression while potentiating anti-PD-1 immunotherapy sensitivity rather than apatinib in HCC. a Schematic of the orthotopic tumor mouse model: Hepa1-6 cells transduced with OE-TKTL1 (TKTL1 overexpression) or OE-VE (vector control) lentiviruses were subcutaneously implanted, followed by tumor transplantation to the liver. Anti-PD-1 treatment was administered, with live imaging at day 21 and sample harvest at day 42. b Tumor growth curves of orthotopic models in OE-VE, OE-TKTL1, OE-VE+Anti-PD-1, and OE-TKTL1+Anti-PD-1 groups (days 21, 28, 35). c Quantification of tumor volume and tumor weight in each treatment group. Data are mean ± SD ( n = 5, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA). d TUNEL immunofluorescence staining of orthotopic tumors to detect apoptotic cells. Scale bar: 50 µm. e Ki67 immunofluorescence staining of orthotopic tumors to assess cell proliferation. Scale bar: 50 µm. f Quantification of TUNEL-positive cells in orthotopic tumors. Data are mean ± SD ( n = 3, *** p < 0.001, **** p < 0.0001; One-way ANOVA). g Quantification of Ki67-positive cells in orthotopic tumors. Data are mean ± SD ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA). h Immunofluorescence staining and analysis of CD8 + (green) and PD-1 + (red) T cells in orthotopic tumors. Nuclei: DAPI (blue). Scale bar: 50 µm. Data are mean ± SD ( n = 3, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA). i Flow cytometry analysis of CD8 + T cell exhaustion markers (PD-1, CTLA-4, TOX) and effector cytokine (IFN-γ) in orthotopic tumor tissues. Data are mean ± SD ( n = 3, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA). j Schematic of the diethylnitrosamine (DEN)/carbon tetrachloride (CCl4)-induced orthotopic HCC model: Mice were treated with DEN+CCl4, followed by AAV-VE (vector control) or AAV-TKTL1 injection at week 20, anti-PD-1 treatment from week 24, MRI scanning, and liver tissue harvest at week 28. k Representative micro-MRI images of mouse livers pre- and post-treatment; representative photographs of excised livers. Scale bar: 1 cm. H&E staining of tumor (T) and peritumoral (P) tissues from DEN/CCl4-induced models. Scale bar: 50 µm. l TUNEL and Ki67 immunofluorescence staining of DEN/CCl4-induced tumor tissues. Scale bar: 50 µm. m Quantification of tumor size changes pre- and post-treatment in DEN/CCl4-induced models related to ( k ). Data are mean ± SD ( n = 5, ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA). n Quantification of TUNEL-positive and Ki67-positive cells in DEN/CCl4-induced models related to ( l ). Data are mean ± SD ( n = 3, ns: not significant, * p < 0.05, *** p < 0.001, **** p < 0.0001; One-way ANOVA). o Immunofluorescence staining of CD8 + and PD-1 + CD8 + T cells in DEN/CCl4-induced tumors. Scale bar: 50 µm. p Quantification of CD8 + and PD-1 + CD8 + T cell proportions in DEN/CCl4-induced tumors. Data are mean ± SD ( n = 3, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001; One-way ANOVA). q Flow cytometry analysis of CD8 + T cell exhaustion markers (PD-1, CTLA-4, TOX) and effector cytokine (IFN-γ) in DEN/CCl4-induced tumor tissues. Data are mean ± SD ( n = 3, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA). r Schematic illustrating the role of TKTL1 in promoting HCC progression and sensitizing tumors to anti-PD-1 immunotherapy via modulating CD8 + T cell function in the TME

Article Snippet: A combined secondary antibody was applied and left to incubate for 1 h. To maintain the integrity of the nucleus, DAPI staining (Beyotime, China) was utilized.

Techniques: Sensitivity, Transduction, Over Expression, Plasmid Preparation, Control, Transplantation Assay, Imaging, TUNEL Assay, Immunofluorescence, Staining, Analysis, Flow Cytometry, Injection, Tissue, Micro-MRI, Cell Function Assay

TKTL1 reprograms glucose metabolism through glycolysis instead of the PPP to stabilize the HIF-1α/HK2/PD-L1 axis and drive anti-PD-1 sensitivity in HCC. a mRNA expression of glycolysis-related genes (SLC2A1, HK2, HKDC1) in TKTL1-knockdown Hep-3B cells, determined by RNA sequencing. b KEGG pathway enrichment analysis of downregulated differentially expressed genes (DEGs) in TKTL1-knockdown Hep-3B cells, highlighting Glycolysis/Gluconeogenesis and HIF-1α signaling pathways. c Bubble plot of KEGG pathway enrichment for downregulated metabolites from non-targeted metabolomics of TKTL1-knockdown cells, focusing on central carbon metabolism. d Integrative analysis of downregulated DEGs (RNA sequencing) and metabolites (non-targeted metabolomics), visualized as a ranked plot showing enrichment in glycolytic pathways. e Western blot analysis of glycolytic enzymes (GLUT1, HK2, PKM2, ENO1, LDHA) and TKTL1 in HCC-LM3 and Hep-3B cells with TKTL1 knockdown or overexpression. β-actin serves as a loading control. f 13 C-labeled glucose metabolic flux tracing analysis, showing the relative abundance of metabolites in glycolysis, PPP and tricarboxylic acid (TCA) cycle in TKTL1-knockdown vs. negative control (NC) cells. g Extracellular acidification rate (ECAR) measurements (left) and lactic acid production (right) in HCC-LM3 and Hep-3B cells under TKTL1 knockdown or overexpression. Data are mean ± SD ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; Student’s t-test). h , i Western blot analysis of TKTL1, HIF-1α, HK2, and PD-L1 protein expression in HCC-LM3 and Hep-3B cells with TKTL1 knockdown or overexpression. β-actin serves as a loading control. Data are mean ± SD ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; Student’s t-test). j Immunofluorescence staining of TKTL1, HIF-1α, and HK2 protein expression in HCC tissues. Nuclei: DAPI (blue). Scale bar: 150 µm

Journal: Signal Transduction and Targeted Therapy

Article Title: Transketolase-like 1 potentiates PD-1 blockade in hepatocellular carcinoma by glycolysis to prime dendritic cell lactylation

doi: 10.1038/s41392-026-02875-2

Figure Lengend Snippet: TKTL1 reprograms glucose metabolism through glycolysis instead of the PPP to stabilize the HIF-1α/HK2/PD-L1 axis and drive anti-PD-1 sensitivity in HCC. a mRNA expression of glycolysis-related genes (SLC2A1, HK2, HKDC1) in TKTL1-knockdown Hep-3B cells, determined by RNA sequencing. b KEGG pathway enrichment analysis of downregulated differentially expressed genes (DEGs) in TKTL1-knockdown Hep-3B cells, highlighting Glycolysis/Gluconeogenesis and HIF-1α signaling pathways. c Bubble plot of KEGG pathway enrichment for downregulated metabolites from non-targeted metabolomics of TKTL1-knockdown cells, focusing on central carbon metabolism. d Integrative analysis of downregulated DEGs (RNA sequencing) and metabolites (non-targeted metabolomics), visualized as a ranked plot showing enrichment in glycolytic pathways. e Western blot analysis of glycolytic enzymes (GLUT1, HK2, PKM2, ENO1, LDHA) and TKTL1 in HCC-LM3 and Hep-3B cells with TKTL1 knockdown or overexpression. β-actin serves as a loading control. f 13 C-labeled glucose metabolic flux tracing analysis, showing the relative abundance of metabolites in glycolysis, PPP and tricarboxylic acid (TCA) cycle in TKTL1-knockdown vs. negative control (NC) cells. g Extracellular acidification rate (ECAR) measurements (left) and lactic acid production (right) in HCC-LM3 and Hep-3B cells under TKTL1 knockdown or overexpression. Data are mean ± SD ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; Student’s t-test). h , i Western blot analysis of TKTL1, HIF-1α, HK2, and PD-L1 protein expression in HCC-LM3 and Hep-3B cells with TKTL1 knockdown or overexpression. β-actin serves as a loading control. Data are mean ± SD ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; Student’s t-test). j Immunofluorescence staining of TKTL1, HIF-1α, and HK2 protein expression in HCC tissues. Nuclei: DAPI (blue). Scale bar: 150 µm

Article Snippet: A combined secondary antibody was applied and left to incubate for 1 h. To maintain the integrity of the nucleus, DAPI staining (Beyotime, China) was utilized.

Techniques: Sensitivity, Expressing, Knockdown, RNA Sequencing, Analysis, Protein-Protein interactions, Western Blot, Over Expression, Control, Labeling, Negative Control, Immunofluorescence, Staining

TKTL1 activates glycolysis pathway by promoting HIF-1α deubiquitination through recruitment of USP9X in HCC. a Cycloheximide (CHX) chase assay in OE-VE (vector control) and OE-TKTL1 (TKTL1 overexpression) cells, assessing HIF-1α protein degradation kinetics over 0, 4, 8, and 12 h. β-actin serves as a loading control. b CHX chase assay in Sh-NC (negative control) and Sh-TKTL1 (TKTL1 knockdown) cells, evaluating HIF-1α degradation with or without proteasome inhibitor MG132 treatment. β-actin serves as a loading control. c Ubiquitination assay of HIF-1α in HCC-LM3 (Sh-NC vs. Sh-TKTL1) and Hep-3B (OE-VE vs. OE-TKTL1) cells treated with MG132. Immunoprecipitation (IP) with HIF-1α antibody and antibody incubation with ubiquitin (Ub) antibody detect ubiquitinated HIF-1α. d Co-immunoprecipitation (Co-IP) assay of endogenous proteins in HCC-LM3 and Hep-3B cells, confirming the interaction between TKTL1 and HIF-1α. IgG serves as a negative control; input lysates verify protein expression. e Co-IP assay of exogenous proteins in HEK-293T cells transfected with TKTL1 and HIF-1α expression plasmids, validating the TKTL1-HIF-1α interaction. IgG serves as a negative control. f Intersection analysis of TKTL1-interacting proteins identified via IP and deubiquitinating enzyme (DUB) family members, pinpointing USP9X as a key overlapping binding partner of TKTL1. g , h Co-IP assay identifying USP9X as a TKTL1-interacting protein. Co-IP assay demonstrating the interaction between USP9X and HIF-1α. IgG serves as a negative control. i GST-pull down assay transfected with Myc-USP9X, HA-TKTL1, and Flag-HIF-1α plasmids, verifying the formation of the TKTL1-USP9X-HIF-1α ternary complex. j AI-predicted structural model of the TKTL1-USP9X-HIF-1α ternary complex, illustrating the binding interface and spatial configuration of the three proteins. k IP assay using HIF-1α domain mutants in HEK-293T cells, confirming that USP9X binds to the inhibitory domain (ID) of HIF-1α. l IP assay using TKTL1 domain mutants in HEK-293T cells, identifying that HIF-1α binds to the T-pyr domain of TKTL1 and USP9X binds to the T-pyr domain of TKTL1. m Confocal laser scanning microscopy showing the cytoplasmic colocalization of TKTL1, HIF-1α, and USP9X in HCC-LM3 and Hep-3B cells. Nuclei are stained with DAPI (blue). n Immunofluorescence staining of HCC tissue microarrays showing the colocalization of TKTL1, HIF-1α, and USP9X. Inserted merge images confirm triprotein colocalization. Correlation analysis of TKTL1 and HIF-1α expression levels in HCC tissues (Pearson’s correlation coefficient: r = 0.1107, p = 0.003). Scale bar: 150 µm

Journal: Signal Transduction and Targeted Therapy

Article Title: Transketolase-like 1 potentiates PD-1 blockade in hepatocellular carcinoma by glycolysis to prime dendritic cell lactylation

doi: 10.1038/s41392-026-02875-2

Figure Lengend Snippet: TKTL1 activates glycolysis pathway by promoting HIF-1α deubiquitination through recruitment of USP9X in HCC. a Cycloheximide (CHX) chase assay in OE-VE (vector control) and OE-TKTL1 (TKTL1 overexpression) cells, assessing HIF-1α protein degradation kinetics over 0, 4, 8, and 12 h. β-actin serves as a loading control. b CHX chase assay in Sh-NC (negative control) and Sh-TKTL1 (TKTL1 knockdown) cells, evaluating HIF-1α degradation with or without proteasome inhibitor MG132 treatment. β-actin serves as a loading control. c Ubiquitination assay of HIF-1α in HCC-LM3 (Sh-NC vs. Sh-TKTL1) and Hep-3B (OE-VE vs. OE-TKTL1) cells treated with MG132. Immunoprecipitation (IP) with HIF-1α antibody and antibody incubation with ubiquitin (Ub) antibody detect ubiquitinated HIF-1α. d Co-immunoprecipitation (Co-IP) assay of endogenous proteins in HCC-LM3 and Hep-3B cells, confirming the interaction between TKTL1 and HIF-1α. IgG serves as a negative control; input lysates verify protein expression. e Co-IP assay of exogenous proteins in HEK-293T cells transfected with TKTL1 and HIF-1α expression plasmids, validating the TKTL1-HIF-1α interaction. IgG serves as a negative control. f Intersection analysis of TKTL1-interacting proteins identified via IP and deubiquitinating enzyme (DUB) family members, pinpointing USP9X as a key overlapping binding partner of TKTL1. g , h Co-IP assay identifying USP9X as a TKTL1-interacting protein. Co-IP assay demonstrating the interaction between USP9X and HIF-1α. IgG serves as a negative control. i GST-pull down assay transfected with Myc-USP9X, HA-TKTL1, and Flag-HIF-1α plasmids, verifying the formation of the TKTL1-USP9X-HIF-1α ternary complex. j AI-predicted structural model of the TKTL1-USP9X-HIF-1α ternary complex, illustrating the binding interface and spatial configuration of the three proteins. k IP assay using HIF-1α domain mutants in HEK-293T cells, confirming that USP9X binds to the inhibitory domain (ID) of HIF-1α. l IP assay using TKTL1 domain mutants in HEK-293T cells, identifying that HIF-1α binds to the T-pyr domain of TKTL1 and USP9X binds to the T-pyr domain of TKTL1. m Confocal laser scanning microscopy showing the cytoplasmic colocalization of TKTL1, HIF-1α, and USP9X in HCC-LM3 and Hep-3B cells. Nuclei are stained with DAPI (blue). n Immunofluorescence staining of HCC tissue microarrays showing the colocalization of TKTL1, HIF-1α, and USP9X. Inserted merge images confirm triprotein colocalization. Correlation analysis of TKTL1 and HIF-1α expression levels in HCC tissues (Pearson’s correlation coefficient: r = 0.1107, p = 0.003). Scale bar: 150 µm

Article Snippet: A combined secondary antibody was applied and left to incubate for 1 h. To maintain the integrity of the nucleus, DAPI staining (Beyotime, China) was utilized.

Techniques: Plasmid Preparation, Control, Over Expression, Negative Control, Knockdown, Ubiquitin Proteomics, Immunoprecipitation, Incubation, Co-Immunoprecipitation Assay, Expressing, Transfection, Analysis, Binding Assay, GST Pulldown Assay, Confocal, Laser-Scanning Microscopy, Staining, Immunofluorescence, Tissue

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