dapi staining  (Beyotime)

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

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

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

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

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

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

Journal: Frontiers in Pharmacology

Article Title: Protectin DX alleviates lumbar radicular pain in rats by suppressing RIP3/MLKL-associated necroinflammation through restoration of autophagic function

doi: 10.3389/fphar.2026.1957244

Figure Lengend Snippet: Effects of GSK’872 on pain behaviours and spinal neuroinflammation in LRP rats. (A, B) GSK’872 significantly improved MWT and TWL in LRP rats (n = 9 rats/group). Data are presented as mean ± SEM. Two-way repeated-measures ANOVA was performed, followed by Tukey’s post hoc test (* p < 0.05 vs. sham group; # p < 0.05 vs. vehicle group). (C) Representative western blots showing the protein expression of RIP3 and MLKL (n = 4 rats/group). (F) Representative Western blots showing protein expression of IL-1β, IL-6, AND TNF-α (n = 3 rats/group). (J) Representative images of PI staining for necrotic cells (red) (n = 3 rats/group); scale bars: 100 µm. (D, E, G-I, K) Quantitative analyses of protein levels of RIP3 (D) , MLKL (E) , IL-1β (G) , IL-6 (H) , and TNF-α (I) , as well as PI-positive cell number (K) . Data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05 vs. sham group; # p < 0.05 vs. vehicle group). Abbreviations: IL-1β, interleukin-1β; IL-6, interleukin-6; LRP, lumbar radicular pain; MLKL, mixed lineage kinase domain-like protein; MWT, mechanical withdrawal threshold; PI, propidium iodide; RIP3, receptor-interacting protein kinase 3; DAPI, 4’,6-Diamidino-2-phenylindole; TNF-α, tumor necrosis factor-α; TWL, thermal withdrawal latency.

Article Snippet: Cryosections (10 μm) were prepared, washed with phosphate-buffered saline (PBS), and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Boster, China) for 5 min at room temperature.

Techniques: Western Blot, Expressing, Staining

Journal: Frontiers in Pharmacology

Article Title: Protectin DX alleviates lumbar radicular pain in rats by suppressing RIP3/MLKL-associated necroinflammation through restoration of autophagic function

doi: 10.3389/fphar.2026.1957244

Figure Lengend Snippet: Effects of Protectin DX on pain behaviors and spinal necroinflammation in LRP rats. (A, B) PDX (10ng, 100ng or 200ng) treatment prominently meliorated the 50% MWT and prolonged TWL of LRP rat models (n = 9 rats/group). Data are presented as the mean ± SEM; Two-way ANOVA was used for comparisons among groups followed by Turkey’s multiple comparisons test (*P < 0.05 versus sham group; #P < 0.05 versus vehicle group; & P < 0.05 versus 10ng group). (C, F) Representative western blots showing protein expression of RIP3, MLKL, IL-1β, IL-6, and TNF-α (n = 4 rats/group). (J) Double immunofluorescence staining for MLKL (green) and PI (red) in the lumbar spinal cord on postoperative day 7 ((n = 3 rats/group). scale bars: 100µm. (D, E, G-I, K) Quantitative analyses of protein levels of RIP3 (D) , MLKL (E) , IL-1β (G) , IL-6 (H) , and TNF-α (I) , as well as MLKL/PI double-positive cell numbers (K) . Data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05 versus sham group; # p < 0.05 versus vehicle group; & p < 0.05 versus 10ng group). Abbreviations: IL-1β, interleukin-1β; IL-6, interleukin-6; LRP, lumbar radicular pain; MLKL, mixed lineage kinase domain-like protein; MWT, mechanical withdrawal threshold; PI, propidium iodide; RIP3, receptor-intracting protein kinase 3; DAPI, 4’ 6-Diamidino-2-phenylindole; TNF-α, tumor necrosis factor-α; TWL, thermal withdrawal latency; Protectin DX, PDX.

Article Snippet: Cryosections (10 μm) were prepared, washed with phosphate-buffered saline (PBS), and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Boster, China) for 5 min at room temperature.

Techniques: Western Blot, Expressing, Double Immunofluorescence Staining