Journal: Stem cell research & therapy

Article Title: TMT-based quantitative proteomic analysis of spheroid cells of endometrial cancer possessing cancer stem cell properties.

doi: 10.1186/s13287-023-03348-x

Figure Lengend Snippet: Fig. 8 Functional assays of selected targets, PFKFB3, GPRC5A and HK2. A–C The mRNA levels of PFKFB3, GPRC5A and HK2 were detected by qRT-PCR. D The expression levels of PFKFB3, GPRC5A and HK2 were detected by Western blot (Full-length blots are presented in supplementary material). E qRT-PCR results for Ishikawa and HEC1A transfected with HK2-siRNA-1, HK2-siRNA-2, HK2-siRNA-3 and NC. F Western blot analysis revealed significant downregulation in HK2-siRNA cells in comparison with the controls (Full-length blots are presented in supplementary material 1 and 2). G CCK-8 assays for Ishikawa and HEC1A cells transfected with HK2-siRNA or NC. H The number of spheroids for Ishikawa and HEC1A transfected with HK2-siRNA or NC in spheroid formation assay. I KEGG biochemical classification for HK2-siRNA endometrial cancer cells (*P < 0.05, **P < 0.01, *** P < 0.001)

Article Snippet: Primary antibodies of western blotting including HK2 (1:1,000, rabbit anti-human, 2A11C3, #66,974–1-Ig; Proteintech Group, INC, USA), GPRC5A (1:1,000, rabbit anti-human, #10,309–1-AP; Proteintech Group, INC, USA), PFKFB3 (1:1,000, rabbit anti-human, #13,763– 1-AP; Proteintech Group, INC, USA), and GAPDH (1:5,000, mouse anti-human, conjugated with HRP, 1E6D9, #60,004–1-Ig; Proteintech Group, INC, USA).

Techniques: Functional Assay, Quantitative RT-PCR, Expressing, Western Blot, Transfection, Comparison, CCK-8 Assay, Tube Formation Assay

Journal: Frontiers in Oncology

Article Title: MiR-125a-5p regulates the radiosensitivity of laryngeal squamous cell carcinoma via HK2 targeting through the DDR pathway

doi: 10.3389/fonc.2024.1438722

Figure Lengend Snippet: Forecasting the target gene by miR-125a-5p, the expression and biological role of HK2 in LSCC: (A) The research involved examining three sets of data, which involved predicting target genes controlled by miR-125a-5p, (B) creating the network of PPI and finding the important genes, (C) pinpointing the exact location where HK2 and miR-125a-5p bind, and (D, E) comparing levels of HK2 in laryngeal cancer tissues to nearby healthy tissues using immunohistochemical analysis. (F, G) Western blotting was performed to assess the HK2 expression levels in laryngeal cancer tissues compared to nearby non-cancerous tissues. (H) RT-PCR was used to assess HK2 expression levels in laryngeal cancer tissues in comparison to nearby non-cancerous tissues. (I, J) Furthermore, RT-PCR was performed to evaluate the effect on HK2 expression levels in AMN-HC-8 cells when either increased or decreased. (K) The ability of proliferation in laryngeal cancer cells was evaluated by using the CCK-8 assay after altering the expression levels of HK2. (L) The clonogenic assay was employed to investigate alterations in clonability observed in laryngeal cancer cells following modulation of HK2. (M) The dose survival curve demonstrated variations in surviving fraction resulting from up-regulation or down-regulation of HK2. (N) The comprehensive analysis was conducted using single hit multi-target model to investigate the impact of modulating HK2 expression levels on radiotherapy sensitivity. (O) Moreover, the luciferase test was used to verify the specific interaction between miR-125a-5p and HK2. (P, Q) Flow cytometry was used to confirm the impact of modified HK2 expression on apoptosis control. ( * P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: The membrane was incubated at room temperature for 2 h and then incubated overnight with specific primary antibodies against HK2 (ProSci, 1:1000), rH2AX (Microporous, 1:1000), H2AX (Abcam, 1:1000), and GAPDH (ZsBio, 1:5000).

Techniques: Expressing, Immunohistochemical staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Comparison, CCK-8 Assay, Clonogenic Assay, Luciferase, Flow Cytometry, Modification, Control

Journal: Frontiers in Oncology

Article Title: MiR-125a-5p regulates the radiosensitivity of laryngeal squamous cell carcinoma via HK2 targeting through the DDR pathway

doi: 10.3389/fonc.2024.1438722

Figure Lengend Snippet: Single hit multi-target model was used to analyze the effect of HK2 on the radiosensitivity of laryngeal cancer.

Article Snippet: The membrane was incubated at room temperature for 2 h and then incubated overnight with specific primary antibodies against HK2 (ProSci, 1:1000), rH2AX (Microporous, 1:1000), H2AX (Abcam, 1:1000), and GAPDH (ZsBio, 1:5000).

Techniques:

Journal: Frontiers in Oncology

Article Title: MiR-125a-5p regulates the radiosensitivity of laryngeal squamous cell carcinoma via HK2 targeting through the DDR pathway

doi: 10.3389/fonc.2024.1438722

Figure Lengend Snippet: MiR-125a-5p regulates LSCC biological function and radiosensitivity by targeting HK2: (A) The regulation of LSCC cell proliferation is specifically mediated by miR-125a-5p through HK2 targeting. (B) This targeting of HK2 by miR-125a-5p impacts the clonogenic potential of LSCC cells. (C) Additionally, miR-125a-5p plays a role in determining the survival of laryngeal cancer cells under varying doses of irradiation by targeting HK2. (D) The single hit multi-target model approach was employed to assess alterations in the radiosensitivity of laryngeal cancer cells. (E, F) MiR-125a-5p modulates apoptosis in laryngeal cancer cells by interacting with HK2. (G–I) MiR-125a-5p regulates the levels of H2AX and rH2AX by targeting HK2. ( * P <0.05, ** P <0.01, and *** P <0.001).

Article Snippet: The membrane was incubated at room temperature for 2 h and then incubated overnight with specific primary antibodies against HK2 (ProSci, 1:1000), rH2AX (Microporous, 1:1000), H2AX (Abcam, 1:1000), and GAPDH (ZsBio, 1:5000).

Techniques: Irradiation

Journal: Frontiers in Oncology

Article Title: MiR-125a-5p regulates the radiosensitivity of laryngeal squamous cell carcinoma via HK2 targeting through the DDR pathway

doi: 10.3389/fonc.2024.1438722

Figure Lengend Snippet: Single hit multi-target model was used to analyze the effect of miR-125a-5p targeting HK2.

Article Snippet: The membrane was incubated at room temperature for 2 h and then incubated overnight with specific primary antibodies against HK2 (ProSci, 1:1000), rH2AX (Microporous, 1:1000), H2AX (Abcam, 1:1000), and GAPDH (ZsBio, 1:5000).

Techniques:

Journal: Biomedicines

Article Title: SMYD2 Promotes Calcium Oxalate-Induced Glycolysis in Renal Tubular Epithelial Cells via PTEN Methylation.

doi: 10.3390/biomedicines12102279

Figure Lengend Snippet: Figure 2. SMYD2 and glycolysis are activated in glyoxylate-induced nephrolithiasis mice. (A) Repre- sentative immunofluorescence images and semi-quantitative analysis of SMYD2 expression. (B) Rep- resentative images of Oil Red O staining and quantification of the positive staining area in renal tissues of CON and Gly groups; black arrows indicate lipid deposition in RTCs. (C,D) Representative images and semi-quantitative immunohistochemistry analysis of HK2, LDHA, PKM2, CPT1, and PPARα in CON and Gly groups. (E,F) Western blot examines HK2, PKM2, and LDHA levels in renal tissues, with quantification by densitometry. Scale bar = 50 µm. Data are expressed as mean ± SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 ◦C with primary antibodies against SMYD2 (Proteintech), HK2 (ABclonal), PKM2 (Proteintech), LDHA (ABclonal), FN (Boster), α-SMA (Proteintech), COL1A1 (Affinity Biologicals, Ancaster, Canada), BAX (ABclonal), BCL-2 (ABclonal), Cleaved caspase3 (Abcam), mTOR (Proteintech), p-mTOR (Proteintech), S6 (ABclonal), p-S6 (ABclonal), AKT Biomedicines 2024, 12, 2279 4 of 20 (Proteintech), p-AKT (Proteintech), PTEN (ABclonal), N-cadherin (ABclonal), Vimentin (Proteintech), Vinculin (ABclonal), and β-Actin (Servicebio).

Techniques: Immunofluorescence, Expressing, Staining, Immunohistochemistry, Western Blot

Journal: Biomedicines

Article Title: SMYD2 Promotes Calcium Oxalate-Induced Glycolysis in Renal Tubular Epithelial Cells via PTEN Methylation.

doi: 10.3390/biomedicines12102279

Figure Lengend Snippet: Figure 5. Glycolysis promotes COM-induced apoptosis, inflammation, and EMT in HK-2 cells. (A) HK-2 cells were stimulated with 100 µg/mL COM for 0, 12, 24, and 48 h, and the levels of HK2, PKM2, and LDHA were measured by Western blotting. (B) The expression of HK2, PKM2, and LDHA was quantified by densitometry. (C,D) Western blot analysis showing the levels of HK2, PKM2, and LDHA after 2-DG treatment and densitometric analysis. (E) Determination of lactate concentration in the supernatant of HK-2 cells. (F–H) Western blot analysis and densitometric analysis of the levels of N-cadherin, Vimentin, FN, COL1A1, α-SMA, BAX, BCL-2, and Cleaved caspase3 expression after 2-DG treatment. (I,J) Flow cytometry indicated that 2-DG inhibited apoptosis and necrosis. (K) The levels of IL-1β, IL-6, and TNF-α in the supernatant of the HK-2 cells in the respective groups were measured by ELISA. Data are expressed as mean ± SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 ◦C with primary antibodies against SMYD2 (Proteintech), HK2 (ABclonal), PKM2 (Proteintech), LDHA (ABclonal), FN (Boster), α-SMA (Proteintech), COL1A1 (Affinity Biologicals, Ancaster, Canada), BAX (ABclonal), BCL-2 (ABclonal), Cleaved caspase3 (Abcam), mTOR (Proteintech), p-mTOR (Proteintech), S6 (ABclonal), p-S6 (ABclonal), AKT Biomedicines 2024, 12, 2279 4 of 20 (Proteintech), p-AKT (Proteintech), PTEN (ABclonal), N-cadherin (ABclonal), Vimentin (Proteintech), Vinculin (ABclonal), and β-Actin (Servicebio).

Techniques: Western Blot, Expressing, Concentration Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: SMYD2 Promotes Calcium Oxalate-Induced Glycolysis in Renal Tubular Epithelial Cells via PTEN Methylation.

doi: 10.3390/biomedicines12102279

Figure Lengend Snippet: Figure 6. Silencing SMYD2 specifically inhibits glycolysis, apoptosis, inflammation, and EMT in HK-2 cells. (A,B) Western blot verified the knockdown efficiency of SMYD2 and quantified it by densitometry. (C–F) Western blot showed that SMYD2-knockdown suppressed HK2, PKM2, and LDHA expression. (G) Measurement of lactate in the supernatant of HK-2 cells from the specified groups (H) Representative immunofluorescence images of HK2, PKM2, LDHA after SMYD2- knockdown. (I,J) Western blot detection of N-cadherin, Vimentin, FN, Collagen l, α-SMA, BAX, BCL-2, Cleaved caspase3 expression after SMYD2 knockdown and quantification by densitometry. (K) The levels of IL-1β, IL-6, and TNF-α in the supernatant of HK-2 cells in the indicated group were measured by ELISA. Scale bar = 50 µm. Data are expressed as mean ± SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 ◦C with primary antibodies against SMYD2 (Proteintech), HK2 (ABclonal), PKM2 (Proteintech), LDHA (ABclonal), FN (Boster), α-SMA (Proteintech), COL1A1 (Affinity Biologicals, Ancaster, Canada), BAX (ABclonal), BCL-2 (ABclonal), Cleaved caspase3 (Abcam), mTOR (Proteintech), p-mTOR (Proteintech), S6 (ABclonal), p-S6 (ABclonal), AKT Biomedicines 2024, 12, 2279 4 of 20 (Proteintech), p-AKT (Proteintech), PTEN (ABclonal), N-cadherin (ABclonal), Vimentin (Proteintech), Vinculin (ABclonal), and β-Actin (Servicebio).

Techniques: Western Blot, Knockdown, Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: SMYD2 Promotes Calcium Oxalate-Induced Glycolysis in Renal Tubular Epithelial Cells via PTEN Methylation.

doi: 10.3390/biomedicines12102279

Figure Lengend Snippet: Figure 7. SMYD2 methylates PTEN and inhibits its expression, thereby promoting glycolysis by acti- vating the mTORC1 pathway in HK-2 cells. (A–D) Western blot and densitometry analysis detected the expression of PTEN and mTORC1 signals after si-SMYD2 in vitro and in vivo. (E) Demonstration of two protein binding models in Surface form. (F) SMYD2 interaction with PTEN in HK-2 cells. (G) In HK-2 cells transfected with si-SMYD2 or si-Ctrl, IP with anti-PTEN antibody, and subsequent blotting with anti-PTEN, anti-methylated lysine antibodies. (H) Western blot analysis of the levels of HK2, PKM2, and LDHA in HK-2 cells. Data are expressed as mean ± SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 ◦C with primary antibodies against SMYD2 (Proteintech), HK2 (ABclonal), PKM2 (Proteintech), LDHA (ABclonal), FN (Boster), α-SMA (Proteintech), COL1A1 (Affinity Biologicals, Ancaster, Canada), BAX (ABclonal), BCL-2 (ABclonal), Cleaved caspase3 (Abcam), mTOR (Proteintech), p-mTOR (Proteintech), S6 (ABclonal), p-S6 (ABclonal), AKT Biomedicines 2024, 12, 2279 4 of 20 (Proteintech), p-AKT (Proteintech), PTEN (ABclonal), N-cadherin (ABclonal), Vimentin (Proteintech), Vinculin (ABclonal), and β-Actin (Servicebio).

Techniques: Expressing, Western Blot, In Vitro, In Vivo, Protein Binding, Transfection, Methylation