Journal: Frontiers in Physiology

Article Title: Alteration in the number, morphology, function, and metabolism of erythrocytes in high-altitude polycythemia

doi: 10.3389/fphys.2024.1359357

Figure Lengend Snippet: (A) WB display figure of erythrocyte CD73/adenosine/S1P/2,3-BPG metabolic pathway and eENT1/adenosine/BPGM/2,3-BPG metabolic pathway–related proteins. Statistical results of adenosine A2B receptor–related (B) , SPHK1-related (C) , P-SPHK1-related (D) , eENT1-related (E) , BPGM-related (F) , and GAPDH-related expressions (G) . Statistical result for plasma CD73 (H) , plasma adenosine (I) , erythrocyte cytoplasmic adenosine content (J) , erythrocyte cytoplasmic S1P content (K) , and erythrocyte cytoplasmic 2,3-BPG contents (L) . WB, Western blotting; CD73, 5′-nucleotidase; S1P, sphingosine-1-phosphate; BPG, 2,3-bisphosphoglycerate; eENT1, erythrocyte equilibrative nucleoside transporter 1; SPHK1, sphingosine kinase 1; P-SPHK1, phospho-SPHK1; BPGM, bisphosphoglycerate mutase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The antibodies used were as follows: anti-adenosine A2b receptor [ADORA2B]/ADORA2B antibody (ab229671, 1:500; Abcam, Cambridge, United States), sphingosine kinase 1 (SPHK1) polyclonal antibody (10670-1-AP, 1:500; Proteintech, Wuhan, China), phospho-SPHK1 (Ser225) rabbit polyclonal antibody (19561-1-AP, 1:500; Proteintech), ENT1 polyclonal antibody (PA5-75507, 1:500; Invitrogen, Waltham, MA, United States), bisphosphoglycerate mutase (BPGM) polyclonal antibody (17173-1-AP, 1:500; Proteintech), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (ab181602, 1:10,000; Abcam), and beta-actin rabbit polyclonal antibody (20536-1-AP, 1:1,000; Proteintech).

Techniques: Western Blot

Journal: Frontiers in Physiology

Article Title: Alteration in the number, morphology, function, and metabolism of erythrocytes in high-altitude polycythemia

doi: 10.3389/fphys.2024.1359357

Figure Lengend Snippet: (A) WB display figure of erythrocyte CD73/adenosine/S1P/2,3-BPG metabolic pathway and eENT1/adenosine/BPGM/2,3-BPG metabolic pathway–related proteins. Statistical results of adenosine A2B receptor–related (B) , SPHK1-related (C) , P-SPHK1-related (D) , eENT1-related (E) , BPGM-related (F) , and GAPDH-related expressions (G) . Statistical result for plasma CD73 (H) , plasma adenosine (I) , erythrocyte cytoplasmic adenosine content (J) , erythrocyte cytoplasmic S1P content (K) , and erythrocyte cytoplasmic 2,3-BPG contents (L) . WB, Western blotting; CD73, 5′-nucleotidase; S1P, sphingosine-1-phosphate; BPG, 2,3-bisphosphoglycerate; eENT1, erythrocyte equilibrative nucleoside transporter 1; SPHK1, sphingosine kinase 1; P-SPHK1, phospho-SPHK1; BPGM, bisphosphoglycerate mutase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The antibodies used were as follows: anti-adenosine A2b receptor [ADORA2B]/ADORA2B antibody (ab229671, 1:500; Abcam, Cambridge, United States), sphingosine kinase 1 (SPHK1) polyclonal antibody (10670-1-AP, 1:500; Proteintech, Wuhan, China), phospho-SPHK1 (Ser225) rabbit polyclonal antibody (19561-1-AP, 1:500; Proteintech), ENT1 polyclonal antibody (PA5-75507, 1:500; Invitrogen, Waltham, MA, United States), bisphosphoglycerate mutase (BPGM) polyclonal antibody (17173-1-AP, 1:500; Proteintech), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (ab181602, 1:10,000; Abcam), and beta-actin rabbit polyclonal antibody (20536-1-AP, 1:1,000; Proteintech).

Techniques: Western Blot

Journal: Biomolecules & Therapeutics

Article Title: Growth Inhibitory and Pro-Apoptotic Effects of Hirsuteine in Chronic Myeloid Leukemia Cells through Targeting Sphingosine Kinase 1

doi: 10.4062/biomolther.2022.023

Figure Lengend Snippet: SPHK1 is highly expressed in CML cells and predicts clinical outcomes. (A) Kaplan-Meier representation of the relapse-free survival (RFS) of the two groups of CML patients with different SPHK1 expression levels (GSE71014). p -value was obtained using Kaplan-Meier analysis and compared with a long-rank test. (B) The protein expression of SPHK1 in PBMCs and various cancer cell lines (n=3).

Article Snippet: Antibody against p-SPHK1 (Ser225) (1:1,000, #19561-1-AP) was from Proteintech (Wuhan, Hubei, China).

Techniques: Expressing

Journal: Biomolecules & Therapeutics

Article Title: Growth Inhibitory and Pro-Apoptotic Effects of Hirsuteine in Chronic Myeloid Leukemia Cells through Targeting Sphingosine Kinase 1

doi: 10.4062/biomolther.2022.023

Figure Lengend Snippet: HST regulates the sphingolipid rheostat, represses SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt in CML cells. (A, B) K562 cells and K562/G01 cells were exposed with HST for 48 h. Cer concentration (A) and extracellular S1P level (B) were detected by ELISA. (C) SPHK1 activity was detected by ELISA. (D) S1PR1 level was analyzed by qRT-PCR. (E) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. Data represent mean value ± SD. * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: Antibody against p-SPHK1 (Ser225) (1:1,000, #19561-1-AP) was from Proteintech (Wuhan, Hubei, China).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Quantitative RT-PCR, Western Blot

Journal: Biomolecules & Therapeutics

Article Title: Growth Inhibitory and Pro-Apoptotic Effects of Hirsuteine in Chronic Myeloid Leukemia Cells through Targeting Sphingosine Kinase 1

doi: 10.4062/biomolther.2022.023

Figure Lengend Snippet: HST targets at SPHK1. (A, B) Molecular docking study of HST with SPHK1. (A) Two-dimensional interaction diagram of HST and SPHK1. Only interacting residues were labeled. (B) The spatial interaction diagram of HST and SPHK1. (C, D) CETSAs were performed on K562 cells and K562/G01 cells, the SPHK1 expression was determined by western blot (C). Relative band intensity of SPHK1 (D). Experiments were repeated at least in triplicate. Data represent mean value ± SD.

Article Snippet: Antibody against p-SPHK1 (Ser225) (1:1,000, #19561-1-AP) was from Proteintech (Wuhan, Hubei, China).

Techniques: Labeling, Expressing, Western Blot

Journal: Biomolecules & Therapeutics

Article Title: Growth Inhibitory and Pro-Apoptotic Effects of Hirsuteine in Chronic Myeloid Leukemia Cells through Targeting Sphingosine Kinase 1

doi: 10.4062/biomolther.2022.023

Figure Lengend Snippet: HST exerts anti-leukemic effect in CML cells through SPHK1 suppression. K562 cells and K562/G01 cells were exposed with HST for 48 h after transfected with SPHK1 vector or empty vector for 6 h. PF543 (30 μM in K562, 40 μM in K562/G01) was used as a positive control. (A) SPHK1 expression was detected by western blot. (B, C) Cell viability was conducted using MTT. (D-F) Cell apoptosis was analyzed using flow cytometry. (G, H) Extracellular S1P level (G) and Cer concentration (H) were detected by ELISA. (I) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. (J) S1PR1 level was determined using qRT-PCR. Data represent mean value ± SD. * p <0.05, *** p <0.001 versus the empty vector-transfected cells; ## p <0.01, ### p <0.001 versus the empty vector-transfected cells treated with HST.

Article Snippet: Antibody against p-SPHK1 (Ser225) (1:1,000, #19561-1-AP) was from Proteintech (Wuhan, Hubei, China).

Techniques: Transfection, Plasmid Preparation, Positive Control, Expressing, Western Blot, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: eLife

Article Title: Protective mitochondrial fission induced by stress-responsive protein GJA1-20k

doi: 10.7554/eLife.69207

Figure Lengend Snippet:

Article Snippet: Transfected construct (human) , siRNA to DRP1 , Thermo Fisher Scientific , ID 19561 , .

Techniques: Plasmid Preparation, Expressing, Transfection, Construct, Recombinant, Negative Control, DC Protein Assay, Isolation, Luminescence Assay, Purification, Software, Imaging, Staining