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: Antibodies against Cyclin B1 (1:1,000, #4135), CDC2 (1:1,000, #9116), Cyclin D1 (1:1,000, #55506), Caspase 3 (1:1,000, #9662), PARP (1:1,000, #9532), Caspase-8 (1:1,000, #9746), Caspase-9 (1:1,000, #9502), Cytochrome c (1:1,000, #4272), BCR-ABL (1:1,000, #2862), p-BCR-ABL (Y412) (1:1,000, #2865), PI3K-p110α (1:1,000, #4249), p-Akt (Ser473) (1:1,000, #3787), Akt (1:1,000, #9272), β-actin (1:1,000, #8457), anti-rabbit and anti-mouse HRP-conjugated secondary antibodies (1:2,000) were purchased from Cell Signaling Technology (Danvers, MA, USA).

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 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: Antibodies against Cyclin B1 (1:1,000, #4135), CDC2 (1:1,000, #9116), Cyclin D1 (1:1,000, #55506), Caspase 3 (1:1,000, #9662), PARP (1:1,000, #9532), Caspase-8 (1:1,000, #9746), Caspase-9 (1:1,000, #9502), Cytochrome c (1:1,000, #4272), BCR-ABL (1:1,000, #2862), p-BCR-ABL (Y412) (1:1,000, #2865), PI3K-p110α (1:1,000, #4249), p-Akt (Ser473) (1:1,000, #3787), Akt (1:1,000, #9272), β-actin (1:1,000, #8457), anti-rabbit and anti-mouse HRP-conjugated secondary antibodies (1:2,000) were purchased from Cell Signaling Technology (Danvers, MA, USA).

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

Journal: International Journal of Molecular Sciences

Article Title: c-Abl Tyrosine Kinase Is Regulated Downstream of the Cytoskeletal Protein Synemin in Head and Neck Squamous Cell Carcinoma Radioresistance and DNA Repair

doi: 10.3390/ijms21197277

Figure Lengend Snippet: Synemin regulates c-Abl kinase activity. ( A ) Heatmap of tyrosine kinase activities in cells depleted of synemin and exposed to 6 Gy X-ray. Kinase activity profiles were generated by PamGene Technology. ( B ) Normalized kinase statistic of un-irradiated controls (grey) and samples 1 h (yellow) or 24 h (orange) post 6 Gy X-ray exposure. ( C ) Top affected tyrosine kinases in synemin-depleted cell cultures 24 h post 6 Gy X-ray irradiation. X -axis indicates the τ value for each kinase (τ < 0 indicates reduced kinase activity relative to control). Dot colors indicate the specificity score. Dot size indicates the number of included peptides. The Y -axis shows the overall score. ( D ) Western blotting of c-Abl expression and phosphorylation from whole cell lysates of synemin-depleted cells. ( E ) Densitometries of Western blots from synemin-depleted, 6-Gy irradiated cells showing phosphorylated forms of c-Abl shown in “D” (Y412 and T735, n = 4). Phosphorylation levels were calculated relative to the total amount of c-Abl. Dots represent each independent experiment. Data are presented as mean ± SD (two-sided t -test; * p < 0.05).

Article Snippet: Antibodies against c-Abl (#2862P), c-Abl T715 (#2846S), and c-Abl Y412 (#2865S) were from Cell Signaling (Frankfurt, Germany); β-actin (#A1978) and Synemin (#S9075, for zebrafish western blot) were from Sigma-Aldrich (Taufkirchen, Germany); γH2AX S139 (#05-636) was from Upstate (Schwalbach, Germany) and 53BP1 (#NB100-904) was from Novus Biologicals (Cambridge, UK).

Techniques: Activity Assay, Generated, Irradiation, Western Blot, Expressing