Journal: Clinical and Translational Medicine

Article Title: SHP2 inhibition and adjuvant therapy synergistically target KIT‐mutant GISTs via ERK1/2‐regulated GSK3β/cyclin D1 pathway

doi: 10.1002/ctm2.70231

Figure Lengend Snippet: Molecular mechanism of SHP2 inhibition in GIST cells. (A) Immunoblotting analysis of whole‐cell lysate from four GIST cell lines treated with SHP099 for 24 h. Statistical analysis of the ratios of p‐mTORC1/mTORC1 and p‐S6K/p70 S6K are presented ( n = 3). (B) Cell cycle and statistical analysis of the four GIST cell lines treated with SHP099 (10 and 20 µM) for 24 h ( n = 3). (C) Immunoblotting analysis of whole cell lysate from four GIST cells treated with SHP099 for 24 h. Statistical analysis of the ratio of p‐cyclin D1/cyclin D1, p‐Rb/Rb and p‐GSK3β/GSK3β are presented ( n = 3). (D) Immunoblotting analysis of whole‐cell lysates from four GIST cell lines under treatment of SHP099 (12 µM) and/or LiCl (20 mM) for 24 h. Statistical analysis is presented ( n = 3). (E) Cell cycle analysis and statistical analysis of the four GIST cell lines treated with SHP099 (12 µM) and/or LiCl (20 mM) for 24 h ( n = 3).

Article Snippet: Lentiviral plasmids harbouring PTPN11 or CCND1 sequences were constructed and subsequently co‐transfected into HEK 293T cells with helper psPAX2 and pMD2.G vectors using the polyethylenimine reagent (MCE).

Techniques: Inhibition, Western Blot, Cell Cycle Assay

Journal: Clinical and Translational Medicine

Article Title: SHP2 inhibition and adjuvant therapy synergistically target KIT‐mutant GISTs via ERK1/2‐regulated GSK3β/cyclin D1 pathway

doi: 10.1002/ctm2.70231

Figure Lengend Snippet: Role of cyclin D1 in GIST cells. (A) Immunoblotting analysis of the indicated proteins in GIST cells following CCND1 knockout using the CRISPR/Cas9 system. Statistical analysis was presented for the ratio of cyclin D1 to β‐ACTIN and phosphorylated Rb to total Rb ( n = 3). (B) Analysis of cell proliferation in GIST cells following CCND1 gene knockout. Cell proliferation was assessed using the CCK‐8 assay ( n = 3). (C) Analysis of the effects of cyclin D1 overexpression in GIST cells. GIST cells were infected with recombinant lentivirus carrying CCND1 or control virus for 2 days. Then CCND1 ‐expressing GIST cells and control GIST cells (1 × 10 4 cells/well) were plated. These cells were co‐cultured with RMC4550 for 3 days. Absorbance was measured daily using the CCK‐8 assay ( n = 3).

Article Snippet: Lentiviral plasmids harbouring PTPN11 or CCND1 sequences were constructed and subsequently co‐transfected into HEK 293T cells with helper psPAX2 and pMD2.G vectors using the polyethylenimine reagent (MCE).

Techniques: Western Blot, Knock-Out, CRISPR, Gene Knockout, CCK-8 Assay, Over Expression, Infection, Recombinant, Control, Virus, Expressing, Cell Culture

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-219a-2-3p modulates the proliferation of thyroid cancer cells via the HPSE/cyclin D1 pathway

doi: 10.3892/etm.2021.10091

Figure Lengend Snippet: HPSE and cyclin D1 expression, cell cycle and cell proliferation are regulated by miR-219a-2-3p mimic via HPSE. (A) HPSE expression increased in B-CPAP and TPC-1 cells following transfection with HPSE plasmids. (B) miR-219a-2-3p mimic suppressed HPSE and cyclin D1 expression. Notably, ectopic HPSE expression reversed the inhibitory effect of miR-219a-2-3p mimic on cyclin D1 expression. (C) Cell cycle analysis indicated a higher number of B-CPAP and TPC-1 cells in the G0/G1 phase and less cells in the S phase following transfection with miR-219a-2-3p mimic. Furthermore, the number of EdU-positive B-CPAP and TPC-1 cells decreased following transfection with miR-219a-2-3p mimic. (D) HPSE was exogenously expressed in miR-219a-2-3p mimic-transfected cells, which decreased the number of cells in the G0/G1 phase and increased the number of cells in the S phase compared with the empty vector group. In addition, the number of proliferative cells was increased in miR-219a-2-3p mimic-transfected cells with HPSE upregulation. Magnification, x200. Red staining, EdU; blue staining, DAPI. *P<0.05; **P<0.01. HPSE, heparanase; miR, microRNA; NC, negative control; Con, control; Vec, empty vector.

Article Snippet: The membranes were then incubated with the following primary antibodies: Rabbit anti-HPSE (1:1,000; cat. no. 24529-1-AP; ProteinTech Group, Inc.), mouse anti-cyclin D1 (1:500; cat. no. TA804673; ZSGB-BIO) and anti-GAPDH (1:500; cat. no. TA-08; ZSGB-BIO) overnight at 4˚C.

Techniques: Expressing, Transfection, Cell Cycle Assay, Plasmid Preparation, Staining, Negative Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Magnolol alleviates hypoxia-induced pulmonary vascular remodeling through inhibition of phenotypic transformation in pulmonary arterial smooth muscle cells.

doi: 10.1016/j.biopha.2022.113060

Figure Lengend Snippet: Fig. 3. Effects of magnolol on the phenotypic transformation of pulmonary arteries in hypoxia-induced PH rats. (A) Representative images of immunohistochemistry staining for Ki67 (cell proliferation markers) in each group; (B-F) The protein expression of SM-22α, CyclinD1, OPN, and PCNA in the pulmonary artery of rats; Data are expressed as mean ± SD. +Mag(L): hypoxia+ magnolol (10 mg/kg/d); +Mag(H): hypoxia+ magnolol (20 mg/kg/d). **P < 0.01 vs Control; #P < 0.05, ##P < 0.01 vs Hypoxia.

Article Snippet: The PVDF membranes were blocked with 5% BSA and then incubated with the following primary antibodies: collagen I (No. ab34710, 1:1000, Abcam), collagen III (No. ab7778, 1:1000, Abcam), SM-22α (No. ab14106, 1:1000, Abcam), α-SMA (No. 19245, 1:1000, Cell Signaling Technology), OPN (No. ab91655, 1:1000, Abcam), PCNA (No. AF0261, 1:1000, Beyotime), calponin (No. AF1393, 1:1000, Beyotime), CyclinD1 (No. BM4272, 1:2000, BOSTER), p-JAK2 (No. ab32101, 1:1000, Abcam), JAK2 (No. AF1489, 1:2000, Beyotime), p-STAT3 (No. 13332, 1:1000, Signalway Antibody), STAT3 (No. 41465, 1:1000, Signalway Antibody), α-tubulin (No. sc-5286, 1:200, Santa Cruz) and β-actin (No. AF5001, 1:1000, Beyotime).

Techniques: Transformation Assay, Immunohistochemistry, Staining, Expressing, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Magnolol alleviates hypoxia-induced pulmonary vascular remodeling through inhibition of phenotypic transformation in pulmonary arterial smooth muscle cells.

doi: 10.1016/j.biopha.2022.113060

Figure Lengend Snippet: Fig. 5. Effects of magnolol on the proliferation, migration, and phenotypic transformation of PASMCs induced by hypoxia. (A) The cell viability was detected by the CCK-8 array. (B-C) Representative pictures of EdU staining. (D-E) The cell migration ability was detected by Transwell and scratch arrays. (F-G) The protein expression of SM-22α, α-SMA, Calponin, OPN, CyclinD1, PCNA, and collagen I in PASMCs. Data are expressed as mean ± SD. +Mag(L): hypoxia+ magnolol (10 mg/ kg/d); +Mag(H): hypoxia+ magnolol (20 mg/kg/d). **P < 0.01 vs Control; #P < 0.05, ##P < 0.01 vs Hypoxia.

Article Snippet: The PVDF membranes were blocked with 5% BSA and then incubated with the following primary antibodies: collagen I (No. ab34710, 1:1000, Abcam), collagen III (No. ab7778, 1:1000, Abcam), SM-22α (No. ab14106, 1:1000, Abcam), α-SMA (No. 19245, 1:1000, Cell Signaling Technology), OPN (No. ab91655, 1:1000, Abcam), PCNA (No. AF0261, 1:1000, Beyotime), calponin (No. AF1393, 1:1000, Beyotime), CyclinD1 (No. BM4272, 1:2000, BOSTER), p-JAK2 (No. ab32101, 1:1000, Abcam), JAK2 (No. AF1489, 1:2000, Beyotime), p-STAT3 (No. 13332, 1:1000, Signalway Antibody), STAT3 (No. 41465, 1:1000, Signalway Antibody), α-tubulin (No. sc-5286, 1:200, Santa Cruz) and β-actin (No. AF5001, 1:1000, Beyotime).

Techniques: Migration, Transformation Assay, CCK-8 Assay, Staining, Expressing, Control