recombinant human tgf-beta 1 (human cell-expressed) protein  (Bio-Techne corporation)

Journal: Cell Death & Disease

Article Title: TGFβ signaling sensitizes MEKi-resistant human melanoma to targeted therapy-induced apoptosis

doi: 10.1038/s41419-024-07305-1

Figure Lengend Snippet: A Dose-response curves of cell growth of six patient-derived melanoma cell lines treated with trametinib as determined in a colorimetric metabolic activity (MTT) assay after 72 h of treatment. Cell lines were considered sensitive with an IC50 lower than 2000 pM. Sensitive lines are shown in green and resistant lines in pink. B , C Flowcytometry quantification of Annexin V negative cells (“Live cells”) after 72 h exposure to various concentrations of TGFβ1 and/or 10 nM trametinib (MEKi) in a putative “sensitive” ( B ) and a “resistant” ( C ) melanoma cell line. D – G Live cell quantification upon TGFβ1 (10 ng/mL) and/or MEKi (10 nM) treatment in another “sensitive” melanoma cell line ( D ) and three further “resistant” cell lines ( E – G ). Experiments were performed in 3–4 independent replicates. P -values were calculated by ordinary one-way ANOVA and multiple comparisons for selected pairs with * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. H , I Western blots for phosho-SMAD2 (pSMAD2), total SMAD2 (tSMAD2), phosho-SMAD1/5 (pSMAD1/5), total SMAD1 (tSMAD1) and ACTB from whole cell lysates of a sensitive ( H ) and a resistant ( I ) melanoma cell line after 1 h of TGFβ1 / MEKi combinatorial treatment.

Article Snippet: After settling, cells were directly incubated in presence of 10 nM trametinib (A-1258, Active Biochemicals), 10 ng/mL TGFβ1 (7754-BH-005/CF, R&D Systems), 0.25 µM Sytox Green (S7020, ThermoFisher Scientific) and 32 nM Hoechst 33342 (62249, ThermoFisher Scientific).

Techniques: Derivative Assay, Activity Assay, MTT Assay, Western Blot

Journal: Cell Death & Disease

Article Title: TGFβ signaling sensitizes MEKi-resistant human melanoma to targeted therapy-induced apoptosis

doi: 10.1038/s41419-024-07305-1

Figure Lengend Snippet: A , B Live cell quantification of MEKi-resistant M170117 ( A ) and M010817 ( B ) cells, after siRNA mediated downregulation of the indicated genes and treatment with DMSO or combinations of 10 ng/mL TGFβ1 or 10 nM MEKi (trametinib) for 72 h. C , D Live cell quantification of M170117 ( C ) and M010817 ( D ) upon genetic knock-out of BCL2L11 with two different sg sequences and one non-targeting control sequence, after treatment with DMSO or combinations of 10 ng/mL TGFβ1 or 10 nM MEKi (trametinib) for 72 h. Experiments in A – D were performed in three independent replicates. Conditions with a significant rescue effect upon TGFβ1 + MEKi treatment are highlighted by red frames. E Live cell quantification of M170117 cells after combinatorial downregulation of BCL2L11 , BAP1 and/or UBE4B and treatment with 10 ng/mL TGFβ1 and 10 nM MEKi (trametinib) for 72 h. F , G Live cell quantification of M170117 ( F ) and M010817 ( G ) cells, 24 h after transfection with in total 200 ng of BCL2L11 , BAP1 and/or UBE4B mRNA as well as parallel treatment with 40 µM ZVAD as indicated below. Experiments in E – G were performed in four, respectively three independent replicates. P -values in all graphs were calculated by one-way ANOVA and multiple comparisons for selected pairs with * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: After settling, cells were directly incubated in presence of 10 nM trametinib (A-1258, Active Biochemicals), 10 ng/mL TGFβ1 (7754-BH-005/CF, R&D Systems), 0.25 µM Sytox Green (S7020, ThermoFisher Scientific) and 32 nM Hoechst 33342 (62249, ThermoFisher Scientific).

Techniques: Knock-Out, Control, Sequencing, Transfection

Journal: Cell Death & Disease

Article Title: TGFβ signaling sensitizes MEKi-resistant human melanoma to targeted therapy-induced apoptosis

doi: 10.1038/s41419-024-07305-1

Figure Lengend Snippet: A Levels of human TGFβ1 in the supernatant of M010817 cells 48 h after transfection with increasing doses of TGFB1 mRNA or control (zsGreen) mRNA detected by ELISA. B Live cell quantification upon MEKi treatment (10 nM) and transfection with increasing doses of TGFB1 mRNA in M010817 cells 72 h after transfection. Experiments were performed in 3 independent replicates. P -values were calculated by one-way ANOVA and multiple comparisons for selected pairs. C Schematic overview of the patient-derived xenograft setup indicating the timeline of tumor growth and applications of oral treatments with trametinib as well as intra-tumoral TGFβ1 or control (fLuc) mRNA injections. D Validation of exogenous TGFB1 expression by RT-qPCR 24 h after transfection with TGFB1 mRNA in M010817 xenograft tumors. P -values were calculated by non-parametric Mann-Whitney test. E Western blots for BCL2L11 and β-ACTIN (ACTB) from M010817 xenograft tumor lysates 24 h after injection with TGFB1 mRNA and/or combinatorial treatment with MEKi (trametinib) Quantifications of BCL2L11 levels are shown below, normalized to the loading control ACTB and to the MEKi only condition (average of 2 animals per blot). F Quantification of BCL2L11 protein levels by western blot in M010817 xenograft tumor lysates 24 h after injection with TGFB1 mRNA and/or combinatorial treatment with MEKi (trametinib). P -values were calculated by one-way ANOVA and multiple comparisons for selected pairs. G Growth curve of M010817 xenograft tumors in nude mice treated with MEKi or vehicle and/or TGFB1 or control (fLuc) mRNA. P -values were calculated by two-way ANOVA and multiple comparisons for selected pairs. For all graphs, p -values are shown by numbers or with * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: After settling, cells were directly incubated in presence of 10 nM trametinib (A-1258, Active Biochemicals), 10 ng/mL TGFβ1 (7754-BH-005/CF, R&D Systems), 0.25 µM Sytox Green (S7020, ThermoFisher Scientific) and 32 nM Hoechst 33342 (62249, ThermoFisher Scientific).

Techniques: Transfection, Control, Enzyme-linked Immunosorbent Assay, Derivative Assay, Expressing, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Injection