Journal: Cell Death and Differentiation

Article Title: Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM

doi: 10.1038/cdd.2016.96

Figure Lengend Snippet: Inhibition of BCL-XL but not inhibition of BCL-2 cooperates with PLX4032 in the killing of BRAFV600E mutant melanoma cell lines. (a) M14, UACC257, Malme3M and SKMEL5 BRAFV600E cell lines were treated with DMSO (vehicle; -) or PLX4032 (+ at 3 μM) in the presence of the pan-caspase inhibitor Q-VD-OPh for 24 h before western blot analysis to detect the indicated pro-survival BCL-2-like proteins. Probing with an antibody to HSP70 was used as a loading control. (b) BRAFV600E mutant melanoma cell lines from panel (a) were treated with DMSO, PLX4032 (3 μM) and/or (c) ABT-737 (1 μM; inhibitor of BCL-2, BCL-XL and BCL-W), ABT-199 (1 μM; inhibitor of BCL-2) or ABT-1155463 (1 μM; inhibitor of BCL-XL). Cell survival was assessed after 5 days of treatment by flow cytometric analysis. Living cells were identified as those negative for Annexin V and propidium iodide. The data are derived from three independent experimental replicates, each comprising the average of three technical replicates and are presented as mean±S.E.M.; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

Article Snippet: Antibodies (clone) were purchased or obtained from the sources indicated: anti-BIM (Enzo Life Sciences, Farmingdale, NY, USA, cat# ADI-AAP-330-E), anti-PUMA (Abcam, Cambridge, UK, cat# 27669), anti-BMF (WEHI, clone 12E10), anti-NOXA (ProSci, Poway, CA, USA, cat# 2437), anti-MCL-1 (WEHI, clone 19C4-15), anti-BCL-2 (WEHI, clone Bcl-2-100), anti-BCL-XL (WEHI, clone 9C9), anti-pERK (Cell Signaling, Danvers, MA, USA, cat# 4370), anti-ERK (Cell Signaling, cat# 9102), anti-HSP70 (gift from Dr. R Anderson PMCC Melbourne, clone N6), anti-TP53 (Santa Cruz, Dallas, TX, USA, cat# 6243).

Techniques: Inhibition, Mutagenesis, Western Blot, Derivative Assay

Journal: Cell Death and Differentiation

Article Title: Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM

doi: 10.1038/cdd.2016.96

Figure Lengend Snippet: HGF inhibits PLX4032-induced upregulation of PUMA and BIM in cMET+ BRAF mutant melanoma cells and thereby protects them against PLX4032-induced killing. (a) cMET− (M14 and UACC257) and cMET+ (Malme3M and SKMEL5) BRAFV600E melanoma cell lines were treated with DMSO (vehicle; black bar) or PLX4032 (grey bars, at 3 μM) with the indicated concentrations of HGF (0, 3, 6, 12, 25, 50, 100, 200 ng/ml) for 5 days and cell survival was assessed by flow cytometry. Living cells were defined as those negative for Annexin V and propidium iodide. The concentration at which HGF causes significant protection from PLX4032-induced killing is indicated. Data are derived from three independent experimental replicates, each comprising the average of three technical replicates and are presented as mean±S.E.M.; *P<0.05, **P<0.01, ****P<0.0001. (b) cMET− (M14 and UACC257) and cMET+ (Malme3M and SKMEL5) BRAF mutant cells were treated with single agents or combinations of DMSO (vehicle), HGF (50 ng/ml) and PLX4032 (3 μM) for 24 h in the presence of Q-VD-OPh. The mRNA of cells was subjected to qRT-PCR analysis to measure the expression of pro-survival (BCL-2, BCL-X, BFL-1, MCL-1) and pro-apoptotic (BIM, PUMA) BCL-2 family members. The mRNA expression levels of the different genes were normalized to the levels of Gapdh and the data are presented as fold change relative to DMSO (vehicle)-treated control cells. The data are derived from three independent experimental replicates and are presented as mean±S.E.M. (c) Cells were treated as described in panel (b) and analyzed by western blotting for the expression of pro-survival (BCL-2, BCL-XL, MCL-1) and pro-apoptotic (BIM, PUMA) BCL-2 family members. Probing for HSP70 was used as a loading control

Article Snippet: Antibodies (clone) were purchased or obtained from the sources indicated: anti-BIM (Enzo Life Sciences, Farmingdale, NY, USA, cat# ADI-AAP-330-E), anti-PUMA (Abcam, Cambridge, UK, cat# 27669), anti-BMF (WEHI, clone 12E10), anti-NOXA (ProSci, Poway, CA, USA, cat# 2437), anti-MCL-1 (WEHI, clone 19C4-15), anti-BCL-2 (WEHI, clone Bcl-2-100), anti-BCL-XL (WEHI, clone 9C9), anti-pERK (Cell Signaling, Danvers, MA, USA, cat# 4370), anti-ERK (Cell Signaling, cat# 9102), anti-HSP70 (gift from Dr. R Anderson PMCC Melbourne, clone N6), anti-TP53 (Santa Cruz, Dallas, TX, USA, cat# 6243).

Techniques: Mutagenesis, Flow Cytometry, Concentration Assay, Derivative Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: Cell Death and Differentiation

Article Title: Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM

doi: 10.1038/cdd.2016.96

Figure Lengend Snippet: The combination of PLX4032 and BCL-XL inhibition can partially overcome HGF-mediated resistance of cMET+ BRAF mutant melanoma cells. (a) Malme3M and SKMEL5 cells were treated with DMSO (vehicle), PLX4032 (3 μM) alone or in combination with ABT-737 (1 μM) (blue bars), ABT-199 (1 μM) (green bars), A-1155463 (1 μM) (red bars), with or without addition of HGF (50 ng/ml) for 5 days and cell survival was assessed by flow cytometry. Living cells were identified as those negative for Annexin V and PI. The data are derived from three independent experimental replicates, each comprising the average of three technical replicates. The data were analyzed using Student's t-test and are presented as mean±S.E.M.; *P<0.05, **P<0.01. (b) Under steady-state conditions, low levels of pro-apoptotic BH3-only proteins are present in the BRAF mutant melanoma cells. Hence, pro-survival proteins are able to neutralize BAX and BAK to cause survival of the cell. In the presence of PLX4032, PUMA and BIM are getting induced, which leads to sequestration of pro-survival proteins, allowing BAX and BAK to induce apoptosis. In the presence of HGF, PLX4032 mediated upregulation of PUMA and BIM is substantially diminished, and this is the underlying molecular mechanism that causes melanoma cells to develop resistance to PLX4032-induced killing. When combining PLX4032 and the BCL-XL-specific inhibitor, A-1155463, HGF-mediated resistance can partially be overcome

Article Snippet: Antibodies (clone) were purchased or obtained from the sources indicated: anti-BIM (Enzo Life Sciences, Farmingdale, NY, USA, cat# ADI-AAP-330-E), anti-PUMA (Abcam, Cambridge, UK, cat# 27669), anti-BMF (WEHI, clone 12E10), anti-NOXA (ProSci, Poway, CA, USA, cat# 2437), anti-MCL-1 (WEHI, clone 19C4-15), anti-BCL-2 (WEHI, clone Bcl-2-100), anti-BCL-XL (WEHI, clone 9C9), anti-pERK (Cell Signaling, Danvers, MA, USA, cat# 4370), anti-ERK (Cell Signaling, cat# 9102), anti-HSP70 (gift from Dr. R Anderson PMCC Melbourne, clone N6), anti-TP53 (Santa Cruz, Dallas, TX, USA, cat# 6243).

Techniques: Inhibition, Mutagenesis, Flow Cytometry, Derivative Assay

Journal: Cellular signalling

Article Title: Vegfr3 activation of Pkd2l1 + CSF-cNs triggers the neural stem cell response in spinal cord injury.

doi: 10.1016/j.cellsig.2025.111675

Figure Lengend Snippet: Fig. 1. Vegfr3 is expressed primarily in Pkd2l1+ CSF-cNs. (A) A diagram illustrating the process of isolating and culturing primary CSF-cNs. (B) Pkd2l1+ CSF-cNs expressed Vegfr1, Vegfr2, and Vegfr3, with Vegfr3 showing the highest expression in vitro. (C) Western blot analysis of Vegfr1, Vegfr2, and Vegfr3 in Pkd2l1+ CSF- cNs. (D) Strategy diagram for the construction of Pkd2l1-2 A-CreERT2 × R26-CAG-LSL-ZsGreen transgenic mice, outlining the genetic modifications and breeding strategy employed. (E) Immunofluorescence colabeling of Pkd2l1 and the inherent ZsGreen fluorescence in Pkd2l1-2 A-CreERT2 × R26-CAG-LSL-ZsGreen transgenic mice. The overlap of the Pkd2l1 and ZsGreen signals confirmed the successful generation of the transgenic model. (F) Coexpression analysis showing Vegfr3 localization in Pkd2l1+ CSF-cNs. Double immunofluorescence staining revealed the colocalization of Vegfr3 with Pkd2l1+ cells, indicating that Vegfr3 is expressed in these neurons. (G) Immunofluorescence images illustrate the lack of colocalization between Vegfr3 and Foxj1, a specific marker for ependymal cells, in both the sham and SCI 7 d groups. These findings indicate that Vegfr3+ cells are distinct from ependymal cells. (H) Immunofluorescence images show the absence of colocalization between Vegfr3 and ALDH1L1, an astrocyte marker, in both the sham and SCI 7 d groups. Scale bar: 50 μm (B, E, F, G, H).

Article Snippet: For mIHC, the following primary antibodies were used: Pkd2l1 (#AB9084, 1:700, Merck Millipore), CD133 (#66666–1-Ig, 1:500, Proteintech), GFAP (#3670, 1:300, Cell Signaling Technology), EGFR (#66863–1-lg, 1:500, Proteintech), and Nestin (Sc-6251, 1:300, Santa Cruz Biotechnology).

Techniques: Expressing, In Vitro, Western Blot, Transgenic Assay, Immunofluorescence, Fluorescence, Double Immunofluorescence Staining, Marker

Journal: Cellular signalling

Article Title: Vegfr3 activation of Pkd2l1 + CSF-cNs triggers the neural stem cell response in spinal cord injury.

doi: 10.1016/j.cellsig.2025.111675

Figure Lengend Snippet: Fig. 3. Role of Vegfr3 in the proliferation and activation of Pkd2l1+ CSF-cNs. (A, B) The overexpression and knockdown efficiencies of Vegfr3 in Pkd2l1+ CSF-cNs were confirmed through qRT-PCR and Western blotting following transfection. (C) Representative images of CSF-cNs and the average diameter of neurospheres with or without Vegfr3 overexpression or inhibition. (D) Representative immu nofluorescence images and quantification of EdU+ Pkd2l1+ CSF-cNs with or without Vegfr3 overexpression or inhibition. (E) Representative immunofluorescence images and quantification of pHH3+ Pkd2l1+ CSF-cNs with or without Vegfr3 overexpression or inhibition. (F) A CCK-8 assay was used to assess the viability of Pkd2l1+ CSF-cNs with either Vegfr3 overexpression or knockdown. (G) Representative Western blots showing the protein levels of EGFR, Ascl, and Sox2 in Pkd2l1+

Article Snippet: For mIHC, the following primary antibodies were used: Pkd2l1 (#AB9084, 1:700, Merck Millipore), CD133 (#66666–1-Ig, 1:500, Proteintech), GFAP (#3670, 1:300, Cell Signaling Technology), EGFR (#66863–1-lg, 1:500, Proteintech), and Nestin (Sc-6251, 1:300, Santa Cruz Biotechnology).

Techniques: Activation Assay, Over Expression, Knockdown, Quantitative RT-PCR, Western Blot, Transfection, Inhibition, Immunofluorescence, CCK-8 Assay