Journal: bioRxiv

Article Title: Laminin-511 and α6 Integrins Regulate the Expression of CXCR4 to Promote Endothelial Morphogenesis

doi: 10.1101/846022

Figure Lengend Snippet: (A-C) RNA was isolated from 6-day bead sprouts and analyzed by qPCR. (A) Shown is the efficiency of α6 depletion and the effects of α6 depletion on the expression of the LM-α4, LM-α5, CXCR4 and ANGPT2 normalized to non-targeting control. (B) Shown is the efficiency of depletion of the LM-α5 chain and the effects of this depletion on the expression of CXCR4 and ANGPT2. (C) Shown is the efficiently of depletion of the LM-α4 chain and the effects of this depletion of the expression of CXCR4 and ANGPT2. n = 3, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Article Snippet: Lentiviruses were produced by co-transfection of HEK293FT cells with the CXCR4 expression vector together with the packaging plasmid, psPAX2, coding for Gag, Pol, Rev, Tat (#12260, Addgene), and the envelope plasmid, pMD2.G, coding for VSV-G (#12259, Addgene).

Techniques: Isolation, Expressing

Journal: bioRxiv

Article Title: Laminin-511 and α6 Integrins Regulate the Expression of CXCR4 to Promote Endothelial Morphogenesis

doi: 10.1101/846022

Figure Lengend Snippet: (A) A dose-curve response using the specific CXCR4 inhibitor, AMD3100, shows a dose-dependent reduction in both number of endothelial sprouts, as well as sprout length in 6-day bead sprout assays. Left panel - Plotted is the number of sprouts on individual beads from 3 fields containing 7-10 beads from each of two independent experiments. Right panel-plotted is the length of individual sprouts on these same beads. Data was analyzed by one-way ANOVA and plotted as the mean length ± s.d. (B) AMD3100 inhibition of CXCR4 in 6-day planar co-culture causes defective morphogenesis in a dose-dependent manner. Six fields were analyzed in each of two independent experiments. Data was analyzed by one-way ANOVA and plotted as the mean length ± s.d. (C) The efficiency of CXCR4 depletion using 2 siRNA targeting sequences determined by qPCR. (D) Average length of tubes/field analyzed from 3 independent experiments with 6 randomly selected fields from each (Left panel). Individual sprout lengths were measured from 3 independent experiments with 10 randomly selected fields each, averaging 6-8 beads per field (Right panel). Data was analyzed by two-tailed Student’s t-test. (E) Immunofluorescence staining of 2-day planar co-cultures showing CXCR4 expression in non-targeting (NT), CXCR4-targeting and α6-targeting endothelial cells. Endothelial cells were stained with CD31 (red). Scale = 40 µm. (F) Expression of CXCR4 in endothelial cells transduced with empty vector (EV) or rCXCR4-expressing vector was measured by flow cytometry. (G) The efficiency of siRNA-mediated knockdown of α6 (Left Panel) and the RNA expression of CXCR4 (Right Panel) in CXCR4- and Empty Vector (EV)-expressing cells was assayed by qPCR for each of the three independent experiments. (H) Plotted are the lengths of individual tubes from 4-day planar co-cultures. Data was from 3 independent experiments with 5-6 randomly selected fields per experiment and analyzed by one-way ANOVA. ns = no significance, ****p ≤ 0.0001.

Article Snippet: Lentiviruses were produced by co-transfection of HEK293FT cells with the CXCR4 expression vector together with the packaging plasmid, psPAX2, coding for Gag, Pol, Rev, Tat (#12260, Addgene), and the envelope plasmid, pMD2.G, coding for VSV-G (#12259, Addgene).

Techniques: Inhibition, Co-Culture Assay, Two Tailed Test, Immunofluorescence, Staining, Expressing, Transduction, Plasmid Preparation, Flow Cytometry, RNA Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Complexity of progranulin mechanisms of action in mesothelioma

doi: 10.1186/s13046-022-02546-4

Figure Lengend Snippet: Progranulin promotes AKT and MAPK activation in mesothelioma cells. A Progranulin (PGRN) protein levels were analyzed by immunoblot in cell lysates and media conditioned from MeT-5A, NCI-H2052, NCI-H2452, NCI-H28 and MSTO-211H cells. B Progranulin levels in media conditioned from MeT-5A, NCI-H2052, NCI-H2452, NCI-H28 and MSTO-211H cells was measured by ELISA assay. N = 3, ± SD, ** p < 0.01, *** p < 0.001. C Levels of total and phosphorylated EphA2 (S897 and S901) were analyzed by immunoblot in cell lysates derived from MeT-5A, NCI-H2052, NCI-H2452, NCI-H28 and MSTO-211H cells serum-starved for 24 h. D Levels of total and phosphorylated EphA2 (S897), AKT and ERK1/2 were assessed by immunoblot in NCI-H2052, NCI-H2452 and MSTO-211H cells serum-starved for 24 h and then treated with 50 nM progranulin for the indicated time. E MSTO-211H cells were transfected with siRNA targeting progranulin or non-targeting control siRNA. At 8 h post-transfection cells were transferred into serum-free medium for 40 h and then analyzed by immunoblot for total and phosphorylated levels of EphA2 (S897), AKT, ERK1/2 and progranulin levels. F Levels of total and phosphorylated EphA2, AKT, ERK1/2 and progranulin were assessed by immunoblot in parental MSTO-211H, MSTO-211H cells with progranulin knock-out by CRISPR/Cas9 ( GRN KO) and GRN KO MSTO-211H cells with reconstituted progranulin expression (left panel) or parental and progranulin overexpressing NCI-H2052 cells (right panel), serum-starved for 24 h. Progranulin levels were also analyzed in conditioned media from cells starved for 48 h. G MSTO-211H and NCI-H2052 cells were serum-starved for 24 h, pre-incubated with either the MEK1/2 inhibitor U0126 (10 µM) or the AKT inhibitor AKTi VIII (5 µM) for 2 h and then treated with the same concentrations of MEK1/2 and AKT inhibitors alone or in combination with 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2, AKT and ERK1/2 were determined by immunoblot

Article Snippet: To generate retroviral particles expressing wild type or mutants EphA2, HEK-293FT cells were transfected with the pMD2-G envelope plasmid, the pUMVC packaging plasmid (a gift of Bob Weinberg, Addgene plasmid #8449) and the pCLXSN retroviral vectors containing the cDNA coding for wild type or mutants EphA2.

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay, Transfection, Control, Knock-Out, CRISPR, Expressing, Incubation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Complexity of progranulin mechanisms of action in mesothelioma

doi: 10.1186/s13046-022-02546-4

Figure Lengend Snippet: Progranulin and EphA2 differ in their ability to modulate mesothelioma cell motility and adhesion. A Migration of parental, GRN KO and progranulin-re-expressing GRN KO MSTO-211H cells was assessed using transwells as described in Material and Methods. B Migration of parental, GRN KO and EphA2 KO MSTO-211H cells as assessed by transwells migration. C Invasion of parental, GRN KO and EphA2 KO MSTO-211H cells was assessed using matrigel-coated transwells. D The ability of parental, GRN KO and EphA2 KO MSTO-211H cells to adhere to plasma fibronectin, collagen and poly-L-Lys was assessed as described in Material and Methods. E The migratory ability of parental and progranulin overexpressing NCI-H2052 cells was assessed using transwells. F Transwell migration of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. G Invasion through matrigel-coated transwells of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. For motility and adhesion assays, data are the average of three independent experiments ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05. H Parental, GRN KO and EphA2 KO MSTO-211H cells were subcutaneously implanted in Rag 2-/- mice and tumor volumes measured at the indicated time post tumor injection. N = 8, ± SD, * p < 0.05, ** p < 0.01

Article Snippet: To generate retroviral particles expressing wild type or mutants EphA2, HEK-293FT cells were transfected with the pMD2-G envelope plasmid, the pUMVC packaging plasmid (a gift of Bob Weinberg, Addgene plasmid #8449) and the pCLXSN retroviral vectors containing the cDNA coding for wild type or mutants EphA2.

Techniques: Migration, Expressing, Clinical Proteomics, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Complexity of progranulin mechanisms of action in mesothelioma

doi: 10.1186/s13046-022-02546-4

Figure Lengend Snippet: Progranulin activates AKT and ERK1/2 in an EphA2-independent manner in mesothelioma cells. A MSTO-211H cells were transfected with siRNA targeting either GRN (progranulin), EphA2 , the combination of the two or non-targeting control (siCTR). At 8 h post-transfection cells were transferred to serum-free media and incubated for additional 40 h. Cells were then treated with 50 nM progranulin for 60 min. Levels of progranulin, total and phosphorylated EphA2 (S897), AKT and ERK1/2 were analyzed by immunoblot. B GRN was depleted using siRNAs targeting progranulin as described in ( A ) in parental MSTO-211H (P), EphA2 KO MSTO-211H and EphA2 KO cells re-expressing wild type EphA2 or the EphA2 mutants EphA2 K646M and EphA2 S897A/S899A/S901A. Levels of progranulin, total and phosphorylated EphA2 (S897), AKT and ERK1/2 were determined by immunoblot. C Total and phosphorylated EphA2 (S897), AKT and ERK1/2 as assessed by immunoblot in EphA2 KO MSTO-211H cells stably transfected with an empty vector or re-expressing wild-type EphA2, serum-starved for 24 h and exposed to 50 nM progranulin for the indicated time. D Parental (P) NCI-H2052 cells, two different EphA2 KO NCI-H2052 clones and a non-targeting control (NTC) NCI-H2052 clone were serum-starved for 24 h and then stimulated with 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2, AKT and ERK1/2 were analyzed by immunoblot

Article Snippet: To generate retroviral particles expressing wild type or mutants EphA2, HEK-293FT cells were transfected with the pMD2-G envelope plasmid, the pUMVC packaging plasmid (a gift of Bob Weinberg, Addgene plasmid #8449) and the pCLXSN retroviral vectors containing the cDNA coding for wild type or mutants EphA2.

Techniques: Transfection, Control, Incubation, Western Blot, Expressing, Stable Transfection, Plasmid Preparation, Clone Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Complexity of progranulin mechanisms of action in mesothelioma

doi: 10.1186/s13046-022-02546-4

Figure Lengend Snippet: FAK mediates progranulin-dependent activation of AKT and ERK1/2 in mesothelioma cells. A , B Parental and EphA2 KO MSTO-211H ( A ) and NCI-H2052 ( B ) cells were serum-starved for 24 h, pre-incubated with the FAK inhibitor PF-573228 (5 µM) for 1 h and then treated with the same concentration of PF-573228 alone or in combination with 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2 (S897), AKT, ERK1/2 and FAK (Y397) were assessed by immunoblot. C NCI-H2052 cells were transfected with siRNA targeting FAK ( PTK2 ) or non-targeting control (siCTR) siRNA. At 48 h post-transfection cells were transferred onto serum-free medium and 24 h later exposed to 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2 (S897), AKT, ERK1/2 and of FAK as assessed by immunoblot

Article Snippet: To generate retroviral particles expressing wild type or mutants EphA2, HEK-293FT cells were transfected with the pMD2-G envelope plasmid, the pUMVC packaging plasmid (a gift of Bob Weinberg, Addgene plasmid #8449) and the pCLXSN retroviral vectors containing the cDNA coding for wild type or mutants EphA2.

Techniques: Activation Assay, Incubation, Concentration Assay, Western Blot, Transfection, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Complexity of progranulin mechanisms of action in mesothelioma

doi: 10.1186/s13046-022-02546-4

Figure Lengend Snippet: EGFR and RYK sustain progranulin signaling in mesothelioma cells. A Total and phosphorylated EphA2 (S897), AKT and ERK1/2 in EphA2 KO MSTO-211H cells stably transduced with an empty vector or re-expressing wild type EphA2 (left panel) and NCI-H2052 cells (right panel) pre-incubated with the EGFR inhibitor gefitinib (10 µM) for 1 h and then exposed to the same concentration of gefitinib alone or in combination with 50 nM progranulin for 15 min. B Total and phosphorylated EGFR (Y1068) in MSTO-211H cells transfected with GRN -specific or non-targeting control (siCTR) siRNA (upper left panel), parental and PGRN overexpressing NCI-H2052 cells (upper right panel) and parental, GRN KO or PGRN-reconstituted GRN KO MSTO-211H cells (lower panel) after serum-starvation for 24 h. C Levels of total and phosphorylated EGFR (Y1068), EphA2 (S897) and AKT in parental or GRN KO MSTO-211H cells serum-starved for 24 h and treated with gefitinib (10 µM) for 1 h 30 min. D Levels of EphA7, total and phosphorylated EphA2 (S897), AKT and ERK1/2 in MSTO-211H cells transfected with EphA7 -specific or control (siCTR) siRNA, transferred onto serum-free 24 h post transfection, incubated for additional 24 h and then treated with 50 nM progranulin for 15 min. E MSTO-211H and NCI-H2052 cells were transfected with siRNA specific for RYK or non-targeting control (siCTR) siRNAs. At 48 h post-transfection cells were transferred onto serum-free medium and incubated for 24 h. Cells were then treated with 50 nM PGRN for 15 min. Total and phosphorylated EphA2 (S897), AKT and ERK1/2 were analyzed by immunoblot. F-G Total and phosphorylated AKT and ERK1/2 in NCI-H2052 transfected with si RYK or control siRNA (siCTR) ( F ) or stably expressing two different RYK- specific or a non-targeting control (shCTR) shRNA treated with 10 µM gefitinib for 1 h and then exposed to the same concentration of gefitinib alone or in combination with 50 nM progranulin for 15 min

Article Snippet: To generate retroviral particles expressing wild type or mutants EphA2, HEK-293FT cells were transfected with the pMD2-G envelope plasmid, the pUMVC packaging plasmid (a gift of Bob Weinberg, Addgene plasmid #8449) and the pCLXSN retroviral vectors containing the cDNA coding for wild type or mutants EphA2.

Techniques: Stable Transfection, Transduction, Plasmid Preparation, Expressing, Incubation, Concentration Assay, Transfection, Control, Western Blot, shRNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Complexity of progranulin mechanisms of action in mesothelioma

doi: 10.1186/s13046-022-02546-4

Figure Lengend Snippet: Progranulin modulates pFAK Y397 levels in a RYK-dependent manner and modulates focal adhesion turn over. A Parental (P), EphA2 KO and EphA2 KO MSTO-211H cells stably transfected with an empty vector, re-expressing wild type EphA2, or EphA2 mutants were transfected with siRNAs targeting progranulin (si GRN ) or non-targeting (siCTR) control. At 8 h post-transfection cells were serum-starved and total and phosphorylated FAK (Y397) were analyzed by immunoblot 48 h post-transfection. B Phosphorylated FAK (Y397) levels in parental, GRN KO, GRN KO MSTO-211H cells with reconstituted progranulin expression (left panel) or parental and PGRN overexpressing NCI-H2052 cells (right panel) serum-starved for 24 h. C Phosphorylated FAK (Y397) in si RYK - or siControl-transfected (siCTR) MSTO-211H and NCI-H2052 cells serum-starved for 24 h, pre-incubated with gefitinib (10 µM) for 1 h and then treated with gefitinib alone or in combination with 50 nM progranulin for 15 min. D Levels of total and phosphorylated FAK (Y397) in EphA2 KO MSTO-211H cells transfected with siRNA targeting RYK or control (siCTR) siRNA, serum-starved for 24 h and stimulated with 50 nM progranulin for 15 min. E Levels of total and phosphorylated FAK (Y397) in MSTO-211H cells transfected with either siRNAs targeting progranulin, RYK , their combination or non-targeting controls (siCTR) and serum-starved for 40 h. F Levels of total and phosphorylated FAK (Y397) in parental and GRN KO MSTO-211H cells transfected with si RYK or control (siCTR) siRNA and serum-starved for 24 h. G-I Parental and GRN KO MSTO-211H cells ( G ), parental MSTO-211H cells 24 h post transfection with siRNA targeting progranulin (si GRN ) or control ( siCTR) siRNA (H), and GRN KO MSTO-211H cells 48 h post transfection with RYK -specific siRNA or control (siCTR) siRNA ( I ) were serum-starved for 24 h and then treated with 10 µM nocodazole (NCZ) for 4 h. Nocodazole was then washed-out and the levels of phosphorylated FAK (Y397) were analyzed by immunoblot in cell lysates at the indicated times after nocodazole washout

Article Snippet: To generate retroviral particles expressing wild type or mutants EphA2, HEK-293FT cells were transfected with the pMD2-G envelope plasmid, the pUMVC packaging plasmid (a gift of Bob Weinberg, Addgene plasmid #8449) and the pCLXSN retroviral vectors containing the cDNA coding for wild type or mutants EphA2.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Control, Western Blot, Incubation