ln 18 gbm cell line  (ATCC)

Journal: Cells

Article Title: Serglycin Cooperates with the Unfolded Protein Response Pathway and Inflammation to Drive Glioblastoma Cell Survival

doi: 10.3390/cells15080660

Figure Lengend Snippet: Loss of SRGN diminishes the expression of UPR markers in GBM cell lines and results in ER stress susceptibility. ( A ) Assessment of the mRNA levels of UPR mediators in LN-18 shSCR and LN-18 shSRGN cells through real-time qPCR. ( B ) Relative mRNA levels of SRGN in LN-18 and U251-MG cell lines. ( C ) SRGN suppression levels in U251-MG siSRGN cells via real-time qPCR and ( D ) phase-contrast microscopy images upon SRGN silencing in U251-MG cells (48 h). ( E ) Relative proliferation rates of U251-MG siControl and U251-MG siSRGN cells after transient SRGN silencing (48 h). ( F ) Evaluation of the gene expression of UPR markers in U251-MG siControl and U251-MG siSRGN cells (48 h). ( G ) IC50 values for LN-18 cell lines upon TM treatment (48 h). ( H ) Wound closure assay in LN-18 cell lines after TM treatment for 24 h and 48 h. Statistically significant differences compared to control are shown by asterisk: * ( p ≤ 0.05).

Article Snippet: The LN-18 GBM cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Microscopy, Gene Expression, Wound Closure Assay, Control

Journal: Cells

Article Title: Serglycin Cooperates with the Unfolded Protein Response Pathway and Inflammation to Drive Glioblastoma Cell Survival

doi: 10.3390/cells15080660

Figure Lengend Snippet: SRGN depletion attenuates the activation profile of UPR in GBM cell lines and renders cells non-responsive to ER stress. ( A ) Western blot analysis of UPR effectors’ protein and phosphorylated levels in TM-treated (48 h) LN-18 cells. ( B ) Immunofluorescence staining for ATF4 (green) and nuclei (blue) depicting the distribution and nuclear accumulation of ATF4 in TM-treated (48 h) LN-18 shSCR cells. ATF4 was not detected in LN-18 shSRGN cells under relative exposure. Scale bar 25 μm. ( C ) Western blot analysis of the basal UPR protein and phosphorylated levels in U251-MG siControl and U251-MG siSRGN cells, following transient SRGN silencing (72 h). All blots are representative of at least three independent experimental repetitions.

Article Snippet: The LN-18 GBM cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Activation Assay, Western Blot, Immunofluorescence, Staining

Journal: Cells

Article Title: Serglycin Cooperates with the Unfolded Protein Response Pathway and Inflammation to Drive Glioblastoma Cell Survival

doi: 10.3390/cells15080660

Figure Lengend Snippet: SRGN-expressing cells possess a pro-survival UPR mechanism and demand global UPR activation. Assessment of LN-18 shSCR and LN-18 shSRGN cells’ viability upon treatment with ( A ) MKC8866 (IRE1 RNase activity inhibitor), ( B ) Kira6 (IRE1 kinase activity inhibitor), ( C ) GSK2606414 (PERK kinase activity inhibitor) and ( D ) Ceapin-A7 (ATF6 TF activity inhibitor) for 24 h incubation at 0.1, 1 and 10 μΜ final concentrations. Statistically significant differences compared to control are shown by bars and asterisk: * ( p ≤ 0.05).

Article Snippet: The LN-18 GBM cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Activation Assay, Activity Assay, Incubation, Control

Journal: Cells

Article Title: Serglycin Cooperates with the Unfolded Protein Response Pathway and Inflammation to Drive Glioblastoma Cell Survival

doi: 10.3390/cells15080660

Figure Lengend Snippet: SRGN suppression provokes an apoptotic phenotype in GBM cells. ( A ) Western blot analysis of the intact and cleaved forms of caspase-3 and PARP-1, as well as the active and total Akt forms in TM-treated (48 h) LN-18 cells. ( B ) caspase-3 and PARP-1 proteolysis assessed through Western blotting in U251-MG cells after transient SRGN knock-down (72 h). ( C ) Zymography assay for determining the active intracellular CTSB isoforms in acidic pH upon TM treatment (48 h) in LN-18 cell lines. Real-time qPCR for evaluating the relative mRNA levels of ( D ) CTSB , ( E ) BAX , ( F ) Bcl-2 and ( G ) Beclin-1 in TM-treated (48 h) LN-18 cell lines. ( H ) Basal mRNA levels of BAX , Bcl-2 and Beclin-1 in U251-MG cells upon SRGN depletion (48 h). All blots and zymography images are representative of at least three independent experimental repetitions. Statistically significant differences compared to control are shown by bars and asterisk: * ( p ≤ 0.05).

Article Snippet: The LN-18 GBM cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Knockdown, Zymography, Control

Journal: Cells

Article Title: Serglycin Cooperates with the Unfolded Protein Response Pathway and Inflammation to Drive Glioblastoma Cell Survival

doi: 10.3390/cells15080660

Figure Lengend Snippet: SRGN orchestrates the inflammatory response in GBM cells. ( A ) Relative mRNA levels of TLRs and TNFRs in LN-18 shSCR and LN-18 shSRGN cell lines assessed through real-time PCR. ( B ) Immunofluorescence staining for P-p65 (green) and nuclei (blue) depicting the distribution and nuclear accumulation of phosphorylated p65 subunit in LN-18 cells at basal conditions. P-p65 was not detected in LN-18 shSRGN cells under relative exposure. Scale bar 25 μm. Real-time qPCR for evaluating the relative mRNA levels of ( C ) TLR2 , ( D ) TLR4 , ( E ) TNFRI , ( F ) TNFRII , ( G ) IL-1β , ( H ) IL-8 and ( I ) CXCL-1 in TM-treated (48 h) LN-18 cell lines. ( J ) Basal mRNA levels of TLR4 and IL-1β in U251-MG cells upon transient SRGN silencing (48 h) assessed through real-time qPCR. ( K ) p65/NF-kB phosphorylated and total forms in TM-treated (48 h) LN-18 cell lines evaluated by Western blotting. All blots are representative of at least three independent experimental repetitions. Statistically significant differences compared to control are shown by bars and asterisk: * ( p ≤ 0.05).

Article Snippet: The LN-18 GBM cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Western Blot, Control