Fig 1: Alendronate reduces the embryonic stem cell signature and activates necrosis in glioblastoma spheres.a DEGs between TS13-20 cells treated with or without alendronate (10 µM) for 7 days were analyzed by RNA sequencing. Genes with expression changes greater than two-fold are shown. DEGs are listed in Supplementary Table S3. b GSEA showed that embryonic stem cell gene signatures were downregulated in alendronate-treated cells. c, d DEGs were analyzed by core analysis using Ingenuity® Pathway Analysis (IPA). c Canonical pathway analysis showed that the most functionally influenced mechanistic network was the complement system. d Downstream effect analysis showed that necrosis- and development-related genes were activated by treatment with alendronate in the disease and functions category. A: cell viability of neurons, B: cell viability, C: neuronal cell death, D–F: apoptosis, G: necrosis, H-J: cellular development, K-M: tissue development, N–O: nervous system development and function, P: embryonic development, Q: organismal development, R: connective tissue development and function. e The network of necrosis-related DEGs induced by treatment with alendronate is shown. f Alendronate caused necrosis-related cell death. TS13-20 cells were treated with staurosporine (0.5 µM, 18 h), hydrogen peroxide (1 mM. 18 h), or alendronate (10 µM, 3 days) and then stained with Hoechst and propidium iodide. Z-stack orthogonal projection images were obtained with an LSM780 confocal microscope and processed using ZEN 2012 analysis software (Carl Zeiss, Germany). g A model linking FDPS with glioblastoma stemness. Glioblastoma TS cells rely on FDPS for maintenance of stemness, and alendronate is a potential candidate drug for glioblastoma treatment
Fig 2: Lower production of FDPS and COL1A1 in murine lungs upon ZA treatment. (A) Mevalonate pathway with key enzymes and intermediates highlighted inset and numbered points indicating PCR measurements (Hmgcs: hydroxymethylglutaryl-CoA synthase; Fdps: farnesyl diphosphate synthase; Dhdds: dehydrodolichol diphosphate synthase; Fdft1: farnesyl-diphosphate farnesyl transferase). (B) Murine lung immunofluorescence analysis showed decreased levels of immunoreactivity for FDPS and collagen 1A1 (COL1A1) in ZA (i.p.)-treated mice compared to bleomycin (Bleo) controls (scale bar = 20 µm), with quantification of green fluorescence. ZA-treated and control samples were compared to bleomycin-treated samples using a one-way ANOVA followed by a Dunnett’s post-hoc test (*p < 0.05; **p < 0.01) and enlarged images of murine airways displaying co-staining of FDPS and COL1A1 (C). (D) Mouse lung homogenate samples analyzed by SDS-PAGE, followed by immunoblotting using rabbit antisera specific to FDPS revealed decreased protein levels within ZA-administered mice. (E) RT-PCR analysis of murine lung lysates reveals decreased levels of Fdft1 and Fdps in ZA-treated samples compared to bleomycin controls. Murine embryonic fibroblast cells stimulated with TGF-ß1 and treated with ZA/PBS for 48 h displayed significantly reduced Fdps levels compared to TGF-ß1 control as confirmed by (F) immunofluorescent staining of TGF-ß1 treated MEF cells (scale bar = 20 µm). (G) FDPS immunoreactivity was measured in human IPF and control resected lung tissues, with IPF tissues showing significantly more immunoreactivity than control tissue (*p = 0.0286) as compared using an unpaired t test with Welch’s correction. Scale bar = 3 mm and 200 µm for inset images.
Fig 3: Intratracheal administration of FDPS siRNA confers protection to bleomycin-challenged mice. (A) Mice were subjected to bleomycin treatment at day 0, administrated FDPS or control siRNA intratracheally on day 15 and sacrificed on day 21. (B) SDS-PAGE followed by western blot analysis using antisera specific for FDPS in mouse lung homogenates confirms significantly decreased levels of FDPS after siRNA administration. Samples were compared using a one-way ANOVA followed by a Dunnett’s post-hoc test (*p < 0.05, **p < 0.01). (C) Measurement of murine weight loss following bleomycin exposure shows a protective effect of FDPS siRNA compared to control siRNA. (D) Bleomycin induced increases in murine lung weights were significantly reduced after FDPS siRNA treatments compared to control siRNA. Comparison between groups were performed using a one-way ANOVA followed by a Dunnett’s post-hoc test (**p < 0.01, ****p < 0.0001). (E) Hydroxyproline levels, which is a measurement of collagen content, was significantly increased in mice treated with bleomycin/control siRNA compared to the FDPS siRNA treated mice. Differences between groups were calculated with a one-way ANOVA followed by a Dunnett’s post-hoc test (***p < 0.001, ****p < 0.0001). (F) Heatmap showing the difference in inflammatory cytokine levels in BALF between treatment groups (blue indicating low value; yellow indicating high value). (G) Bleomycin/control siRNA mice showed increased lung damage, determined by H&E, and more intense collagen staining (picrosirius red). Lungs from mice receiving FDPS siRNA were resembling lungs from control mice regarding both H&E and collagen. (H) Detection and quantification of the fibrotic proteins a-smooth muscle actin (a-SMA) and collagen (COL1A1) using SDS-PAGE followed by western blot revealed a significant increase in both proteins in lungs from bleomycin/control siRNA mice, which was reduced significantly with FDPS siRNA treatment. (I) Immunofluorescence staining of lung sections with anti FDPS antibodies displaying an increase immunoreactivity in bleomycin/control siRNA mice, whereas lungs from mice administered FDPS siRNA were similar to lungs from control mice.
Fig 4: Qki regulates transcription of the genes involved in myelin cholesterol biosynthesis.(A) Bar graph showing the five canonical pathways most affected by Qki on the basis of differentially expressed genes in Qk-Plp-iCKO mice and control mice (n = 2 mice/group). Blue and red indicate pathways whose activity decreased or increased, respectively, in Qk-Plp-iCKO mice. (B) Overlapping canonical pathway networks for the top 20 canonical pathways with a minimum of three common molecules in different pathways. GGPP: geranylgeranyl diphosphate. (C) Bar graph showing the 10 upstream regulators most enriched in Qk-Plp-iCKO mice. (D) Schema of the cholesterol biosynthesis pathway. (E) Quantification of expression of representative enzymes involved in cholesterol biosynthesis in the corpus callosum tissues in Qk-Nestin-iCKO mice and control mice 2 weeks after tamoxifen injection according to real-time qPCR (n = 4 mice/group). (F, G) Representative images of and quantification of immunofluorescent staining of Hmgcs1 (F) and Fdps (G) in Aspa+Qki- oligodendrocytes in the corpus callosum of Qk-Nestin-iCKO mice (n = 3) and Aspa+Qki+ oligodendrocytes in the corpus callosum of control mice (n = 4) 2 weeks after tamoxifen injection. Scale bars, 50 µm. (H) Immunoblots of and quantification of the levels of expression of the representative enzymes involved in cholesterol biosynthesis in the corpus callosum tissues in Qk-Nestin-iCKO mice and control mice 2 weeks after tamoxifen injection (n = 3 mice/group). (I) Quantification of the cholesterol levels in the corpus callosum tissues in Qk-Nestin-iCKO mice (n = 6) and control mice (n = 5) 2 weeks after tamoxifen injection. Data are shown as mean ± s.d. and were analyzed using Student's t test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.Figure 5—source data 1.Exact p-values for statistical analysis.
Fig 5: Knockdown of farnesyl diphosphate synthase (FDPS) suppresses glioblastoma sphere formation.a The protein levels of FDPS between TS13-20 and TS13-18 cell spheres (D0) and differentiated counterparts (D7) were analyzed by western blot. ACTB was used as the loading control. b TS13-20 cells were infected with lentivirus harboring short hairpin RNAs (shRNAs) against FDPS. Two days after infection, the infected cells were selected for 2 days and then maintained an additional 4 days. Knockdown of the FDPS protein was assessed by immunofluorescence staining and confocal microscopy. Images were obtained at ×20 using an LSM510 META or LSM780 confocal microscope (Carl Zeiss, Jena, Germany). c Knockdown of FDPS inhibited secondary sphere formation. The same number of control and FDPS knockdown cells were subjected to secondary sphere formation. Bright field images were obtained using a Cytation 3 microplate reader (Bio-Tek). d FDPS gene expression was significantly upregulated in glioblastoma cells compared with normal cells and in samples with a dead status at 3 years compared with samples with a live status. Three independent microarray-based data sets were median centered and normalized to the unit standard deviation to compare the gene expression level according to the P-value
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