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MedChemExpress mlt 748
Mlt 748, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mlt+748/bio_rxiv__2024__09__26__614808-292-10-16?v=MedChemExpress
Average 92 stars, based on 3 article reviews

mlt 748 - by Bioz Stars, 2026-07

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(A) Workflow of the dual approach to characterize the effects of MALT1 inhibition in patient-derived glioblastoma stem-like cells (GSCs). Patient-derived GSC#9 were treated with vehicle (DMSO) and the MALT1 inhibitor mepazine (MPZ, 20µM, 4h) and analyzed by RNA sequencing. Similarly, control and MPZ (20µM, 6h) treated GSC#9 were processed for proteome-wide label-free quantification (LFQ) analysis. (B) (Top) REAC enrichment analysis of the top upregulated pathways from RNAseq analysis of DMSO versus MPZ treated GSC#9. Upregulated genes (fold change > 1.5) upon MPZ treatment were analyzed. (Bottom) Gene set enrichment analysis (GSEA) plot of ‘’reactome cholesterol biosynthesis’’ and ‘’Horton SREBF targets’’ signatures. (C) Differentially upregulated proteins from DMSO versus MPZ-treated GSC#9 were analyzed with the Pantherdb pathway browser. Four main GO term signatures were identified as follows: 1. actin (1.a: actin filament network formation, 1.b: actin filament bundle assembly, 1.c: actin filament bundle organization, 1.d: positive regulation of actin filament polymerization), 2. spindle (2.a mitotic spindle organization, 2.b: mitotic spindle organization, 2.c: microtubule cytoskeleton organization involved in mitosis, 2.d: spindle assembly, 2.e: spindle organization), 3. lipid (3.a fatty acid oxidation, 3.b lipid oxidation), and, 4. RNA (4.a transcription elongation from RNA polymerase II promoter). (D) Heatmap of cholesterol synthesis gene expression (RNA, purple) and protein level (protein, blue) from GSC#9 treated as described in (A). The enzymes involved in the cholesterol synthesis pathway (black) with intermediate metabolites (purple) are shown on the left. RNA and protein selected hits from the RNAseq and proteome analysis are shown in log 2 fold change. Non-identified genes and proteins are represented with a cross. Data are presented as the log 2 fold change of MPZ vs DMSO treated GSC#9. RNAseq n=3, LFQ n=4. (E) qRT-PCR analysis of the indicated targets in GSC#9 treated for 4h with DMSO and MALT1 inhibitors (MPZ, 20µM and MLT748, 5µM). Alternatively, cells received non-silencing RNA duplexes (si ctl ) and 2 independent duplexes targeting MALT1 (si.2 MALT1 and si.3 MALT1 ) for 3 days. Data were normalized to 2 housekeeping genes (HPRT1, ACTB) and are presented as the mean + s.d. of 3 independent biological replicates. (F) qRT-PCR analysis of HSD17B7 in GSC#4 and GSC#6 treated as described in (E). Data were processed as described in (E). (G) GSC#9 were transfected with luciferase reporter plasmids for either wild-type SREBP2 promoter activity (SRE WT ) or a mutated version (SRE Mut ), to which SREBP2 cannot bind, in combination with Renilla under the control of neutral HSV-thymidine kinase promoter. GSC#9 were next treated for 16h with DMSO, MPZ (20µM), and MLT748 (5µM). Luminescence values were calculated as the ratio SRE WT /SRE Mut , and further normalized to Renilla intensities. Data are presented as the mean + s.d. of 3 independent biological replicates. (H) qRT-PCR analysis of LDLR in GSC#9, GSC#4, and GSC#6, treated as described in (E). Data were processed as described in (E). (I) Western-blot analysis of the levels of SREBP2 and LDLR in GSC#9, GSC#4, and GSC#6, treated for 3h and 24h, respectively, with DMSO and MPZ (20µM). GAPDH served as a loading control. FL (full length) and cleaved SREBP2 forms are indicated with green and red arrowheads, respectively. All panels are representative of at least n=3, unless otherwise specified. t-test and ANOVA, *p<0.05, **p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping

doi: 10.1101/2023.02.27.530259

Figure Lengend Snippet: (A) Workflow of the dual approach to characterize the effects of MALT1 inhibition in patient-derived glioblastoma stem-like cells (GSCs). Patient-derived GSC#9 were treated with vehicle (DMSO) and the MALT1 inhibitor mepazine (MPZ, 20µM, 4h) and analyzed by RNA sequencing. Similarly, control and MPZ (20µM, 6h) treated GSC#9 were processed for proteome-wide label-free quantification (LFQ) analysis. (B) (Top) REAC enrichment analysis of the top upregulated pathways from RNAseq analysis of DMSO versus MPZ treated GSC#9. Upregulated genes (fold change > 1.5) upon MPZ treatment were analyzed. (Bottom) Gene set enrichment analysis (GSEA) plot of ‘’reactome cholesterol biosynthesis’’ and ‘’Horton SREBF targets’’ signatures. (C) Differentially upregulated proteins from DMSO versus MPZ-treated GSC#9 were analyzed with the Pantherdb pathway browser. Four main GO term signatures were identified as follows: 1. actin (1.a: actin filament network formation, 1.b: actin filament bundle assembly, 1.c: actin filament bundle organization, 1.d: positive regulation of actin filament polymerization), 2. spindle (2.a mitotic spindle organization, 2.b: mitotic spindle organization, 2.c: microtubule cytoskeleton organization involved in mitosis, 2.d: spindle assembly, 2.e: spindle organization), 3. lipid (3.a fatty acid oxidation, 3.b lipid oxidation), and, 4. RNA (4.a transcription elongation from RNA polymerase II promoter). (D) Heatmap of cholesterol synthesis gene expression (RNA, purple) and protein level (protein, blue) from GSC#9 treated as described in (A). The enzymes involved in the cholesterol synthesis pathway (black) with intermediate metabolites (purple) are shown on the left. RNA and protein selected hits from the RNAseq and proteome analysis are shown in log 2 fold change. Non-identified genes and proteins are represented with a cross. Data are presented as the log 2 fold change of MPZ vs DMSO treated GSC#9. RNAseq n=3, LFQ n=4. (E) qRT-PCR analysis of the indicated targets in GSC#9 treated for 4h with DMSO and MALT1 inhibitors (MPZ, 20µM and MLT748, 5µM). Alternatively, cells received non-silencing RNA duplexes (si ctl ) and 2 independent duplexes targeting MALT1 (si.2 MALT1 and si.3 MALT1 ) for 3 days. Data were normalized to 2 housekeeping genes (HPRT1, ACTB) and are presented as the mean + s.d. of 3 independent biological replicates. (F) qRT-PCR analysis of HSD17B7 in GSC#4 and GSC#6 treated as described in (E). Data were processed as described in (E). (G) GSC#9 were transfected with luciferase reporter plasmids for either wild-type SREBP2 promoter activity (SRE WT ) or a mutated version (SRE Mut ), to which SREBP2 cannot bind, in combination with Renilla under the control of neutral HSV-thymidine kinase promoter. GSC#9 were next treated for 16h with DMSO, MPZ (20µM), and MLT748 (5µM). Luminescence values were calculated as the ratio SRE WT /SRE Mut , and further normalized to Renilla intensities. Data are presented as the mean + s.d. of 3 independent biological replicates. (H) qRT-PCR analysis of LDLR in GSC#9, GSC#4, and GSC#6, treated as described in (E). Data were processed as described in (E). (I) Western-blot analysis of the levels of SREBP2 and LDLR in GSC#9, GSC#4, and GSC#6, treated for 3h and 24h, respectively, with DMSO and MPZ (20µM). GAPDH served as a loading control. FL (full length) and cleaved SREBP2 forms are indicated with green and red arrowheads, respectively. All panels are representative of at least n=3, unless otherwise specified. t-test and ANOVA, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The following chemical compounds were used: mepazine (Chembridge, 5216177), MLT748 (Selleckchem, S8898), U18666A (Selleckchem, S9669), cerivastatin (Sigma, SML00005), LLOMe (Sigma, L7393), clemastine (Selleckchem, S1847), and raptinal (Sigma, SML-1745).

Techniques: Inhibition, Derivative Assay, RNA Sequencing, Control, Quantitative Proteomics, Gene Expression, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay, Western Blot

(A) Schematic representation of the different pathways which control the intracellular level of cholesterol via i) capture of cholesterol-filled LDL upon binding to the LDLR and trafficking through the endocytic/endosomal pathway, ii) de novo cholesterol synthesis into the mevalonate pathway, iii) handling the pool of free cholesterol via storage into lipid droplets, and, iv) export through different transporters such as ABCA1. (B) Total cholesterol level in GSC#9 transfected with non-silencing RNA duplexes (si ctl ) and 2 independent duplexes targeting MALT1 (si.2 MALT1 and si.3 MALT1 ) for 3 days. Data are expressed as the ratio cholesterol/proteins (µM/µg) and presented as the mean + s.d. of 3 independent biological replicates. (C) Flow cytometry analysis of the filipin III cholesterol probe in GSC#9 treated with vehicle (DMSO) and MALT1 Inhibitors (mepazine, MPZ, 20µM, and MLT748, 5µM) for 24h. Alternatively, cells received si ctl , si.2 MALT1 , and si.3 MALT1 for 3 days. Data are presented as the mean + s.d. of 3 independent biological replicates. (D) (Left) Confocal analysis of the filipin III cholesterol probe (gray) in GSC#9 treated as in (C). Scale bar: 10µm. (Right) Violin representation of the quantification of filipin III signal intensity per cell. n=38. (E) (Left) Confocal analysis of dil-LDL uptake (5µg/mL, 2h, red) in GSC#9 treated as in (C) for 16h. Nuclei are shown in blue (DAPI). Scale bar: 10µm. (Right) Violin representation of the quantification of dil-LDL signal intensity per cell. n>70. All panels are representative of at least n=3, unless otherwise specified. t-test and ANOVA, *p<0.05, **p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping

doi: 10.1101/2023.02.27.530259

Figure Lengend Snippet: (A) Schematic representation of the different pathways which control the intracellular level of cholesterol via i) capture of cholesterol-filled LDL upon binding to the LDLR and trafficking through the endocytic/endosomal pathway, ii) de novo cholesterol synthesis into the mevalonate pathway, iii) handling the pool of free cholesterol via storage into lipid droplets, and, iv) export through different transporters such as ABCA1. (B) Total cholesterol level in GSC#9 transfected with non-silencing RNA duplexes (si ctl ) and 2 independent duplexes targeting MALT1 (si.2 MALT1 and si.3 MALT1 ) for 3 days. Data are expressed as the ratio cholesterol/proteins (µM/µg) and presented as the mean + s.d. of 3 independent biological replicates. (C) Flow cytometry analysis of the filipin III cholesterol probe in GSC#9 treated with vehicle (DMSO) and MALT1 Inhibitors (mepazine, MPZ, 20µM, and MLT748, 5µM) for 24h. Alternatively, cells received si ctl , si.2 MALT1 , and si.3 MALT1 for 3 days. Data are presented as the mean + s.d. of 3 independent biological replicates. (D) (Left) Confocal analysis of the filipin III cholesterol probe (gray) in GSC#9 treated as in (C). Scale bar: 10µm. (Right) Violin representation of the quantification of filipin III signal intensity per cell. n=38. (E) (Left) Confocal analysis of dil-LDL uptake (5µg/mL, 2h, red) in GSC#9 treated as in (C) for 16h. Nuclei are shown in blue (DAPI). Scale bar: 10µm. (Right) Violin representation of the quantification of dil-LDL signal intensity per cell. n>70. All panels are representative of at least n=3, unless otherwise specified. t-test and ANOVA, *p<0.05, **p<0.01, ***p<0.001.

Techniques: Control, Binding Assay, Transfection, Flow Cytometry

(A) Cell viability was measured using CellTiter-Glo assay in GSC#4, GSC#6, and GSC#9 that were pre-treated for 1h with vehicle (DMSO) and mepazine (MPZ, 20µM), and then further challenged for 48h with vehicle (H 2 O), MβCD (0.1%), and either cholesterol alone (50 and 250µM) or in complex with Mβ . Data are presented as the mean + s.d. of at least 3 independent biological replicates. Stars refer to the comparison with the MPZ pretreated and vehicle-challenged condition. (B) (Left) Schematic representation of the cell death processes induced by two lysosomal-destabilizing drugs (LLOMe and clemastine), and by the mitochondrial-mediated intrinsic apoptosis activator (raptinal). (Right) Cell viability was measured using CellTiter-Glo assay in GSC#9 that were pre-treated for 1h with DMSO, LLOMe (1µM), clemastine (20µM), and raptinal (2µM), and then further challenged for 48h with H 2 O, Mβ D (0.1%), and either cholesterol alone (50 and 250µM) or in complex with MβCD. Data are presented as the mean + s.d. of 5 independent biological replicates. (C) Western-blot analysis of the level of SREBP2 in GSC#9 that were pre-treated for 1h with DMSO and MALT1 inhibitors (MPZ,20µM, and MLT748, 5µM), and then further challenged for 3h with vehicle (H 2 O) and with MβCD-complexed cholesterol (chol/MβCD, 250µM). GAPDH served as a loading control. FL (full length) and cleaved SREBP2 forms are indicated with green and red arrowheads, respectively. (D) (Left) Western-blot analysis of the levels of LAMP2, P62, and LC3B in GSC#9 treated as described in (C) for 24h. GAPDH served as a loading control. Unlipidated (LC3B-I) and lipidated (LC3B-II) LC3B forms are indicated with red and green arrowheads, respectively. (Right) Densitometric analysis of the level of P62 and LC3B-II (lipidated form) normalized to GAPDH. Data are presented as the mean + s.d. of 5 independent experiments. (E) (Left) Confocal analysis of P62 staining (red) in GSC#9 treated as described in (D). Nuclei are shown in blue (DAPI). Scale bar: 10µm. (Right) Violin representation of the quantification of P62 punctae per cell. n>39. All panels are representative of at least n=3, unless otherwise specified. t-test and ANOVA, *p<0.05, **p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping

doi: 10.1101/2023.02.27.530259

Figure Lengend Snippet: (A) Cell viability was measured using CellTiter-Glo assay in GSC#4, GSC#6, and GSC#9 that were pre-treated for 1h with vehicle (DMSO) and mepazine (MPZ, 20µM), and then further challenged for 48h with vehicle (H 2 O), MβCD (0.1%), and either cholesterol alone (50 and 250µM) or in complex with Mβ . Data are presented as the mean + s.d. of at least 3 independent biological replicates. Stars refer to the comparison with the MPZ pretreated and vehicle-challenged condition. (B) (Left) Schematic representation of the cell death processes induced by two lysosomal-destabilizing drugs (LLOMe and clemastine), and by the mitochondrial-mediated intrinsic apoptosis activator (raptinal). (Right) Cell viability was measured using CellTiter-Glo assay in GSC#9 that were pre-treated for 1h with DMSO, LLOMe (1µM), clemastine (20µM), and raptinal (2µM), and then further challenged for 48h with H 2 O, Mβ D (0.1%), and either cholesterol alone (50 and 250µM) or in complex with MβCD. Data are presented as the mean + s.d. of 5 independent biological replicates. (C) Western-blot analysis of the level of SREBP2 in GSC#9 that were pre-treated for 1h with DMSO and MALT1 inhibitors (MPZ,20µM, and MLT748, 5µM), and then further challenged for 3h with vehicle (H 2 O) and with MβCD-complexed cholesterol (chol/MβCD, 250µM). GAPDH served as a loading control. FL (full length) and cleaved SREBP2 forms are indicated with green and red arrowheads, respectively. (D) (Left) Western-blot analysis of the levels of LAMP2, P62, and LC3B in GSC#9 treated as described in (C) for 24h. GAPDH served as a loading control. Unlipidated (LC3B-I) and lipidated (LC3B-II) LC3B forms are indicated with red and green arrowheads, respectively. (Right) Densitometric analysis of the level of P62 and LC3B-II (lipidated form) normalized to GAPDH. Data are presented as the mean + s.d. of 5 independent experiments. (E) (Left) Confocal analysis of P62 staining (red) in GSC#9 treated as described in (D). Nuclei are shown in blue (DAPI). Scale bar: 10µm. (Right) Violin representation of the quantification of P62 punctae per cell. n>39. All panels are representative of at least n=3, unless otherwise specified. t-test and ANOVA, *p<0.05, **p<0.01, ***p<0.001.

Techniques: Glo Assay, Comparison, Western Blot, Control, Staining

$(A) Workflow of the Lyso-IP approach to characterize the effects of MALT1 inhibition on the lysosomal proteome in patient-derived GSCs. GSC#9 stably expressing the lysosomal protein TMEM192 tagged with either 3xHA (HA-lyso) or 2xFLAG (FLAG-lyso) were mechanically lysed. Lysosomes were immunoprecipitated with anti-HA magnetic beads. These immunopurified lysosome fractions were subsequently analyzed by western-blot and label-free quantification (LFQ) proteomic. Lysosome-associated proteins were defined as the hits with ≥1.5 fold change (HA/Flag) and p-value ≤ 05. (B) Western-blot analysis of the levels of TMEM192-3xHA (HA) and TMEM192-2xFLAG (FLAG) in lentivirally infected GSC#9. Tubulin served as a loading control. (C) Confocal analysis of TMEM192-3xHA (HA) staining (red) and LAMP2 staining (green) in HA-lyso GSC#9. Nuclei are shown in blue (DAPI). Scale bar: 10µm. (D) Western-blot analysis of the levels of LAMP2, CTSD (PP: pre-pro, m: mature), TMEM192-3xHA (HA), EEA1, CALRETICULIN (CalR), GM130, and VDAC in flag-lyso and HA-lyso GSC#9 immunopurified lysosomes (lyso, light purple square) and whole cell lysate (WCL, dark purple square). Organelles to which belong the proteins are indicated on the right. (E) Comparison of the GO:CC enrichment analysis of organelles (Golgi, peroxisomes, endoplasmic reticulum, lysosomes, and mitochondria) between WCL (dark purple square) and immunopurified lysosomes (lyso, light purple square). -log 10 p-values are reported. (F) KEGG enrichment analysis of proteins defined as lysosomes-associated proteins (fold change >1.5 and p-value ≤0.05) in HA-lyso GSC#9 immunopurified lysosomes as compared to FLAG-lyso immunopurified lysosomes. (G) Heatmap of KEGG: Cholesterol Metabolism candidates from WCL (dark purple square) and lysosomes (lyso, light purple square) in HA-lyso GSC#9, treated with vehicle (DMSO) and mepazine (MPZ, 20µM, 6h). Data are presented as the log 2 fold change. Candidates not identified are shown with a cross. (H) (Left) Western-blot analysis of the level of NPC1 from FLAG-lyso and HA-lyso GSC#9 immunopurified lysosomes (lyso, light purple square) and WCL (whole cell lysate, dark purple square). (Top) FLAG-lyso and HA-lyso GSC#9 were transfected with non-silencing RNA duplexes (si ctl ) and 2 independent duplexes targeting MALT1 (si.2 MALT1 and si.3 MALT1 ) for 3 days. (Bottom) FLAG-lyso and HA-lyso GSC#9 were treated with DMSO, MPZ (20µM), and MLT748 (5µM) for 6h. TMEM192-3xHA (HA) served as a loading control. (Right) Densitometric analysis of NPC1 level normalized to TMEM192-3xHA (HA). Data are presented as the mean + s.d. of 3 independent experiments. (I) (Left) Confocal analysis of Proximity Ligation Assay (PLA) between TMEM192-3xHA and NPC1. HA-lyso GSC#9 were treated as described in (H). Fluorescent signal (green) reflects a <40nM proximity. Nuclei are shown in blue (DAPI). Scale bar: 10µm. (Right) Violin representation of the quantification of the PLA signal intensity per cell. n>108. All panels are representative of at least n=3, unless otherwise specified. t-test and ANOVA, *p<0.05, **p<0.01, ***p<0.001.$

Journal: bioRxiv

Article Title: The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping

doi: 10.1101/2023.02.27.530259

Figure Lengend Snippet: (A) Workflow of the Lyso-IP approach to characterize the effects of MALT1 inhibition on the lysosomal proteome in patient-derived GSCs. GSC#9 stably expressing the lysosomal protein TMEM192 tagged with either 3xHA (HA-lyso) or 2xFLAG (FLAG-lyso) were mechanically lysed. Lysosomes were immunoprecipitated with anti-HA magnetic beads. These immunopurified lysosome fractions were subsequently analyzed by western-blot and label-free quantification (LFQ) proteomic. Lysosome-associated proteins were defined as the hits with ≥1.5 fold change (HA/Flag) and p-value ≤ 05. (B) Western-blot analysis of the levels of TMEM192-3xHA (HA) and TMEM192-2xFLAG (FLAG) in lentivirally infected GSC#9. Tubulin served as a loading control. (C) Confocal analysis of TMEM192-3xHA (HA) staining (red) and LAMP2 staining (green) in HA-lyso GSC#9. Nuclei are shown in blue (DAPI). Scale bar: 10µm. (D) Western-blot analysis of the levels of LAMP2, CTSD (PP: pre-pro, m: mature), TMEM192-3xHA (HA), EEA1, CALRETICULIN (CalR), GM130, and VDAC in flag-lyso and HA-lyso GSC#9 immunopurified lysosomes (lyso, light purple square) and whole cell lysate (WCL, dark purple square). Organelles to which belong the proteins are indicated on the right. (E) Comparison of the GO:CC enrichment analysis of organelles (Golgi, peroxisomes, endoplasmic reticulum, lysosomes, and mitochondria) between WCL (dark purple square) and immunopurified lysosomes (lyso, light purple square). -log 10 p-values are reported. (F) KEGG enrichment analysis of proteins defined as lysosomes-associated proteins (fold change >1.5 and p-value ≤0.05) in HA-lyso GSC#9 immunopurified lysosomes as compared to FLAG-lyso immunopurified lysosomes. (G) Heatmap of KEGG: Cholesterol Metabolism candidates from WCL (dark purple square) and lysosomes (lyso, light purple square) in HA-lyso GSC#9, treated with vehicle (DMSO) and mepazine (MPZ, 20µM, 6h). Data are presented as the log 2 fold change. Candidates not identified are shown with a cross. (H) (Left) Western-blot analysis of the level of NPC1 from FLAG-lyso and HA-lyso GSC#9 immunopurified lysosomes (lyso, light purple square) and WCL (whole cell lysate, dark purple square). (Top) FLAG-lyso and HA-lyso GSC#9 were transfected with non-silencing RNA duplexes (si ctl ) and 2 independent duplexes targeting MALT1 (si.2 MALT1 and si.3 MALT1 ) for 3 days. (Bottom) FLAG-lyso and HA-lyso GSC#9 were treated with DMSO, MPZ (20µM), and MLT748 (5µM) for 6h. TMEM192-3xHA (HA) served as a loading control. (Right) Densitometric analysis of NPC1 level normalized to TMEM192-3xHA (HA). Data are presented as the mean + s.d. of 3 independent experiments. (I) (Left) Confocal analysis of Proximity Ligation Assay (PLA) between TMEM192-3xHA and NPC1. HA-lyso GSC#9 were treated as described in (H). Fluorescent signal (green) reflects a <40nM proximity. Nuclei are shown in blue (DAPI). Scale bar: 10µm. (Right) Violin representation of the quantification of the PLA signal intensity per cell. n>108. All panels are representative of at least n=3, unless otherwise specified. t-test and ANOVA, *p<0.05, **p<0.01, ***p<0.001.

Techniques: Inhibition, Derivative Assay, Stable Transfection, Expressing, Immunoprecipitation, Magnetic Beads, Western Blot, Quantitative Proteomics, Infection, Control, Staining, Comparison, Transfection, Proximity Ligation Assay

(A) Kaplan-Meier curve of the probability of survival for >240 GB patients with low (green) and high (red) NPC1 RNA level based on the TCGA Agilent-4502A dataset. (B) Western-blot analysis of the levels of SREBP2 and LDLR in GSC#9 treated for 3h and 24h, respectively, with vehicle (DMSO) and U18666A (2µg/mL). GAPDH served as a loading control. FL (full length) and cleaved SREBP2 forms are indicated with green and red arrowheads, respectively. (C) GSC#9 were transfected with luciferase reporter plasmids for either wild-type SREBP2 promoter activity (SRE WT ) or a mutated version (SRE Mut ), in combination with TK-Renilla. GSC#9 were next treated for 16h with DMSO and U18666A (2µg/mL). Luminescence values were calculated as the ratio SRE WT /SRE Mut , and further normalized to Renilla intensities. Data are presented as the mean + s.d. of 3 independent biological replicates. (D) Flow cytometry analysis of the filipin III cholesterol probe in GSC#9 transfected with non-silencing (si ctl ) and NPC1 (si NPC1 ) targeting RNA duplexes for 3 days. Histogram is representative of at least 3 independent biological replicates. Mean fluorescence is indicated. E) Confocal analysis of cholesterol (filipin III) staining (gray) and TMEM192-3xHA (HA) staining (red) in HA-lyso GSC#9 treated with DMSO, MALT1 inhibitors (MPZ, 20µM and MLT748, 5µM), and NPC1 inhibitor (U18666A, 2µg/mL) for 16h. Alternatively, cells received si ctl , si NPC1 , and 2 independent RNA duplexes targeting MALT1 (si.2 MALT1 and si.3 MALT1 ). Scale bar: 10µm. (F) (Left) Confocal analysis of lysobisphosphatidic acid (LBPA) staining (green) in GSC#9 treated for 24h as described in (E). Nuclei are shown in blue (DAPI). Scale bar: 10µm. (Right) Violin representation of the quantification of LBPA signal intensity per cell. n>112. (G) Western-blot analysis of the levels of LAMP2 and P62 in GSC#9 treated for 24h, as described in (C). GAPDH served as a loading control. (H) Western-blot analysis of the levels of NPC1, LAMP2, and P62 in GSC#9, transfected as in (D). GAPDH served as a loading control. (I) Cell viability was measured using CellTiter-Glo assay in GSC#9 treated for 48h with DMSO and U18666A at the indicated doses. Data are presented as the mean + s.d. of 3 independent biological replicates. (J) Cell viability was measured using CellTiter-Glo assay in GSC#9 transfected for 3 days with si ctl , si NPC1 , and si NPC2 . Data are presented as the mean + s.d. of 3 independent biological replicates. All panels are representative of at least n=3, unless otherwise specified. t-test and ANOVA, *p<0.05, **p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping

doi: 10.1101/2023.02.27.530259

Figure Lengend Snippet: (A) Kaplan-Meier curve of the probability of survival for >240 GB patients with low (green) and high (red) NPC1 RNA level based on the TCGA Agilent-4502A dataset. (B) Western-blot analysis of the levels of SREBP2 and LDLR in GSC#9 treated for 3h and 24h, respectively, with vehicle (DMSO) and U18666A (2µg/mL). GAPDH served as a loading control. FL (full length) and cleaved SREBP2 forms are indicated with green and red arrowheads, respectively. (C) GSC#9 were transfected with luciferase reporter plasmids for either wild-type SREBP2 promoter activity (SRE WT ) or a mutated version (SRE Mut ), in combination with TK-Renilla. GSC#9 were next treated for 16h with DMSO and U18666A (2µg/mL). Luminescence values were calculated as the ratio SRE WT /SRE Mut , and further normalized to Renilla intensities. Data are presented as the mean + s.d. of 3 independent biological replicates. (D) Flow cytometry analysis of the filipin III cholesterol probe in GSC#9 transfected with non-silencing (si ctl ) and NPC1 (si NPC1 ) targeting RNA duplexes for 3 days. Histogram is representative of at least 3 independent biological replicates. Mean fluorescence is indicated. E) Confocal analysis of cholesterol (filipin III) staining (gray) and TMEM192-3xHA (HA) staining (red) in HA-lyso GSC#9 treated with DMSO, MALT1 inhibitors (MPZ, 20µM and MLT748, 5µM), and NPC1 inhibitor (U18666A, 2µg/mL) for 16h. Alternatively, cells received si ctl , si NPC1 , and 2 independent RNA duplexes targeting MALT1 (si.2 MALT1 and si.3 MALT1 ). Scale bar: 10µm. (F) (Left) Confocal analysis of lysobisphosphatidic acid (LBPA) staining (green) in GSC#9 treated for 24h as described in (E). Nuclei are shown in blue (DAPI). Scale bar: 10µm. (Right) Violin representation of the quantification of LBPA signal intensity per cell. n>112. (G) Western-blot analysis of the levels of LAMP2 and P62 in GSC#9 treated for 24h, as described in (C). GAPDH served as a loading control. (H) Western-blot analysis of the levels of NPC1, LAMP2, and P62 in GSC#9, transfected as in (D). GAPDH served as a loading control. (I) Cell viability was measured using CellTiter-Glo assay in GSC#9 treated for 48h with DMSO and U18666A at the indicated doses. Data are presented as the mean + s.d. of 3 independent biological replicates. (J) Cell viability was measured using CellTiter-Glo assay in GSC#9 transfected for 3 days with si ctl , si NPC1 , and si NPC2 . Data are presented as the mean + s.d. of 3 independent biological replicates. All panels are representative of at least n=3, unless otherwise specified. t-test and ANOVA, *p<0.05, **p<0.01, ***p<0.001.

Techniques: Western Blot, Control, Transfection, Luciferase, Activity Assay, Flow Cytometry, Fluorescence, Staining, Glo Assay

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