Journal: Cell reports

Article Title: TRPML1 Promotes Protein Homeostasis in Melanoma Cells by Negatively Regulating MAPK and mTORC1 Signaling

doi: 10.1016/j.celrep.2019.07.086

Figure Lengend Snippet: (A) Western blot analysis of TRPML1 and β-actin in melanoma cells derived from 7 patients (melanoma patient-derived xenografts [PDXs]: M214, M405, M481, M491, UT10, M528, and M597) and normal melanocytes derived from 3 donors (hMEL). The blot reflects one representative experiment of two performed. (B–D) Western blot analysis of TRPML1 and b-actin in parental cells (P) from three melanomas (A375 cell line (B), and M214 (C) and M481 (D) patient-derived melanomas) as well as in 3 clones per melanoma in which MCOLN1 had been deleted using CRISPR. The blot reflects one representative experiment (of two performed) per melanoma. (E–G) Growth in culture of parental cells (black) versus MCOLN1 -deficient clones (red, blue, and green) in A375 (E), M214 (F), and M481 (G). (H and I) Frequencies of activated caspase 3/7 + cells (H) and Ki-67 + cells (I) among cultured parental cells versus MCOLN1 -deficient clones. (J–L) Growth of subcutaneous tumors in mice transplanted with parental cells versus MCOLN1 -deficient clones in A375 (J), M214 (K), and M481 (L). Data show mean ± SD from one representative experiment (of two performed) with 5 mice per clone. (M and N) Frequencies of activated caspase 3/7 + cells (M) and Ki-67 + cells (N) in subcutaneous tumors grown from parental cells versus MCOLN1 -deficient clones. Data represent mean ± SD from two independent experiments with 5 mice per clone per melanoma per experiment. Statistical significance was assessed using one-way ANOVA or Welch’s one-way ANOVA followed by Dunnett’s or Dunnett’s T3 multiple comparisons tests, respectively, for the last time point measured (E–G and J–L), or one-way ANOVAs followed by Dunnett’s multiple comparisons tests (H and I, M and N). Mean ± SD from two independent experiments with 3 replicate cultures per clone per experiment (E)–(I). ns, not significant; **p < 0.01; ***p < 0.001. See also and .

Article Snippet: A375 (ATCC; CRL-1619; female), M214 (female), M481 (male), and M491 (male) melanoma cells and melanocytes from three donors (hMEL1 (ATCC; PCS-200–013), hMEL2 (Invitrogen; C-024–5C), hMEL3 (Lonza; CC-2586)) were cultured in tissue-culture-treated plates (Corning) with high-glucose DMEM (GIBCO), 10% fetal bovine serum (FBS; Gemini) and 1% penicillin/streptomycin (Fisher).

Techniques: Western Blot, Derivative Assay, Clone Assay, CRISPR, Cell Culture

Journal: Cell reports

Article Title: TRPML1 Promotes Protein Homeostasis in Melanoma Cells by Negatively Regulating MAPK and mTORC1 Signaling

doi: 10.1016/j.celrep.2019.07.086

Figure Lengend Snippet: (A–C) Western blots of p-ERK1/2 (T202/Y204), ERK1/2, p-Akt (S473), Akt, p-S6K (T389), S6K, and b-actin in cultured parental cells (P) or MCOLN1 -deficient cells from A375 (A), M214 (B), or M481 (C) melanomas. (D–F) Western blots of p-ERK1/2, ERK1/2, p-TSC2 (S664), TSC2, p-S6K, S6K, and β-actin in cultured parental cells or MCOLN1 -deficient clones from A375 (D), M214 (E), and M481 (F) treated with DMSO (−) or 5 nM trametinib (MEKi; +). (G–I) Western blots of p-MET (Y1234/1235), MET, p-ERBB3 (Y1289), ERBB3, p-ERK1/2, ERK1/2, p-S6K, S6K, and b-actin in cultured parental cells or MCOLN1 -deficient cells from A375 (G), M214 (H), and M481 (I). All blots reflect one representative experiment (of two performed) per melanoma. See also .

Article Snippet: A375 (ATCC; CRL-1619; female), M214 (female), M481 (male), and M491 (male) melanoma cells and melanocytes from three donors (hMEL1 (ATCC; PCS-200–013), hMEL2 (Invitrogen; C-024–5C), hMEL3 (Lonza; CC-2586)) were cultured in tissue-culture-treated plates (Corning) with high-glucose DMEM (GIBCO), 10% fetal bovine serum (FBS; Gemini) and 1% penicillin/streptomycin (Fisher).

Techniques: Western Blot, Cell Culture, Clone Assay

Journal: Cell reports

Article Title: TRPML1 Promotes Protein Homeostasis in Melanoma Cells by Negatively Regulating MAPK and mTORC1 Signaling

doi: 10.1016/j.celrep.2019.07.086

Figure Lengend Snippet: (A–C) Colocalization of MET with the endosomal marker, Rab7, in parental versus MCOLN1 -deficient cells from A375 (A), M214 (B), and M481 (C) melanomas. (D) Manders colocalization coefficients reporting the fraction of MET staining that co-localized with Rab7 staining. (E–G) Colocalization of MET with the lysosomal marker, LAMP1, in parental versus MCOLN1 -deficient cells from A375 (E), M214 (F), and M481 (G) melanomas. (H) Manders colocalization coefficients reporting the fraction of MET staining that co-localized with LAMP1 staining. All data represent mean ± SD from two independent experiments with 3 quantified cells per clone per experiment. Statistical significance was assessed using unpaired two-tailed t tests; ns, not significant; *p < 0.05; ***p < 0.001. Scale bars represent 10μm. See also .

Article Snippet: A375 (ATCC; CRL-1619; female), M214 (female), M481 (male), and M491 (male) melanoma cells and melanocytes from three donors (hMEL1 (ATCC; PCS-200–013), hMEL2 (Invitrogen; C-024–5C), hMEL3 (Lonza; CC-2586)) were cultured in tissue-culture-treated plates (Corning) with high-glucose DMEM (GIBCO), 10% fetal bovine serum (FBS; Gemini) and 1% penicillin/streptomycin (Fisher).

Techniques: Marker, Staining, Two Tailed Test

Journal: Cell reports

Article Title: TRPML1 Promotes Protein Homeostasis in Melanoma Cells by Negatively Regulating MAPK and mTORC1 Signaling

doi: 10.1016/j.celrep.2019.07.086

Figure Lengend Snippet: (A–C) Western blots of p-Akt (S473), Akt, p-S6K (T389), S6K, and β-actin in cultured parental cells or MCOLN1 -deficient clones from A375 (A), M214 (B), and M481 (C) treated with DMSO (−) or 5 nM Torin1 (+). The blot reflects one representative experiment (of three performed) per melanoma. (D–F) Growth in culture of parental cells (P; black) versus MCOLN1 -deficient clones (red, blue, and green) from A375 (D), M214 (E), and M481 (F) treated with DMSO control (solid bars) or 5 nM Torin1 (striped bars) for 21 days. Torin1 treatment significantly rescued the growth of MCOLN1 -deficient clones. The data represent mean ± SD from two experiments with 3 replicate cultures per clone per experiment. (G–I) Growth of subcutaneous tumors in mice transplanted with parental or MCOLN1 -deficient cells from A375 (G), M214 (H), and M481 (I) and treated with DMSO control (Ctrl; solid lines) or 5 mg/kg/day Rapamycin (Rapa; dotted lines). The data represent mean ± SD from one representative experiment (of two performed) with 5 mice per clone. (J–L) Frequency of activated caspase 3/7 + cells in subcutaneous tumors grown from parental cells or MCOLN1 -deficient clones from A375 (J), M214 (K), and M481 (L) treated with DMSO control (solid bars) or rapamycin (striped bars). These data represent mean ± SD from two independent experiments with 4 to 5 mice per clone per experiment. Statistical significance was assessed using two-way ANOVAs followed by Sidak’s and Dunnett’s multiple comparisons tests (D–F), two-way ANOVAs followed by Dunnett’s multiple comparisons tests for the last time points measured (G–I), or Welch’s one-way ANOVAs or one-way ANOVAs followed by Tamhane’s T2, Dunnett’s T3, or Dunnett’s or Sidak’s multiple comparisons tests (J–L); ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: A375 (ATCC; CRL-1619; female), M214 (female), M481 (male), and M491 (male) melanoma cells and melanocytes from three donors (hMEL1 (ATCC; PCS-200–013), hMEL2 (Invitrogen; C-024–5C), hMEL3 (Lonza; CC-2586)) were cultured in tissue-culture-treated plates (Corning) with high-glucose DMEM (GIBCO), 10% fetal bovine serum (FBS; Gemini) and 1% penicillin/streptomycin (Fisher).

Techniques: Western Blot, Cell Culture, Clone Assay, Control

Journal: Cell reports

Article Title: TRPML1 Promotes Protein Homeostasis in Melanoma Cells by Negatively Regulating MAPK and mTORC1 Signaling

doi: 10.1016/j.celrep.2019.07.086

Figure Lengend Snippet: (A–C) Relative rate of incorporation of a pulse of OP-Puro in cultured parental cells (P; black) versus MCOLN1 -deficient clones (red, blue, and green) from A375 (A), M214 (B), and M481 (C) treated with DMSO control (solid bars) or 5 nM Torin1 (striped bars). Data represent mean ± SD from two independent experiments with 3 replicate cultures per treatment per clone per experiment. (D–I) Intracellular protein aggregates were visualized by staining with Proteostat dye (D–F) and then quantified (G–I) in parental cells (black) versus MCOLN1 -deficient clones (red) treated with DMSO control (solid bars) or 5 nM Torin1 (striped bars). Data represent mean ± SD from two independent experiments with 3 replicate cultures per treatment per clone per experiment. (J–L) Western blot analysis of BiP, p-eIF2 α (S51), total eIF2 α , ATF4, CHOP, p-IRE1 α (S724), total IRE1 α , and β-actin in cultured parental cells or MCOLN1 -deficient cells from A375 (J), M214 (K), and M481 (L) treated with DMSO (−) or 5 nM Torin1 (+). The blot reflects one representative experiment (of two performed) per melanoma. Statistical significance was assessed using two-way ANOVAs followed by Sidak’s and Dunnett’s multiple comparisons tests (A–C) or two-way ANOVAs followed by Sidak’s multiple comparisons tests (G–I); ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars represent 10μm. See also .

Article Snippet: A375 (ATCC; CRL-1619; female), M214 (female), M481 (male), and M491 (male) melanoma cells and melanocytes from three donors (hMEL1 (ATCC; PCS-200–013), hMEL2 (Invitrogen; C-024–5C), hMEL3 (Lonza; CC-2586)) were cultured in tissue-culture-treated plates (Corning) with high-glucose DMEM (GIBCO), 10% fetal bovine serum (FBS; Gemini) and 1% penicillin/streptomycin (Fisher).

Techniques: Relative Rate, Cell Culture, Clone Assay, Control, Staining, Western Blot

Journal: Cell reports

Article Title: TRPML1 Promotes Protein Homeostasis in Melanoma Cells by Negatively Regulating MAPK and mTORC1 Signaling

doi: 10.1016/j.celrep.2019.07.086

Figure Lengend Snippet: (A–D) Macropinocytosis of extracellular protein was measured in cultured parental cells (black) or MCOLN1 -deficient clones (red) treated with either DMSO control (solid bars) versus 5 nM trametinib (MEKi; striped bars) (A and C) or DMSO control (solid bars) versus 5 nM Torin1 (striped bars) (B and D) by incubating cells with DQ-BSA for 6 h, followed by confocal imaging. The data represent mean ± SD from two independent experiments with 3 replicate cultures per treatment per clone per experiment. (E and F) Relative intracellular serine levels in cultured cells (E) or subcutaneous tumors (F) formed by parental cells versus MCOLN1 -deficient cells treated with DMSO control (solid bars), Torin1 (E; striped bars), or rapamycin (F; striped bars). The data reflect two independent experiments with 3 replicate cultures per clone per treatment per experiment (E) or 5 mice per clone per treatment per experiment (F). (G–I) Growth of parental versus MCOLN1 -deficient cells from A375 (G), M214 (H), and M481 (I) in cultures containing 0.4 mM L-serine (solid bars) or 2 mM L-serine (striped bars) for 21 days. These data represent mean ± SD from two independent experiments with 3 replicate cultures per treatment per clone per experiment. (J) Model of TRPML1 function in melanoma cells. Statistical significance was assessed using one-way ANOVA or Welch’s one-way ANOVA followed by Sidak’s or Tamhane’s T2 multiple comparisons tests (C and F) or two-way ANOVAs followed by Sidak’s and/or Dunnett’s multiple comparisons tests (D, E, and G–I); ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars represent 10μm. See also .

Article Snippet: A375 (ATCC; CRL-1619; female), M214 (female), M481 (male), and M491 (male) melanoma cells and melanocytes from three donors (hMEL1 (ATCC; PCS-200–013), hMEL2 (Invitrogen; C-024–5C), hMEL3 (Lonza; CC-2586)) were cultured in tissue-culture-treated plates (Corning) with high-glucose DMEM (GIBCO), 10% fetal bovine serum (FBS; Gemini) and 1% penicillin/streptomycin (Fisher).

Techniques: Cell Culture, Clone Assay, Control, Imaging

Journal: Cell reports

Article Title: TRPML1 Promotes Protein Homeostasis in Melanoma Cells by Negatively Regulating MAPK and mTORC1 Signaling

doi: 10.1016/j.celrep.2019.07.086

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A375 (ATCC; CRL-1619; female), M214 (female), M481 (male), and M491 (male) melanoma cells and melanocytes from three donors (hMEL1 (ATCC; PCS-200–013), hMEL2 (Invitrogen; C-024–5C), hMEL3 (Lonza; CC-2586)) were cultured in tissue-culture-treated plates (Corning) with high-glucose DMEM (GIBCO), 10% fetal bovine serum (FBS; Gemini) and 1% penicillin/streptomycin (Fisher).

Techniques: Virus, Generated, Recombinant, Reverse Transcription, Saline, Cloning, Gel Extraction, Western Blot, Stripping, Bicinchoninic Acid Protein Assay, shRNA, Software, SDS-Gel

Journal: Cell Genomics

Article Title: Optimization of Cas12a for multiplexed genome-scale transcriptional activation

doi: 10.1016/j.xgen.2023.100387

Figure Lengend Snippet: Nanobody recruitment improves CRISPRa activity (A) Schematic representing dCas12a nanobody-based recruitment of the transactivation domain (top). Plasmid maps depicting one vector containing the ALFA tag fused to dCas12a and a second vector containing the ALFA nanobody, TAD, and targeting guide (bottom). (B) Heatmaps displaying comparative performance between the p65, VP64, and Activ TADs when recruited via nanobody to dCas12a with increasing numbers of ALFA tags (1×, 3×, 5×) at either the N or the C terminus (N′ or C′). Variable vector components are indicated by dashed lines. Experiments conducted in HT29, HCC2429, and A375 cell lines. Color scale reflects levels of normalized MFI of CD4 expression within each cell line. (C) Bar plot illustrating combinatorial effects emerging from the direct tethering of TADs to the N terminus of dCas12a and nanobody-based recruitment of varying TAD configurations to the N and/or C termini in HT29 cells. The x axis shows normalized MFI values of CD4 expression on a log10 scale. (D) Bar plot comparing CRISPRa activity when a nanobody-TAD combination is recruited to the N′ or C′ terminus of Cas12a tethered to VP64 (N′ 5× tag, C′ 5× tag). Normalized CD4 MFI values are shown for three cell lines (A375, HT29, and HCC2429). See also Figure S2 .

Article Snippet: A375 (female), HCC2429 (female), HT29 (female), and MelJuSo (female) cells were obtained from the Cancer Cell Line Encyclopedia at the Broad Institute.

Techniques: Activity Assay, Plasmid Preparation, Expressing

Journal: Cell Genomics

Article Title: Optimization of Cas12a for multiplexed genome-scale transcriptional activation

doi: 10.1016/j.xgen.2023.100387

Figure Lengend Snippet: Effective multiplexing with a nanobody-based system (A) Schematic depicting single-gene targeting guide cassette architecture (top). Histograms show expression levels of CD4 (APC), CD274 (APC), CD97 (FITC), and CD26 (FITC) in HT29, A375, and HCC2429 cells expressing 5×-tag-dCas12a-VP64 when targeted individually by three guides per gene paired with nanobody-VP64, nanobody-p65, or nanobody-p65-HSF1 (bottom). Data from one representative replicate shown; data for all replicates are included in . (B) Schematic depicting multiplexed targeting guide cassette architecture (top). Heatmaps of normalized MFI values for CD26, CD4, CD97, and CD274 in HT29, A375, and HCC2429 cells expressing 5×-tag-dCas12a-VP64 when targeted simultaneously by one guide per gene paired with different TADs, same as those used in (A). MFI values were normalized to basal expression within each cell line/gene combination, as in the color scale (bottom). (C) Comparison of RNA expression levels across samples expressing 5×-tag-dCas12a-VP64 and either nanobody-VP64 or nanobody-p65 with or without three CD4-targeting guides. Shrunken LFC in the CD4-targeting population is plotted against mean normalized read counts of all replicates for baseline expression (n = 3). See also Figure S3 .

Article Snippet: A375 (female), HCC2429 (female), HT29 (female), and MelJuSo (female) cells were obtained from the Cancer Cell Line Encyclopedia at the Broad Institute.

Techniques: Multiplexing, Expressing, Comparison, RNA Expression

Journal: Cell Genomics

Article Title: Optimization of Cas12a for multiplexed genome-scale transcriptional activation

doi: 10.1016/j.xgen.2023.100387

Figure Lengend Snippet:

Article Snippet: A375 (female), HCC2429 (female), HT29 (female), and MelJuSo (female) cells were obtained from the Cancer Cell Line Encyclopedia at the Broad Institute.

Techniques: Recombinant, Biomarker Discovery, Library Amplification, Software

Journal: Structure (London, England : 1993)

Article Title: Crystal Structures of Anti-Apoptotic BFL-1 and its Complex with a Covalent Stapled Peptide Inhibitor

doi: 10.1016/j.str.2017.11.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-His 6 Abcam Cat# Ab18184; RRID: AB_444306 Mouse anti-BFL-1 Abcam Cat# 125259; RRID: AB_10971364 Sheep anti-mouse IgG:HRP Biorad Cat# AAC10P; RRID: AB_321929 Goat polyclonal anti-GST GE Healthcare Cat# 27-4577-01; RRID: AB_771432 Donkey anti-goat IgG:HRP Santa Cruz Biotech Cat# Sc-2020; RRID: AB_631728 Bacterial and Virus Strains LOBSTR BL21(DE3) Competent Cells Kerafast Cat# EC001 One Shot BL21(DE3) Competent Cells Invitrogen Cat# C600003 Chemicals, Peptides, and Recombinant Proteins His 6 BFL-1ΔC WT, C55S, C4S/C19S, C4S/C19S/C55S Walensky Lab N/A GST MCL-1ΔNΔC Walensky Lab N/A GST BCL-X L ΔC Walensky Lab N/A D-NA-NOXA SAHB Walensky Lab N/A Ac-NOXA SAHB Walensky Lab N/A Critical Commercial Assays Q5 Site Directed Mutagenesis Kit New England Biolabs Cat# E0554S Pierce Silver Stain Kit ThermoFisher Scientific Cat# 24612 Pierce BCA Protein Assay Kit ThermoFisher Scientific Cat# 23225 Deposited Data Apo BFL-1 Crystal Structure Protein databank 5WHI BFL-1/D-NA-NOXA Crystal Structure Protein databank 5WHH Experimental Models: Cell Lines Human: A375P (female) ATCC CRL-3224; RRID: CVCL_6233 Recombinant DNA Plasmid PET17b Novagen Cat# 69663 Plasmid PET17B_BFL1ΔC_WT Walensky Lab N/A Plasmid PET17b_BFL1ΔC_C4S/C19S Walensky Lab N/A Plasmid PET17b_BFL1ΔC_C4S/C19S/C55S Walensky Lab N/A Plasmid PET17b_BFL1ΔC_C55S Walensky Lab N/A Plasmid PGEX-4T-1 GE Healthcare Cat# 28-9545-49 Plasmid PGEX-4T-1_MCL-1ΔNΔC Walensky Lab N/A Plasmid PGEX-4T-1_BCL-X L ΔC Walensky Lab N/A Software and Algorithms GraphPad Prism Graphpad Software Inc. https://www.graphpad.com/scientific-software/prism/ XIA2 Winter, 2010 https://xia2.github.io POINTLESS Evans, 2006 http://www.ccp4.ac.uk XDS Kabsch, 2010 http://xds.mpimf-heidelberg.mpg.de/ Phaser McCoy et al., 2007 https://www.phenix-online.org/documentation/reference/phaser.html Buster Smart et al., 2012 https://www.globalphasing.com/buster/ Rhofit Smart et al., 2012 https://www.globalphasing.com/buster/manual/rhofit/manual/ Phenix Adams et al., 2010 https://www.phenix-online.org/ Coot Emsley and Cowtan, 2004 https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ Differences Distance Matrix Program Center for Structural Biology at Yale University http://www.csb.yale.edu/userguides/datamanip/ddmp/ Pymol The PyMol Molecular Graphics System, Version 1.7.4.0 Schrodinger, LLC Open in a separate window KEY RESOURCES TABLE

Techniques: Virus, Recombinant, Mutagenesis, Silver Staining, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Software