Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Schematic representing the initial events of the metastatic cascade that can be measured using the CRC-OOC. Tumor cells in the top channel (1) can be visualized and analyzed separately from tumor cells that have invaded and adhered in the endothelial compartment (2). Additionally, tumor cells that are found in the endothelial effluent (3; circulating tumor cells (“CTC-like” cells)) can also be collected and analyzed. (B.) Circulating tumor cells were collected from the endothelial effluent of stretched and not stretched HCT116 CRC-Chips and RNAseq was performed. GO Pathway analysis was performed on a subset of genes with either a 2-fold difference between stretched and not stretched CTCs or an FDR-adjusted p-value <0.1. N=2 biological replicates with 3 pooled chips in each replicate. (C.) Gene expression of neurotransmitter-related genes were measured by a neurotransmitter-specific PCR array. HCT116 tumor cells of stretched and not stretched chips were harvested on day 6 from the top epithelial channel and isolated via FACs. Data is displayed as the gene expression fold change of stretch versus not stretch conditions Expression was normalized to the average of 5 housekeeping genes. Genes that had a 1.5-fold increase or decrease in the stretched condition are displayed. N=3 biological replicates with 3 chips pooled per biological replicate. (D.) Schematic of the production of GABA from glutamate by the enzyme GAD1. In this figure, experiments related to GABA are indicated in purple and experiments related to GAD1 are indicated in light blue. (E.) Representative confocal immunofluorescent images of the epithelial (top; 1) or endothelial (bottom; 2) channel of the CRC-Chips stained for GABA (purple) on day 6. Invaded HCT116 H2B-GFP stain positive for GABA, while HCT116 H2B-GFP tumor cells that are in the top channel stain weakly for GABA. DAPI stains the nuclei of Caco2 C2BBe1 cells in the top channel and endothelial cells in the bottom channel. Scale bars represent 200 μm in the top channel image and 100 μm in the bottom channel images. Top channel images are maximum projections that span a 35 μm Z-height with a 5 μm step size. Bottom channel images are maximum projections that span a 10 μm Z-height with a 5 μm step size. (F.) RNAseq analysis was performed on CRC organoids and normalized GAD1 expression is shown. N=5 independent donors with 2-3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test; ***p<0.001 ( G.) CRC organoids were isolated from stretched chips and qPCR analysis of GAD1 gene expression was performed. N=5 independent doners with 3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test p<0.05. ( H.) GAD1 mRNA expression from TCGA in KRAS, NRAS, or BRAF mutant primary colon cancer tumors. N=196 patients with KRAS, NRAS, or BRAF mutant tumors; N=201 patients with KRAS, NRAS, or BRAF wildtype tumors. Individual data points are shown and median with interquartile range is represented. Data was analyzed with an unpaired t-test; ****p<0.0001. ( I.) Kaplan-Meier curve with univariate analysis of the survival of patients with KRAS, NRAS, or BRAF mutated CRC tumors based on high versus low expression of GAD1 (defined as above or below the median GAD1 mRNA expression z-score of 0.3). Data was extracted from the TCGA. N=254 patients. Data was analyzed using a log-rank (Mantel-Cox test). ( J.) Effluent from the epithelial channel of the patient-derived organoids was collected on day 0 (D0) and day 6 (D6). GABA intensity was analyzed from extracted metabolites N=6 chips per timepoint per patient; n=4 on D0 and 2 on D6 for UK. Data was analyzed using a two-way ANOVA; ***p<0.001; ****p<0.0001. ( K .) US-H2B-GFP (top) and UP-H2B-GFP (bottom) stretched tumor-chips were stained for GABA (purple). Scale bars represent 200 μm. L. Representative 10x immunofluorescence images of the 5 tumors stained for EpCAM (green), CK20 (red), and GABA (purple). Scale bars represent 500 μm and 200 μm for UK. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Gene Expression, Isolation, Expressing, Staining, Mutagenesis, Derivative Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Invasion of HCT116 tumor-chips in the presence or absence of exogenous GABA (flowed through the epithelial channel) was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=6 chips. Individual data are shown, with mean ± SEM represented and analyzed using a one-way ANOVA; ****p<0.0001. (B.) Intracellular [ 13 C 4 ]GABA or unlabeled GABA was measured via mass spectrometry-based metabolomics in the HCT116 tumor-chips after the addition of exogenous GABA for six days. N=3 chips. (C.) Schematic of GABA catabolism by ABAT, subsequent entry into the TCA cycle, and inhibition of ABAT activity by vigabatrin. In this figure, experiments related to GABA are indicated as purple, and experiments related to ABAT are indicated as teal. (D.) Western blot analysis of ABAT in shRNA control or ABAT shRNA HCT116 tumor cells. Cropped western blot (left) and quantification (right) confirm knockdown of ABAT. (E.) Growth rate of ABAT-knockdown or control HCT116 tumor cells when grown in traditional cell culture methods. N=3. Individual data are shown and mean ± SEM are represented. Data was analyzed using a t-test; **p<0.01. (F.) Numbers of ABAT-knockdown or control HCT116 tumor cells in the top channel on-chip as measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (D6). N=5-6 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; *p<0.05;***p<0.001. (G.) Invasion of ABAT knockdown (KD) or control shRNA HCT116 tumor-chips in the presence or absence of stretching was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=5-6 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; *p<0.05. (H.) Numbers of HCT116 tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). Individual data are shown and mean ± SEM are represented. N=4 chips. Data was analyzed using a two-way ANOVA; **p<0.01. (I.) Invasion of HCT116 tumor-chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; ***p<0.001. (J.) Numbers of US-H2B-GFP (red) or UP-H2B-GFP (blue) tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; ns=p>0.05. (K.) Invasion of US-H2B-GFP or UP-H2B-GFP organoid-tumor chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; **p<0.01. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Mass Spectrometry, Inhibition, Activity Assay, Western Blot, shRNA, Control, Knockdown, Cell Culture, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis

doi: 10.1101/2021.01.31.428633

Figure Lengend Snippet: A) Overview of recovery conditions. Following AY9944 sterol depletion, HEK293T hCLTA-Tq2 EN cells were exposed to cholesterol-loaded MβCD (MβCD-Chol, and chased in LPDS media) [Top] , supplemented continuously with lipoprotein-rich 15% FBS in the presence of AY9944 [Middle] , or incubated continuously in 7.5% LPDS media in the absence of AY9944 to allow endogenous cholesterol synthesis [Bottom] . B) Mid-plane live-cell confocal microscopy of HEK293T hCLTA-Tq2 EN following addition of transferrin-conjugated to AF-555 (Tfn-555) during recovery as described in (A). Representative images from different fields of view shown. Tfn and clathrin distributions in normal and arrested CME [Inset] . Scale bar = 10 μm. C) Corresponding cellular sterol profiles quantified by GC/MS (Mean ± SD). N = 3 biological replicates from independent experiments. Dotted and dashed lines represent total sterol abundance of untreated FBS and LPDS cultured cells, respectively.

Article Snippet: HEK293T cells were then incubated with transferrin (Tfn) conjugated to Alexa FluorTM 555 (25 ug/mL, Invitrogen) for 30 mins at 37°C, followed by rinsing in ice-cold PBS, and fixation in 4% paraformaldehyde for 20 min. For high-content imaging analysis, 24 well glass bottom plates (Cellvis) were imaged using CX7 Rescue High-Content Screening (HCS) Navigator software (PerkinElmer, Waltham, MA) and standard HCS imaging protocol.

Techniques: Incubation, Confocal Microscopy, Gas Chromatography-Mass Spectrometry, Cell Culture

Journal: bioRxiv

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis

doi: 10.1101/2021.01.31.428633

Figure Lengend Snippet: The ability of structurally diverse sterols to rescue CME inhibition due to sterol depletion by AY9944 treatment was evaluated by direct delivery of sterols to the PM via MβCD carrier. A) Summary of physical properties and phase separation behavior of sterols. Topological Polar Surface Area (TPSA) computed surface sum over all polar atoms. Ordered lipid ( L o ) domain (raft) stabilizing (+) or disrupting (−) sterols indicated relative to cholesterol (++). Refer to Table S2 for additional details. B) Sterol profiles of AY9944 treated HEK293T cells following 1h incubation with sterol loaded MβCD (Mean ± SD). N = 4 independent biological replicates from two MβCD-sterol preparations. C) Tfn uptake relative to controls cultured in 7.5% LPDS for 48 h (Mean ± SD). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA (F(12, 51) = 11.23, p < 0.0001) and Dunnett’s test versus LPDS control (N = 5 biological replicates from 3 independent experiments, ~1,500 cells per replicate). Cholesterol, desmosterol, 7DHC, and lathosterol support formation of L o domains in model membranes as designated. D) Representative confocal images taken mid-plane following 30 min incubation with AF-555 conjugated transferrin (Tfn). Scale bar = 20 μm. (c), Commercially available, pre-loaded MβCD-Chol.

Article Snippet: HEK293T cells were then incubated with transferrin (Tfn) conjugated to Alexa FluorTM 555 (25 ug/mL, Invitrogen) for 30 mins at 37°C, followed by rinsing in ice-cold PBS, and fixation in 4% paraformaldehyde for 20 min. For high-content imaging analysis, 24 well glass bottom plates (Cellvis) were imaged using CX7 Rescue High-Content Screening (HCS) Navigator software (PerkinElmer, Waltham, MA) and standard HCS imaging protocol.

Techniques: Inhibition, Incubation, Cell Culture, Control

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , S -acylation is mediated by ZDHHC loading of long-chain acyl-CoA derived from lipid biosynthesis followed by acyl transfer to a proximal Cys of a protein substrate and regeneration of apo-ZDHHC. The reversible cycle is closed by thioester hydrolysis by APTs. b , X-ray structure of human ZDHHC20 irreversibly inhibited by lipid mimic 2-bromopalmitate (PDB ID: 6BML ). Inset, sterically demanding residues in the ZDHHC20 lipid-binding pocket contact the acyl chain distal to the DHHC catalytic site. c , Steric complementation between a ZDHHC ‘hole’ mutant and an alkyne-tagged ‘bumped’ lipid substrate probe enables selective loading and tag transfer to ZDHHC substrates, bypassing endogenous (WT) ZDHHCs. Fluorescence visualization and chemical proteomics are enabled by bioorthogonal conjugation to multifunctional capture reagents.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Derivative Assay, Binding Assay, Mutagenesis, Fluorescence, Conjugation Assay

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , Fatty acid probes containing an alkynyl click-handle (blue), varying chain length L = 16, 18 or 20 heavy atoms in the chain (carbons + nitrogen) and an R ‘bump’ group (red)—Ac, c Pr or Bz. b , Two-stage pairing strategy for a designed ZDHHC20 mutant optimizes probe chain length and then bump size to match the new binding cavity, with probe activation, selectivity over ZDHHC20 WT and transfer to a known ZDHHC20 substrate (IFITM3) optimized in parallel. c – f , Bump-hole loading analysis of C-terminal FLAG-tagged ZDHHC20 WT and mutants in HEK293T cells treated with 15 μM YnPal ( c , d ) or 18-Ac ( e , f ) for 4 h (D, catalytic-dead ZDHHC20(C156S); E, empty vector; n = 3 independent biological replicates average ± s.d.). g , Probe bump-size optimization by transfer assays with HA-IFITM3 and either WT ZDHHC20 (W) or ZDHHC20(Y181G) (M) co-expression in HEK293T cells ( n = 3 independent biological replicates average ± s.d.). h , Average loading and transfer activity relative to highest fluorescent/input ratio ( n = 3 independent biological replicates average ± s.d.). i , j , Enzyme kinetics for WT ZDHHC20 and ZDHHC20(Y181G) treated with Pal-CoA ( i ) or 18-Bz-CoA ( j ) using a KDH assay ( 3 ). Michaelis–Menten plots generated from average reaction rate (NADH generated μM min −1 , n = 3 independent experiments) ± s.d. versus lipid concentration (μM). d , f , h , The two-tailed unpaired t test of Prism 9.0 was used to determine P values and noted above relevant comparisons.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Mutagenesis, Binding Assay, Activation Assay, Plasmid Preparation, Expressing, Activity Assay, Generated, Concentration Assay, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a-b ) Catalytically dead ZDHHC20 is appreciably labeled by YnPal at peripheral cysteine sites. FLAG-tagged WT and ZDHHC20[C156S] constructs were transfected in HEK293T cells and treated with the indicated concentration of YnPal for 4 h. After lysis and IP with anti-FLAG resin, samples were subjected to CuAAC with TAMRA azide and separated by SDS-PAGE. ZDHHC20 loading and input were measured by in-gel fluorescence and anti-ZDHHC20 immunoblot (n = 3 independent biological replicates). ( c-d ) Thioester dependence of ZDHHC20 labeling was demonstrated upon treatment of YnPal and C18-Bz treated samples with 0.8 M neutralized NH 2 OH following IP and CuAAC with TAMRA azide (n = 3 independent biological replicates). ( e-f ) Time-course measuring 15 µM YnPal labeling of ZDHHC20 WT expressing HEK293T cells (n = 3 independent biological replicates). ( g-h ) Labeling activity of the indicated concentrations of YnPal in FLAG-tagged ZDHHC20[Y181G] and ZDHHC20[Y181G/C156S] expressing HEK293T cells (n = 3 independent biological replicates). The average (n = 3 independent biological replicates) loading ( b , d , f and h ) was reported as a percent of the maximal fluorescent: input ratios ± S.D. between treatments with and without hydroxylamine. ( i-l ) Probe chain-length was optimized against ZDHHC20[Y181G] using cell-based loading ( i-j ) and transfer ( k-l ) assays in HEK293T using ZDHHC20 WT (W) and ZDHHC20[Y181G] (M). ( i ) HEK293T cells were treated with 15 µM acetyl bumped probes of L = 16, 18 and 20 for 4 h and enzyme loading assessed by in-gel fluorescence following anti-FLAG IP and CuAAC with TAMRA azide (n = 3 independent biological replicates). ( k ) HEK293T cells co-expressing ZDHHC20[Y181G] and HA-Ifitm3 were treated with 15 µM 18-Ac or 20-Ac for 4 h with loading and transfer of the probe assessed following by anti-FLAG/anti-HA IP and CuAAC with TAMRA azide (n = 3 independent biological replicates). ( j-l ) The average (n = 3 independent biological replicates) loading and transfer activity were reported as a percent of the maximal fluorescent/input ratios ± S.D. The two tailed unpaired t-test of Prism 9.0 was used to determine p-values and are note above relevant comparisons.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Labeling, Construct, Transfection, Concentration Assay, Lysis, SDS Page, Fluorescence, Western Blot, Expressing, Activity Assay, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Wild-type (WT), Y181G (YG), C156S (CS) and Y181G/C156S (YGCS) FLAG-tagged ZDHHC20 constructs were transfected into HEK293T cells and purified by anti-FLAG agarose affinity chromatography. After enzyme elution with 3X FLAG-peptide, buffer was exchanged using 50 kDa M.W. cut-off protein concentrator tubes and sample concentration determined using a BSA standard curve. All samples were run on SDS-PAGE gels and protein visualized by Coomassie staining (n = 2 independent experiments). ( b ) An enzyme-coupled assay monitoring ZDHHC20 autoacylation was established using commercial α-ketoglutarate dehydrogenase enzyme (KDH) along with its substrates α-ketoglutarate (α-KG), thiamine pyrophosphate (TPP) and NAD+. Optimization of α-ketoglutarate dehydrogenase (KDH) ( c ) and WT ZDHHC20 ( d ) concentrations. Pal-CoA ( e ) and 18-Bz-CoA ( f ) KDH activities were determined in the absence of ZDHHC20, to establish background rates for each probe. ( g ) 18-Bz-CoA displayed significant background activity in the KDH assay without ZDHHC20. Reaction rates for ZDHHC20[C156S] ( h ) and ZDHHC20[Y181G, C156S] ( i ) treated with Pal-CoA or 18-Bz-CoA. Michaelis-Menten plots generated by plotting average (n = 3 independent experiments) reaction rates (NADH generated (µM)/min) ± S.D.) versus lipid concentration (µM) using Prism 9.0. For reactions with 18-Bz-CoA, the basal rates at all concentrations tested were subtracted from the corresponding total reaction rates.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Construct, Transfection, Purification, Affinity Chromatography, Concentration Assay, SDS Page, Staining, Activity Assay, Generated

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: FLAG-tagged ZDHHC20 WT and ZDHHC20[Y181G] expressing HEK293T cells were treated with the indicated concentration of 18-Bz ( a-b ) for 4 h in cell-based loading assays (n = 3 independent biological replicates). ( c-d ) FLAG-tagged ZDHHC20[Y181G] and HA-Ifitm3 expressing HEK293T cells were treated with 15 µM 18-Bz for the indicated time in cell-based loading and transfer assays (n = 3 independent biological replicates). ( e-f ) FLAG-tagged ZDHHC20 WT and ZDHHC20[Y181G] expressing HEK293T cells were treated with 15 µM 18-Bz for the indicated times (n = 3 independent biological replicates). Lysates were clicked with TAMRA azide then analyzed by in-gel fluorescence and SDS-PAGE; note YG-dependent labeling of substrate protein bands (*). Input was assessed by anti-ZDHHC20 (D20) immunoblot. The average (n = 3 independent biological replicates) loading ( b, d & f ) and transfer ( d ) were reported as a percent of the maximal fluorescent: input ratios ± S.D. ( g-h ) The effect of FBS concentration on ZDHHC20 loading and transfer. ( g ) FLAG-tagged wild-type (WT) or ZDHHC20[Y181G] (M) and HA-Iftim3 expressing HEK293T cells were treated with 15 µM YnPal or 18-Bz in the presence of 0.5 or 10% FBS for 4 h in cell-based transfer assays. ( h ) The average (n = 3 independent biological replicates) loading and transfer were reported as a percent of the maximal fluorescent: input ratio ± S.D. The two tailed unpaired t-test of Prism 9.0 was used to determine p-values and noted above the relevant comparisons.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Expressing, Concentration Assay, Fluorescence, SDS Page, Labeling, Western Blot, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Left: representative confocal microscopy images showing average signal of Z-stacks of HEK293T cells transiently co-expressing ZDHHC20 WT HA-tagged and Y181G mutant FLAG-tagged. Each image shows signal for HA (magenta), FLAG (green), p-cadherin as plasma membrane marker (yellow), nucleus (blue) and a composite image of all signals. Scale bar at the bottom right marks 20 mm, while the other white line highlights the region of interest (ROI) used for image analysis. Right: plot showing normalized fluorescence signal for each of the channels in the ROI (1 biological replicate). ( b ) As a , but with using Gm130 as Golgi marker (1 biological replicate).

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Confocal Microscopy, Expressing, Mutagenesis, Membrane, Marker, Fluorescence

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , Chemical proteomic OBH workflow for enrichment and identification of S -acyltransferase substrates and S -acylation sites by LC–MS/MS. b , Chemical proteomic analysis of ZDHHC20 substrates in HEK293T cells (15 µM 18-Bz, 8 h). Enrichment in ZDHHC20(Y181G) cells over WT ZDHHC20 reveals selective ZDHHC20 loading (red triangle), and significantly enriched substrates (green circles) selected for further validation (red circles), with site identification data (blue triangles; Student’s two-tailed unpaired t test, S 0 = 0.5, adjusted FDR = 0.01, n = 4 independent biological replicates per condition). c , d , LC–MS/MS spectrum corroborating reported sites of CD151 ( c ) S -acylation at Cys11 and Cys15 and of STX7 ( d ) S -acylation at Cys28 (see also Extended Data Fig. ). e , S -acylated proteome profiling using YnPal. HEK293T cells transiently transfected with WT ZDHHC20 or ZDHHC20(Y181G) were treated with 15 µM YnPal for 8 h before processing using the on-bead digestion workflow. Substrates highlighted in green had been identified using a chemical–genetic system (Student’s two-tailed unpaired t test, S 0 = 0.5, adjusted FDR = 0.01, n = 4 independent biological replicates per condition). f , g , Validation of S -acylation for substrates at endogenous levels. HEK293T cells transiently transfected with WT ZDHHC20 (W) or ZDHHC20(Y181G) (M) were treated with 15 µM 18-Bz ( f ) or 15 µM YnPal ( g ) for 24 h. Lysates were clicked with biotin azide before enrichment on neutravidin magnetic beads. Representative immunoblots are shown for input and pull-down signals ( n = 2 independent biological replicates). h , Venn diagram of putative ZDHHC20 substrates identified in HEK293T, MDA-MB231 and PANC1 cells. i , Statistical overrepresentation analysis of putative ZDHHC20 substrate cellular compartment (Slim)-GO terms compared to the full human genome using the PANTHER classification system showing terms with >9 −log ( P value) from an FDR-adjusted two-tailed Fisher’s exact test.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Transfection, Magnetic Beads, Western Blot

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Full gel and western blots of all replicates corresponding to the chemical proteomics ZDHHC20 substrate identification (Fig. ) performed in HEK293T cells. A portion of the lysate was clicked with TAMRA azide for analysis by in-gel fluorescence. The bands present at ~35 kDa in ZDHHC20[Y181G] (M) lanes but absent in WT ZDHHC20 lanes indicate selective loading of 18-Bz on ZDHHC20[Y181G] over WT-ZDHHC20. Anti-FLAG WB indicates similar expression levels of WT construct compared to ZDHHC20[Y181G] construct. Vinculin is used as loading control (n = 4 independent biological replicates). ( b-e ) Chemical proteomics ZDHHC20 substrate detection with 18-Bz probe (15 µM) in ( b ) PANC1 cells and (C) MDA-MB-231 cells. Cells were transiently transfected with WT ZDHHC20 versus ZDHHC20[Y181G] (M) then clicked with biotin azide and enriched on neutravidin agarose for proteomic processing. Significantly enriched putative substrates (Student’s two tailed unpaired t-test S0 - 0.5, adjusted FDR - 0.01) are shown as green circles, hits with site identification data are shown in as blue triangles and other validated substrates are highlighted as red circles. 200 putative ZDHHC20 substrates are identified in (B) PANC1 cells and 50 putative substrates in ( c ) MDA-MB-231 cells. ( d-e ) Gel and western blots corresponding to the volcano plot in ( a-b ) where a portion of the lysate was clicked with TAMRA azide as described in B (n = 4 independent biological replicates). (f) Statistical over/underrepresentation analysis of putative ZDHHC20 substrate biological process GO-terms compared to a reference list containing reported S -acylated proteins (SwissPalm) using the PANTHER classification system showing terms with >1.5 -Log 10 (p-value) from an FDR adjusted Fisher’s exact two tailed test. (g-h) PTRH2 Site ID analysis and quantification. (g ) Validation of HA-PTRH2 S -acylation by ZDHHC20 using the bumped probe 18-Bz and S -acylation site mutants. Representative images (n = 3 independent biological replicates) for TAMRA signal are shown, as well as for HA and FLAG immunoblots for HA pull down and input. Calnexin was used as loading control. (h ) Bar plot showing the ratio of TAMRA fluorescence and HA pulldown signal of PTRH2 cysteine mutants as a percentage of WT PTRH2 ratio. The two tailed unpaired t-test statistical module of Prism 9.0 was used to calculate p-values and noted above relevant comparisons. (i) Profiling of Flp-in T-Rex substrates ZDHHC20 cell lines. The average (n = 3 independent biological replicates) Fold change of FLAG signal is reported as a percent of the maximal ratios ± S.D. The unpaired t-test statistical module of Prism 9.0 was used to determine p-values and noted above relevant comparisons. Related to main Fig. .

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Western Blot, Fluorescence, Expressing, Construct, Control, Transfection, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a-b ) Gels and corresponding volcano plots for HEK293T cells treated with 15 μM 20- c Pr for 8 h in the presence of ZDHHC15 WT or ZDHHC15[Y184G]. ( a ) Lysates were subjected to CuAAC with TAMRA azide to assess probe incorporation and expression levels of FLAG-tagged ZDHHC and the loading control vinculin. ( b ) Volcano plot showing enrichment of putative ZDHHC15 substrates by ZDHHC15[Y184G] (Student’s two tailed unpaired T-test, S 0 0.5, adjusted FDR 0.01, n = 4 independent biological replicates) of matched lysates processed by OBH workflow and analyzed by LC-MS/MS. The positive control ZDHHC15 (red triangle) shows enrichment and many sites of modification (blue triangle) were identified through our OBH workflow. ( c-d ) Gels and corresponding volcano plots for PANC1 cells treated as described in ( a-b ). ( e-f ) Gels and corresponding volcano plots for HEK293T cells treated with 15 μM 20- Bz for 8 h in the presence of ZDHHC7 WT or ZDHHC7[L57G]. ( e ) Lysates were subjected to CuAAC with TAMRA azide to assess probe incorporation and expression levels of FLAG-tagged ZDHHCs and the loading control vinculin. ( f ) Volcano plot showing enrichment of putative ZDHHC7 substrates by ZDHHC7[L57G] (Student’s two tailed unpaired T-test, S 0 0.5, adjusted FDR 0.05, n = 4 independent biological replicates) of matched lysates processed by OBH workflow and analyzed by LC-MS/MS. ( g-h ) Overlap among ZDHHC substrate profiles for ZDHHC7, ZDHHC15, and ZDHHC20 (Student’s two tailed unpaired t-test, S 0 0.5, adjusted FDR 0.05, n = 4 independent biological replicates). ( g ) Volcano plot of ZDHHC20 OBH shown in Fig. , with unique putative substrates; putative substrates shared with ZDHHC7 and/or ZDHHC15 highlighted. ( h ) Volcano plot of ZDHHC7 OBH shown in Supplementary Fig. with unique putative substrates; putative substrates shared with ZDHHC15 and/or ZDHHC20 highlighted.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Expressing, Control, Two Tailed Test, Liquid Chromatography with Mass Spectroscopy, Positive Control, Modification

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , b , ZDHHC20(Y181G) retains exquisite selectivity for specific cysteines on substrates IFITM3 ( a ) and PI4K2A ( b ; n = 3 independent biological replicates average ± s.d.), matching previously reported labeling, with the 18-Bz bumped probe. c , Validation of HA-STX7 S -acylation by ZDHHC20 using the bumped probe 18-Bz and S -acylation site mutants (C28A) and (C239A). Representative images ( n = 3 independent biological replicates average ± s.d.) for TAMRA signal are shown, as well as for HA and FLAG immunoblots for HA pull down and input. Calnexin was used as loading control. d , e , Validation of HA-VAMP3 and HA-BCAP31 site S -acylation by ZDHHC20 using the bumped probe 18-Bz and S -acylation site mutants, VAMP3(C76A) and BCAP31(C23A). d , Cell-based transfer assays were performed without FLAG-ZDHHC20 and HA-VAMP3 enrichment, but rather with direct labeling of cell lysates by TAMRA-azide click followed by SDS–PAGE and anti-HA, anti-FLAG and anti-vinculin immunoblot analysis. e , FLAG-ZDHHC20 and HA-BCAP31 constructs were enriched before TAMRA-azide click labeling. f , Confirmation of trans -auto- S -acylation in peripheral cysteines on a catalytically dead C-HA-ZDHHC20(C156S) (D) by a mutant C-FLAG-ZDHHC20(Y181G) (M) with 15 μM 18-Bz. Catalytically dead C-FLAG-ZDHHC20(Y181G) (DM) did not transfer the probe to D. Cells transfected with an empty vector (E) were used as negative control. HA- and FLAG-tagged ZDHHC20 constructs were transiently cotransfected into HEK293T cells and treated with 15 μM 18-Bz for 4 h. After cell lysis, constructs were separately enriched on anti-HA and anti-FLAG resins, clicked with TAMRA-azide and separated by SDS–PAGE. Loading and input were visualized by in-gel fluorescence and immunoblot, respectively. The average ( n = 3 independent biological replicates) loading and transfer activity were reported as a percent of the maximal fluorescent:input ratios ± s.d. a , c , f , The two-tailed unpaired t test of Prism 9.0 was used to determine P values and noted above relevant comparisons.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Labeling, Western Blot, Control, SDS Page, Construct, Mutagenesis, Transfection, Plasmid Preparation, Negative Control, Lysis, Fluorescence, Activity Assay, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , Profile of WT ZDHHC20 (W) or ZDHHC20(Y181G) Flp-In 293 T-REx cell lines treated with 18-Bz (15 µM, 24 h). Lysates were clicked with TAMRA azide and then analyzed by in-gel fluorescence and SDS–PAGE. Note that the asterisk represents YG-dependent labeling of substrate protein bands. b , Comparison of protein expression levels between doxycycline induction of Flp-In 293 T-REx cells and overexpression by transient expression in HEK293T cells. Representative immunoblots are shown for FLAG at high or low exposure, to probe for ZDHHC20 WT versus ZDHHC20(Y181G), and calnexin as loading control ( n = 3 independent biological replicates). c , In Flp-In 293 T-REx cells ZDHHC20(Y181G) retains exquisite selectivity for its substrate IFITM3 with the 18-Bz bumped probe, as seen in prior experiments. d , Chemical proteomic analysis of ZDHHC20 substrates in Flp-In 293 T-REx cells (15 µM 18-Bz, 24 h). Enrichment in T-REx ZDHHC20(Y181G) cells over T-REx WT ZDHHC20 reveals selective ZDHHC20 modification of substrates (green) (Student’s two-tailed unpaired t test, S 0 = 0.5, adjusted FDR = 0.01, n = 4 independent biological replicates per condition). e , f , Validation of S -acylation for T-REx ZDHHC20(Y181G) substrates at endogenous levels. Flp-In 293 T-REx cells, WT ZDHHC20 (W) or ZDHHC20(Y181G) (M), induced with doxycycline for 24 h, were treated with 15 µM 18-Bz ( e ) or YnPal ( f ) for 24 h. Lysates were clicked with biotin azide before enrichment on neutravidin magnetic beads. Representative immunoblots are shown for input and pull-down signals ( n = 2 independent replicates).

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Fluorescence, SDS Page, Labeling, Comparison, Expressing, Over Expression, Western Blot, Control, Modification, Two Tailed Test, Magnetic Beads

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Untreated (UT) or gRNA/CAS9 treated (pSpCas9(BB)-2A-Puro, PX459 plasmid) HEK293T cells were probed with anti-ZDHHC20 (D20) and –vinculin antibodies. Cells treated with gRNA1/CAS9 resulted in knockdown (KD); whereas cells treated with gRNA2/CAS9 yielded two ZDHHC20-knockout (D20-KO) clones: KO1 and KO2 (n = 2 independent biological replicates). ( b ) WT or KO2 HEK293T cells were transfected with HA-IFITM3 and empty vector or C-FLAG-tagged ZDHHC20. Cells were then treated with 15 mM YnPal for 4 h before being harvested and lysed. IFITM3 and D20 were enriched in one pot with a mix of anti-HA and –FLAG resins before being treated with TAMRA-azide and click reagents. Tagged proteins were eluted from beads with 1X Laemmli buffer and separated by SDS-PAGE. YnPal ZDHHC20-loading and transfer to IFITM3 and input were visualized by in-gel fluorescence and anti-HA and -FLAG immunoblot, respectively (n = 2 independent biological replicates). ( c ) The average (n = 3 independent biological replicates) loading and transfer activity was reported as a percent of the maximal D20 fluorescent: input ratio and as a percent of the WT IFITM3 (empty vector) fluorescent: input ratio ± S.D. The two tailed unpaired t-test of Prism 9.0 was used to determine p-values and noted above relevant comparisons ( d-g ) WT HEK293T cells, two ZDHHC20 KO clones, and one partial knockdown (KD) clone were treated with 15 μM YnPal for 8 h. As a control for lipidation, HEK293T cells were treated with palmitic acid (Pal) and also taken through the experiment. Samples were then clicked with biotin-TAMRA-azide, 10% of which was analyzed by SDS-PAGE, in-gel fluorescence, and anti-tubulin western blot ( d ) (n = 3 independent biological replicates). The remainder was enriched on dimethylated neutravidin beads and digested for LC-MS/MS LFQ analysis. (E-G) Whilst a small number of proteins are identified as being significantly enriched/depleted (Student’s two tailed unpaired T-test S 0 – 0.1, adjusted FDR – 0.05), they are few in number and none are consistently found which correspond to our putative chemical genetic substrates found in HEK293T cells. ( f ) Analysis of YnPal treated cells against Pal shows a large number a potentially lipidated proteins have been identified, with many well validated S -acylation proteins identified, some of which have been highlighted in blue.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Plasmid Preparation, Knockdown, Knock-Out, Clone Assay, Transfection, SDS Page, Fluorescence, Western Blot, Activity Assay, Two Tailed Test, Control, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Schematic representation of TurboID fusion proteins used for proximity labeling experiments. ( b-c ) Confirmation of the expression of each fusion protein by western blot after generation of ‘Jump-in’ cell lines using either anti-V5 antibody ( b ) (n = 2 independent biological replicates) or an anti-GFP antibody ( c ) (n = 2 independent biological replicates). The labeling efficiency of the TurboID biotin ligase was confirmed by the addition of 500 μM biotin for the indicated times. Only those cells expressing the ligase show an increase in the biotinylation of proteins, as determined by Streptavidin conjugated HRP, compared to the UT HEK293T cells, and also in a time dependent manner. ( d ) Volcano plot showing the enrichment of proteins when comparing the C-terminally tagged ZDHHC20 with the N-terminally tagged construct (Student’s two tailed unpaired t-test S 0 – 0.1, adjusted FDR 0.01). There does appear to be a preference for either the N- or C- terminus for some interactors, none of these correspond to our chemical genetic hits. ( e ) Volcano plot showing the enrichment of proteins when comparing the N-terminally tagged ZDHHC20 with the Turbo GFP construct (Student’s two tailed unpaired t-test S 0 – 0.1, adjusted FDR 0.01). ( f ) TurboID-based proximity-labeing enabled detection of ZDHHC20 (D20) interactors. Volcano plot showing the mean log 2 difference in protein group intensities between N-TurboID-ZDHHC20 and TurboID-GFP clones (Student’s two tailed unpaired T-test S 0 – 0.1, adjusted FDR 0.01).

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Labeling, Expressing, Western Blot, Construct, Two Tailed Test, Clone Assay

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , Structure-guided ZDHHC engineering exemplified for ZDHHC7 (see also Extended Data Figs. and ). ZDHHC7 homology model (yellow/orange) overlayed on experimental ZDHHC20 structure (dark green) identifies a potential hole-generating amino acid (Leu57) on an adjacent helix in the vicinity of ZDHHC20 Tyr181; lipid density (blue mesh), and length/size probe analysis identifies a mutant/probe pair (ZDHHC7(L57G)/20-Bz) with optimal activity and selectivity over WT ZDHHC7. b , Bump-hole analysis of N-FLAG-tagged WT ZDHHCs or mutant ZDHHCs ZDHHC3(I182G) (D3), ZDHHC7(L57G) (D7), ZDHHC11(M181A) (D11) and ZDHHC15(Y184G) (D15) in HEK293T cell-based loading assays using 15 µM corresponding optimized probe. c , Average ( n = 3 independent biological replicates) loading reported as a percent of maximal fluorescent:input ratio ± s.d. P values determined by Prism 9.0 two-tailed unpaired t test statistical module and noted above relevant comparisons. d , ZDHHC15 substrate discovery in HEK293T cells treated with 15 µM 20- c Pr in HEK293T cells using the OBH workflow. In total, 107 chemical–genetic ZDHHC15 substrates were identified (Student’s two-tailed unpaired t test, S 0 = 0.5, adjusted FDR = 0.01, n = 4 independent biological replicates). Substrates unique or in common with parallel analyses for DHHC7 and DHHC20 in HEK293T cells are highlighted (Extended Data Fig. ). e , Overlap of chemical–genetic ZDHHC substrates identified in HEK293T cells. Of 301 total substrates, only 87 are shared by 2 or more family members, suggesting distinct substrate pools for each ZDHHC.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Mutagenesis, Activity Assay, Two Tailed Test

Journal: Cell reports

Article Title: Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina

doi: 10.1016/j.celrep.2024.114005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Micro BCA TM Protein Assay Kit , ThermoFisher Scientific , Cat# #23235 RRID: N/A.

Techniques: Control, Affinity Purification, Recombinant, Bicinchoninic Acid Protein Assay, Western Blot, Electron Microscopy, RNAscope, Multiplex Assay, Mass Spectrometry, Mutagenesis, Software, Microscopy

Journal: The EMBO Journal

Article Title: Limited oxygen in standard cell culture alters metabolism and function of differentiated cells

doi: 10.1038/s44318-024-00084-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Maintenance of iPSCs: Human induced pluripotent stem cells (iPSCs) were maintained on vitronectin XFTM (10 μg/mL, StemCell Technologies)-coated plates and in Essential 8 (E8) medium consisting of DMEM/F12 (Gibco), l -ascorbic acid 2-phosphate (1%), insulin-transferrin-selenium solution (2%, Life Technologies), sodium bicarbonate (0.7%), and Penicillin/Streptomycin (P/S) (1%), freshly supplemented with TGFβ (10 ng/mL, R&D) and FGF2 (12 ng/mL, Qkine).

Techniques: Recombinant, Affinity Purification, Sequencing, Saline, Bicinchoninic Acid Protein Assay, Reverse Transcription, SYBR Green Assay, Software, Imaging, Gas Chromatography, Mass Spectrometry, Microscopy