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 Proliferation

Article Title: Establishment of a porcine pancreatic stem cell line using T‐REx ™ system‐inducible Wnt3a expression

doi: 10.1111/cpr.12188

Figure Lengend Snippet: The expression of GFP and Wnt3a was regulated by Dox in a time‐ and dose‐dependent manner. (a) The expression of GFP in PSCs was observed under fluorescence microscope after treatment with different concentrations of Dox (0, 0.1, 1, 5, 10 μg/ml) for different times (0, 24, 48 and 72 h) (bar = 200 μm). (b) The relative Wnt3a mRNA expression level in PSCs after Dox treatment for different times analyzed by QRT‐PCR. (c) The relative Wnt3a mRNA expression level in PSCs after treatment with different concentrations of Dox analysed by QRT‐PCR. (d) GFP‐positive PSCs were quantified by flow cytometric after induced by Dox. (e) The histogram of statistical result for Fig. d. (f) The Wnt3a protein expression in PSCs after Dox addition for different times detected by Western blotting. (g) The Wnt3a protein expression in the supernate of cultured PSC after Dox treatment for 72 h. (h) Immunostaining of Wnt3a and active β‐catenin in PSCs after treatment with or without Dox (bar = 100 μm).

Article Snippet: Wnt3a was amplified from mouse tail vein‐derived samples using Vazyme Blood Direct PCR Kit (Vazyme, Nanjing, China), by PCR.

Techniques: Expressing, Fluorescence, Microscopy, Quantitative RT-PCR, Western Blot, Cell Culture, Immunostaining

Journal: Oncology letters

Article Title: Luteolin induces apoptosis by impairing mitochondrial function and targeting the intrinsic apoptosis pathway in gastric cancer cells.

doi: 10.3892/ol.2023.13913

Figure Lengend Snippet: Figure 1. Viability of HGC‑27 and MFC cells is affected by luteolin. HGC‑27 and MFC cells were treated with different doses of luteolin for 24 h. Human gastric cancer HGC‑27 cells viability was inhibited by luteolin. (A) Morphological changes of HGC‑27 cells were observed under a light microscope (magni‑ fication, x100). (B) HGC‑27 cell viability was detected by the Cell Counting Kit‑8 assay. Mouse forestomach carcinoma MFC cells viability was inhibited by luteolin. (C) Morphological changes of MFC cells were observed under a light microscope (magnification, x200). (D) MFC cell viability was detected using the Cell Counting Kit‑8 assay. The decrease in the viability rate was analyzed using the GraphPad Prism 6.0 software. Experiments were repeated at least three times. **P<0.01 vs. DMSO group. HGC‑27, human gastric cancer HGC‑27 cell line; MFC, mouse forestomach carcinoma cell line.

Article Snippet: Luteolin was dissolved in dimethyl sulfoxide (DMSO) (D8371, Solarbio, Beijing, China) and diluted with complete medium to the required concentration.

Techniques: Light Microscopy, CCK-8 Assay, Software

Journal: Oncology letters

Article Title: Luteolin induces apoptosis by impairing mitochondrial function and targeting the intrinsic apoptosis pathway in gastric cancer cells.

doi: 10.3892/ol.2023.13913

Figure Lengend Snippet: Figure 2. HGC‑27 and MFC cells apoptosis are induced by luteolin. Morphological changes indicative of (A) HGC‑27 cells and (B) MFC cells apoptosis were observed using Hoechst 33258 staining method under an inverted fluorescence microscope (magnification, x100). Red arrows indicated the apparent apoptotic morphological features, such as karyopyknosis, nucleosome and chromosome condensation. Following double‑staining with Annexin‑V FITC and PI, the flow cytometry was used to test the apoptosis in (C) HGC‑27 cells and (E) MFC cells, and the quantitative determination of apoptosis of (D) HGC‑27 cells and (F) MFC cells was showed on the histogram. Experiments were repeated at least three times. *P<0.05 and **P<0.01 vs. DMSO group. HGC‑27, human gastric cancer HGC‑27 cell line; MFC, mouse forestomach carcinoma cell line.

Article Snippet: Luteolin was dissolved in dimethyl sulfoxide (DMSO) (D8371, Solarbio, Beijing, China) and diluted with complete medium to the required concentration.

Techniques: Staining, Fluorescence, Microscopy, Flow Cytometry

Journal: Oncology letters

Article Title: Luteolin induces apoptosis by impairing mitochondrial function and targeting the intrinsic apoptosis pathway in gastric cancer cells.

doi: 10.3892/ol.2023.13913

Figure Lengend Snippet: Figure 3. ROS accumulation in HGC‑27 and MFC cells is induced by luteolin. The fluorescence intensity of (A) HGC‑27 cells and (B) MFC cells were visualized under a fluorescence inverted microscope (magnification, x100). Luteolin‑induced ROS levels were detected using DCFH‑DA staining and flow cytometry in (C) HGC‑27 cells and (E) MFC cells. The quantitative analysis of ROS levels in (D) HGC‑27 cells and (F) MFC cells was showed on the histo‑ gram. (G) SOD activity was assessed in HGC‑27 and MFC cells using a microplate reader at an absorbance of 560 nm. Experiments were repeated at least in triplicate. Data were presented as mean ± SD. **P<0.01 vs. DMSO group. ROS, reactive oxygen species; SOD, superoxide dismutase; HGC‑27, human gastric cancer HGC‑27 cell line; MFC, mouse forestomach carcinoma cell line.

Article Snippet: Luteolin was dissolved in dimethyl sulfoxide (DMSO) (D8371, Solarbio, Beijing, China) and diluted with complete medium to the required concentration.

Techniques: Fluorescence, Inverted Microscopy, Staining, Flow Cytometry, Activity Assay

Journal: Oncology letters

Article Title: Luteolin induces apoptosis by impairing mitochondrial function and targeting the intrinsic apoptosis pathway in gastric cancer cells.

doi: 10.3892/ol.2023.13913

Figure Lengend Snippet: Figure 4. Mitochondrial membrane potential, ATP levels and some enzyme activities in HGC‑27 and MFC cells induced by luteolin. The luteolin‑treated (A) HGC‑27 cells and (C) MFC cells were stained with JC‑1, and analyzed using flow cytometry. Quantitative statistics of the mitochondrial membrane potential was based on flow cytometry in (B) HGC‑27 cells and (D) MFC cells. The (E) ATP levels, (F) Na+/K+‑ATPase activities and (G) Ca2+/Mg2+‑ATPase activities were showed based on the microplate system at the absorbance values of 660 nm. The data were presented as mean ± SD. The experiments were repeated in triplicate. *P<0.05, **P<0.01 vs. DMSO group. HGC‑27, human gastric cancer HGC‑27 cell line; MFC, mouse forestomach carcinoma cell line.

Article Snippet: Luteolin was dissolved in dimethyl sulfoxide (DMSO) (D8371, Solarbio, Beijing, China) and diluted with complete medium to the required concentration.

Techniques: Membrane, Staining, Flow Cytometry

Journal: Oncology letters

Article Title: Luteolin induces apoptosis by impairing mitochondrial function and targeting the intrinsic apoptosis pathway in gastric cancer cells.

doi: 10.3892/ol.2023.13913

Figure Lengend Snippet: Figure 5. Luteolin induces the enzyme activities of the METC complexes in HGC‑27 and MFC cells. Complexes (A) Ⅰ, (B) Ⅲ and (C) Ⅴ were assessed by testing kits. The data were obtained using a microplate reader at 340, 550 and 660 nm, respectively. The data were presented as mean ± SD. The experiments were performed at least in triplicate. *P<0.05 and **P<0.01 vs. DMSO group; HGC‑27, human gastric cancer HGC‑27 cell line; MFC, mouse forestomach carcinoma cell line.

Article Snippet: Luteolin was dissolved in dimethyl sulfoxide (DMSO) (D8371, Solarbio, Beijing, China) and diluted with complete medium to the required concentration.

Techniques:

Journal: Oncology letters

Article Title: Luteolin induces apoptosis by impairing mitochondrial function and targeting the intrinsic apoptosis pathway in gastric cancer cells.

doi: 10.3892/ol.2023.13913

Figure Lengend Snippet: Figure 6. Luteolin unbalanced Bcl‑2 and Bax protein expression in HGC‑27 and MFC cells. Bcl‑2 and Bax protein levels in (A) HGC‑27 cells and (B) MFC cells were examined using western blot. (C) Ratio between Bcl‑2 and Bax protein expression levels is showed in the histogram. The data were presented as mean ± SD. Experiments were repeated at least in triplicate. *P<0.05 and **P<0.01 vs. DMSO group. Bcl‑2, B cell lymphoma‑2; Bax, Bcl‑2‑associated X; HGC‑27, human gastric cancer HGC‑27 cell line; MFC, mouse forestomach carcinoma cell line.

Article Snippet: Luteolin was dissolved in dimethyl sulfoxide (DMSO) (D8371, Solarbio, Beijing, China) and diluted with complete medium to the required concentration.

Techniques: Expressing, Western Blot

Journal: Cancers

Article Title: Targeted Disruption of E6/p53 Binding Exerts Broad Activity and Synergism with Paclitaxel and Topotecan against HPV-Transformed Cancer Cells

doi: 10.3390/cancers14010193

Figure Lengend Snippet: The cell-cycle arrest induced by Cpd12 occurs in the G1 phase. ( A ) The effects of Cpd12, the inactive small molecule Cpd3, or DMSO, on cell cycle modulation were evaluated by cytofluorimetric analysis of HPV-positive cervical (ME180 and MS751) and head-and-neck (SCC152) cancer cells treated for 24 h and then stained with PI. Bar graphs represent the percentage of cells in the different phases of the cell cycle (G0/G1, M, G2/S) as the mean of three independent experiments. ( B ) Cell-cycle distributions of HPV-positive cervical (ME180, MS751, HeLa, CaSki, and SiHa) and head-and-neck (SCC152) cancer cells treated with Cpd12 (50 µM) or DMSO for 24 h were determined through immunofluorescence assays. Cells in either the G0, G1, S, G2, or M phase were determined as a function of Ki67 and nuclear staining, and fluorescence intensities were quantified from maximum intensity projections of Z-stack images acquired with a 600× magnification. Total numbers of cells were: n DMSO = 463 and n Cpd12 = 304 for ME180 cells, n DMSO = 303 and n Cpd12 = 316 for MS751 cells, n DMSO = 314 and n Cpd12 = 267 for SCC152 cells, n DMSO = 303 and n Cpd12 = 270 for HeLa cells, n DMSO = 388 and n Cpd12 = 330 for CaSki cells, and n DMSO = 336 and n Cpd12 = 244 for SiHa cells. Nuclei were stained with DRAQ5. 2N: diploid DNA content, 4N: tetraploid DNA content.

Article Snippet: HPV-positive HeLa (HPV18), CaSki (HPV16), SiHa (HPV16), ME180 (HPV68), MS751 (HPV45), and HPV-negative C33A cervical cancer cells, HPV-positive SCC152 (HPV16) and HPV-negative FaDu head-and-neck cancer cells, and human foreskin fibroblasts (HFF) were all from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Staining, Immunofluorescence, Fluorescence

Journal: Cancers

Article Title: Targeted Disruption of E6/p53 Binding Exerts Broad Activity and Synergism with Paclitaxel and Topotecan against HPV-Transformed Cancer Cells

doi: 10.3390/cancers14010193

Figure Lengend Snippet: The Cpd12-induced rescue of p53 affects the migration capacity of HPV-transformed cells. ( A ) Confluent monolayers of HeLa, CaSki, and ME180 cervical cancer cells were scraped up two times and then treated with increasing concentration of Cpd12 (10, 25, and 50 µM) in low serum medium. The wound closure was monitored before treatment and after 12, 24, and 48 h, and representative images were taken using a bright-field inverted microscope (100× magnification). ( B ) HeLa cells were treated with Cpd12 (50 μM) for 24 h. Then, cell migration was evaluated by transwell assays after an incubation of other 24 h in Cpd12-free medium. Representative images of migrated cells fixed and colored with crystal violet were taken using a bright-field inverted microscope (100× magnification). In both panels, cells treated with DMSO or Cpd3 (50 μM) were included as negative controls.

Article Snippet: HPV-positive HeLa (HPV18), CaSki (HPV16), SiHa (HPV16), ME180 (HPV68), MS751 (HPV45), and HPV-negative C33A cervical cancer cells, HPV-positive SCC152 (HPV16) and HPV-negative FaDu head-and-neck cancer cells, and human foreskin fibroblasts (HFF) were all from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Migration, Transformation Assay, Concentration Assay, Inverted Microscopy, Incubation

Journal: Cancers

Article Title: Targeted Disruption of E6/p53 Binding Exerts Broad Activity and Synergism with Paclitaxel and Topotecan against HPV-Transformed Cancer Cells

doi: 10.3390/cancers14010193

Figure Lengend Snippet: Analysis of the effect of the drug combination on HPV-positive cervical and head-and-neck cancer cells viability.

Article Snippet: HPV-positive HeLa (HPV18), CaSki (HPV16), SiHa (HPV16), ME180 (HPV68), MS751 (HPV45), and HPV-negative C33A cervical cancer cells, HPV-positive SCC152 (HPV16) and HPV-negative FaDu head-and-neck cancer cells, and human foreskin fibroblasts (HFF) were all from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques:

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Western bloting analysis of total PCLP1 expression in B cell lines (Raji, Ramos and Daudi), B cells from four healthy donors (B cells 1–4), and a T cell line (Jurkat). Actin is shown as a loading control. ( B ) PCLP1 expression on the surface of various B cell lines and Jurkat T cell line by flow cytometry. The grey line and the red line in the histograms represent isotype control (mouse IgG) and PCLP1 staining (anti-PCLP1 mAb), respectively. ( C ) Raji cells were stably transfected with pEGFP-PCLP1 (Raji-PCLP1) or pEGFP (Raji-Ctrl) and whole cell extracts from three different clones of each cell type were analyzed for the expression of PCLP1 by Western-blotting using an anti-hPCLP1 mAb. PCLP1: endogenous PCLP1. PCLP1-GFP: ectopic PCLP1. Actin is shown as a loading control. ( D ) Representative fluorescence microscope images of Raji cells stably expressing PCLP1-GFP fusion protein showing the localization of PCLP1-GFP (green) to cell surface and vesicles (I), and centrosome (II), using a 60× objective. Nuclei were stained with Hoechst 33342 (blue). ( E ) Representative fluorescence microscope images of Raji-PCLP1 cells showing the centrosomal localization of PCLP1-GFP fusion protein (green) at interphase (top) and mitosis (bottom). Cells were stained with an anti-γ-tubulin mAb to visualize the pericentriolar area of the centrosome (red). Nuclei were stained with DAPI (blue). Insets at lower right corner show amplified images of the centrosomal region. The size bar indicates 5 µm. ( F ) PCLP1 expression was determined on the surface of malignant and normal B cells from 11 patients with different lymphoma subtypes and 3 patients with ALL by flow cytometry. Median fluorescence intensity (MFI) of PCLP1 expression on malignant B cells was normalized to that detected on normal B cells from the same patient. N: normal B cells; M: malignant B cells; B-ALL: B-cell acute lymphoblastic leukemia; FL: follicular lymphoma; WM: Waldenström macroglobulinemia; HCL: hairy cell leukemia; CLL: chronic lymphocytic leukemia.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Western Blot, Expressing, Control, Flow Cytometry, Staining, Stable Transfection, Transfection, Clone Assay, Fluorescence, Microscopy, Amplification

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Cell proliferation of Raji-PCLP1 and Raji-Ctrl cell clones was measured counting the number of viable cells at different time points of culture in complete medium using the trypan blue exclusion assay. ( B ) Colony formation of Raji-PCLP1 and Raji-Ctrl cells cultured in methylcellulose medium for 11 days. Colonies were stain with crystal violet and counted using an inverted microscope. The graph depicts the number of colonies per well from four independent experiments performed in triplicate. Representative microscope pictures and enlarged images of colony formation after crystal violet staining are shown. Statistical analysis of mean ± SD was calculated using two-tailed paired Student´s t -test. *** P < 0.001. ( C ) Cell-to-cell adhesion was calculated as the number of cell aggregates formed at 72 h of culture. Representative microscope images of cell aggregates using a 10× objective are shown. The size bar indicates 100 µm. ( D ) Cells were incubated in the presence of anti-CD62L mAb or isotype control (mIgG1) (left) or in the presence of anti-CD18 mAb or mIgG1 (right) and the number of aggregates were counted at 2 h. Cells were cultured in the presence of 1 mM EDTA, 1 mM EGTA or vehicle and the number of aggregates were counted at 24 h (middle). The graphs in C and D represents the mean ± SD of the number of aggregates per field containing more than 20 cells from 3 independent experiment and analyzing 10 fields in each experiment. Statistical analysis was calculated using no-paired Student´s t -test (C) and Mann-Whitney U test (D). ** P < 0.01 *** P < 0.001. ( E ) Chemotactic migration of Raji-PCLP1 or Raji-Ctrl toward 100 ng/ml CXCL12 using Transwell chambers. Data represents the mean chemotactic index ± SD normalized to control cells from six independent experiments. Statistical analysis was calculated using Mann-Whitney U test. ** P < 0.01.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Clone Assay, Trypan Blue Exclusion Assay, Cell Culture, Staining, Inverted Microscopy, Microscopy, Two Tailed Test, Incubation, Control, MANN-WHITNEY, Migration

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Raji-Ctrl and Raji-PCLP1 cells were treated with increasing concentrations of vehicle or dexamethasone for 72 hours and cell death was analyzed after Annexin V-PE/7´AAD staining by flow cytometry. The graph on the left shows the mean percentages ± SD of total specific cell death (including early apoptosis, late apoptosis and necrotic cells) from six independent experiments after subtracting the spontaneous cell death (vehicle). The percentage of specific cell death was calculated as follows: [(% lysis of target cell – % spontaneous cell death)/(100% – % spontaneous cell death)] × 100. Representative dot plots showing Annexin V/7AAD staining in cells treated with vehicle or 1000 µM dexamethasone are shown (right). As displayed in the dot plots, the spontaneous cell death (Annexin V PE positive/7ADD negative, Annexin V PE positive/7ADD positive and Annexin V PE negative/7ADD positive cells) in cells cultured with the vehicle was approximately 7.5% in Raji-Ctrl and 5.5% in Raji-PCLP1 cells. ( B ) Expression of PCLP1 on Annexin V and 7ADD double-negative population of cells treated with dexamethasone or vehicle by flow cytometry. ( C ) Raji-Ctrl and Raji-PCLP1 cells were treated with increasing concentrations of vehicle or H 2 O 2 for 6 h and cell death was analyzed as indicated in A. The graph on the left shows the mean percentages ± SD of total cell death from five independent experiments after subtracting the spontaneous cell death (vehicle). Representative dot plots showing Annexin V/7AAD staining in cells treated with vehicle or 200 µM H 2 O 2 are shown (left). As displayed in the dot plots, the spontaneous cell death in the presence of vehicle was approximately 17.0% in Raji-Ctrl and 5.9% in Raji-PCLP1 cells. ( D ) Cells were incubated overnight with increasing concentrations of obinutuzumab or vehicle and cell viability was measured based on Annexin V/7AAD double negative staining relative to vehicle-treated cells. Data show the mean percentages ± SD of viable cells from six independent experiments. ( E ) A representative image of the homotypic adhesion induced by obinutuzumab as visualized by light microscopy is shown. The size bar indicates 100 µm. Statistical analysis was calculated using two-tailed paired Student´s t -test. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Staining, Flow Cytometry, Lysis, Cell Culture, Expressing, Incubation, Negative Staining, Light Microscopy, Two Tailed Test

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Measurement of pH of Raji-PCLP1 or Raji-Ctrl cell culture medium containing glutamine (complete medium) (left) or in the absence of glutamine (right) at the indicated time points. ( B ) Proliferation of Raji-PCLP1 and Raji-Ctrl cells in glucose-free culture medium (left) or glutamine-free culture medium (middle) at different time points. The number of viable cells was normalized to their counterparts grown in complete medium. Data show the mean ± SD of five independent experiments. Proliferation of Raji-PCLP1 and Raji-Ctrl cells in glucose-free conditions and decreasing concentrations of glutamine at 72 h of culture (right). The number of viable cells was normalized to their counterparts grown in complete medium. Data show the mean ± SD of six independent experiments. ( C ) Death of Raji-PCLP1 and Raji-Ctrl cells under glutamine-deprivation conditions. Cells were incubated in the presence of glucose and decreasing concentration of glutamine and the percentage of cell death was determined at 72 h of culture after Annexin V-PE/7´AAD staining by flow cytometry. Data show the mean ± SD of five independent experiments. Representative dot plots showing Annexin V/7AAD staining in cells treated with complete medium (2 mM glutamine/11 mM glucose) or glutamine free medium (0 mM glutamine/11 mM glucose) are depicted. ( D ) Effect of glucose depletion on PCLP1 expression in Raji cells. Cells were cultured in high-glucose medium (11 mM) or low-glucose medium (0.5 mM) for 72 h and the expression of PCLP1 on cell surface was determined by flow cytometry. Data show MFI of PCLP1 expression from five independent experiments. Statistical analysis was calculated using two-tailed paired Student´s t -test. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Cell Culture, Incubation, Concentration Assay, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: Determination of lipid droplet content in Raji-PCLP1 and Raji-Ctrl cells ( A ) or Jurkat-PCLP1 and Jurkat-Ctrl cells ( B ) by fluorescence microscopy. Cells were cultured in complete medium for the indicated times, fixed and stained with Nile Red for the detection of neutral lipid droplets. Ten fields were randomly selected using a 60× oil objective and the total area of lipid droplets per cell was calculated employing ImageJ software. Bar graphs represent the quantitative analysis of lipid droplets content per cell. Mean ± SD of lipid droplet area per cell is shown. Statistical analysis was calculated using Mann-Whitney U test. * P < 0.05. ** P < 0.01. *** P < 0.001. Representative fluorescence microscopy images depicting lipid droplets stained with Nile Red (right panels, yellow). Nuclei were labeled with DAPI (left panels, blue). Digital zoom of a selected area is shown. The size bar indicates 10 µm.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Fluorescence, Microscopy, Cell Culture, Staining, Software, MANN-WHITNEY, Labeling

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Schematic representation of cancer cell metabolism. Once transported into cells, glucose is phosphorylated to glucose 6-phosphate by hexokinases. Glucose 6-phosphate can then be metabolized via aerobic glycolysis or PPP. Through the aerobic glycolysis, glucose 6-phosphate is sequentially converted to pyruvate. Afterwards, pyruvate is transformed to acetyl-CoA via TCA or to lactate. Acetyl-CoA can be used for the synthesis of fatty acids and lipids for biomembrane generation. Glucose 6-phosphate that enters PPP generates ribose, for nucleotides synthesis, and NADPH, for FA synthesis. Glutamine provides intermediates to maintain TCA function and contributes to lipid synthesis and the production of purines and pyrimidines for nucleotide synthesis. Glucose and glutamine also provide the carbon and nitrogen necessary for the synthesis of nonessential amino acids. Metabolic inhibitors are depicted in red color. 2DG: 2-deoxyglucose; 6AN: 6 aminonicotinamide. ( B ) Raji-PCLP1 and Raji-Ctrl cells were cultured for 72 h in complete medium in the absence or in the presence of increasing concentrations of the 2-DG (glycolysis inhibitor). ( C ) To determine the effect of compound 968 (glutaminase inhibitor) on cell proliferation, Raji-PCLP1 and Raji-Ctrl cells were cultured for 7 days in complete medium in the absence or in the presence of increasing concentrations of the inhibitor. ( D ) Raji-PCLP1 and Raji-Ctrl cell were cultured for 72 h in complete medium in the absence or in the presence of increasing concentrations of cerulenin (fatty acid synthase inhibitor). ( E ) Raji-PCLP1 and Raji-Ctrl cells were cultured for 72 h in complete medium in the absence or in the presence increasing concentrations of 6AN (PPP inhibitor). Cell proliferation was assessed by counting live cells on a hemocytometer using trypan blue exclusion dye. The number of viable cells was normalized to their counterparts grown in the absence of the inhibitor. Data show the mean ± SD percentage relative to non-treated cells of five independent experiments. Statistical analysis was calculated using two-tailed paired Student´s t -test. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Transformation Assay, Cell Culture, Two Tailed Test

Journal: Molecules

Article Title: A Litopenaeus vannamei Hemocyanin-Derived Antimicrobial Peptide (Peptide B11) Attenuates Cancer Cells’ Proliferation

doi: 10.3390/molecules23123202

Figure Lengend Snippet: Peptide B11 affects the cell morphology and induces the apoptosis of HeLa cells. ( A ) Changes in cell morphology following treatment with PBS, peptide B11, and 5-FU for 24 h. Micrographs were obtained using an inverted microscope (20×). ( B ) Changes in cell nuclei morphology following treatment with PBS, peptide B11, and 5-FC for 24 h. The 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) stained nuclei were observed with a fluorescence microscope (20×). ( C ) Flow cytometric analysis of apoptosis in HeLa cells after 8 h to 48 h of treatment with PBS, peptide B11, and 5-FU, and staining with Annexin V/propidium iodide (Annexin V/PI). Quadrants: lower-left represent live cells (Annexin V negative/PI negative); lower-right represent early apoptotic/primary apoptotic cells (Annexin V positive/PI negative); upper-right represent late apoptotic/secondary apoptotic cells (Annexin V positive/PI positive); upper-left represent necrotic cells (Annexin V negative/PI positive). The numbers in the respective quadrants indicate the percentage of cells present in that area. Data shown represent one of three independent experiments.

Article Snippet: The ability of peptide B11 to induce cell death in terms of apoptosis was determined using flow cytometry with Annexin V-FITC apoptosis detection kit (Beijing Beyotime Corp, Beijing, China) following the manufacturers’ instructions.

Techniques: Inverted Microscopy, Staining, Fluorescence, Microscopy

Journal: Molecules

Article Title: A Litopenaeus vannamei Hemocyanin-Derived Antimicrobial Peptide (Peptide B11) Attenuates Cancer Cells’ Proliferation

doi: 10.3390/molecules23123202

Figure Lengend Snippet: Localization of peptide B11 in the mitochondria, its effect on mitochondrial membrane potential (∆Ψm), and apoptosis induction in HeLa cells. ( A ) Microscopic images showing the intracellular localization of rhodamine-labeled B11 in HeLa cells. Cells were treated with 50 μg/mL of rhodamine-labeled B11 for 8 h, washed with PBS and stained with 200 nM of MitoTracker Green. Images were captured with a confocal microscope under a 40× objective (scale bar = 10 µm). ( B ) Mitochondrial membrane potential (∆Ψm) of HeLa cells treated with peptide B11. Cells were treated for 24 h with peptide B11 (50 μg/mL) and PBS (0.01 M, pH 7.4), followed by staining with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole-carbocyanide iodine (JC-1) working solution and incubated for 20 min at 37 °C protected from light. Images were observed under confocal microscopy (scale bar = 20 µm). For positive control, cells were treated with 10 µM of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Red fluorescence represents the mitochondrial aggregate form of JC-1, indicating an intact mitochondrial membrane potential. Green fluorescence represents the monomeric form of JC-1, indicating dissipation of the ∆Ψm. ( C ) Immunoblots of ( i ) caspase-9 and caspase-3, ( ii ) Bax, and ( iii ) Bcl-2 protein levels in peptide B11-treated HeLa cells analyzed by Western blot. Cell lysates from HeLa cells treated with peptide B11 (50 μg/mL) or PBS (0.01 M, pH 7.4) for 24 h were analyzed using the appropriate antibodies, with β-actin used as a loading control. Numbers below the blots represent the relative gray values determined using ImageJ program.

Article Snippet: The ability of peptide B11 to induce cell death in terms of apoptosis was determined using flow cytometry with Annexin V-FITC apoptosis detection kit (Beijing Beyotime Corp, Beijing, China) following the manufacturers’ instructions.

Techniques: Membrane, Labeling, Staining, Microscopy, Incubation, Confocal Microscopy, Positive Control, Fluorescence, Western Blot, Control

Journal: American Journal of Translational Research

Article Title: TGF-β 1 induces human aortic vascular smooth muscle cell phenotype switch through PI3K/AKT/ID2 signaling

doi:

Figure Lengend Snippet: Cell morphology by Coomassie blue staining (×400). A. Blank control group; B. TGF-β1 group; C. TGF-β1 inhibitor group (SB525334); D. AKT inhibitor group (MK2206-2HCL); E. Combined inhibitors group (SB525334+MK2206-2HCL).

Article Snippet: Reagents and instruments The main reagents and instruments of this study included the following: human aortic VSMCs (T/G HA-VSMCs, ATCC, US); high-glucose DMEM, trypsin, penicillin-streptomycin, PBS (all from Hyclone, US); fetal bovine serum (FBS, Zhejiang Tianhang Biological Technology Stock Co., Ltd., China); recombinant human TGF-β 1 (PeproTech, US); SB525334 and MK2206-2HCL (Selleck, US); an inverted microscope (OLYMPUS CK2-TR, Japan); a biological safety cabinet (Haier HR40-IIA2, China); a CO2 incubator (SANYO MCO-175, Japan); a microplate reader (Rayto RT-6500, US); a real-time PCR system (ABI7900, US); and a fluorescence microscope (OLYMPUS BX51, Japan).

Techniques: Staining, Control