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: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: SIRPα and FcγRI are arranged in discrete nanoclusters at macrophage surfaces. (A and B) TIRF and dSTORM images of SIRPα (A) and FcγRI (B) at the surface of human macrophages seeded onto PLL- (nonactivated, top) or hIgG-coated slides (bottom) for 10 min and stained with fluorescently labeled specific antibodies. Bars, 5 µm. Regions delineated by white squares are zoomed-in and shown with corresponding density maps (pseudocolor scale), thresholded binary maps and Ripley’s K analysis of the molecules in the selected regions. Bars, 1 µm. L(r)-r represents the degree of clustering relative to simulated random distributions, indicated by the 99% confidence intervals (CIs); r is the radial scale. (C–E) Nanocluster areas (C), density (D), and percentage of localizations in nanoclusters (E) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions were calculated by subjecting dSTORM data to spatial point-pattern analysis and thresholding. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from three independent donors. ns, not significant; ****, P < 0.0001; two-tailed t test assuming unequal variance. (F and G) Label-density variation analysis for SIRPα (F) and FcγRI (G) yields characteristic normalized ρ/η curves for clustered proteins. Cells were stained with anti–SIRPα-AF647 (F) or anti–FcγRI-AF488 (G) at different labeling concentrations and imaged by dSTORM. Each data point represents a single cell, color-coded by antibody concentration used for labeling. Red lines indicate reference curves for a random distribution of molecules.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Staining, Labeling, Two Tailed Test, Concentration Assay

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: SIRPα and FcγRI nanoclusters are constitutively associated in nonactivated human macrophages but segregate upon activation with hIgG. (A) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with PLL (nonactivated, top) or hIgG (middle) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares (middle column) are shown enlarged (right columns) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. As a positive control, macrophages seeded onto PLL-coated slides were stained with anti–FcγRI-AF488 mAb followed by anti–mouse IgG1-AF647 secondary antibody (bottom). (B) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRI in cells seeded onto PLL- (gray) or hIgG-coated (red) slides for 10 min or for positive control data (green). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (C) Nearest-neighbor (NN) analysis from data shown in (B). Each symbol represents the median NN of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ****, P < 0.0001; two-tailed t test assuming unequal variance. (D) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥ 20,000 clusters from a minimum of 10 cells per condition) from cells seeded onto PLL- (light gray), hIgG-coated (light red) slides, or positive control data (green). Corresponding simulated data are also shown, in which the centroid positions of SIRPα nanoclusters in both nonactivating (dark gray) and hIgG-activating conditions (dark red) were randomized within the cell area.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Activation Assay, Incubation, Staining, Fluorescence, Positive Control, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: SIRPα and the low-affinity Fc receptor, FcγRII, are segregated on a nanometer scale. (A) TIRF and dSTORM images showing FcγRII (green) and SIRPα (red) at the surface of human macrophages incubated for 10 or 30 min on slides coated with PLL (nonactivated) or hIgG and stained with anti–FcγRII-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (B) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRII in cells seeded onto PLL- or hIgG-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively) or for positive control data (green). The positive control data in this figure is the same as in . Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (C) NND analysis from data shown in B. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ***, P < 0.001; one-way analysis of variance (ANOVA) with Tukey’s post-hoc test. (D) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). a.u. arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Incubation, Staining, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: FcγRs reorganize into concentric rings upon activation. (A) TIRF images of FcγRI (top) and FcγRII (bottom) at the surface of human macrophages incubated for 10 or 30 min on slides coated with PLL (nonactivated) or hIgG and stained with fluorescently labeled specific antibodies. Bars, 10 µm. (B) TIRF and dSTORM images of FcγRI (green) and FcγRII (red) at the surface of macrophages incubated for 10 or 30 min on slides coated with PLL or hIgG and stained with anti–FcγRI-AF488 and anti–FcγRII-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and FcγRII in cells seeded onto PLL- or hIgG-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively) or for positive control data (green). The positive control data in this figure are the same as in . Data are from a minimum of 10 cells from three independent donors. Bars represent mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (E) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). a.u., arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Activation Assay, Incubation, Staining, Labeling, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Specific activation of FcγRI is required for its reorganization into concentric rings and segregation from SIRPα nanoclusters. (A and B) TIRF (bars, 10 µm) and dSTORM (bars, 5 µm) images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 (A) or 30 min (B) on slides coated with hIgG1 or hIgG2 and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα in cells seeded onto hIgG1- or hIgG2-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (E) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). a.u., arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Activation Assay, Incubation, Staining, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Rearrangement of macrophage surface receptors triggered by mobile hIgG. (A) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on SLBs loaded with streptavidin (nonactivating) or with streptavidin-hIgG (activating) and stained with a fluorescently labeled specific antibody. Two example images are shown for each condition. Bars, 10 µm. (B) dSTORM images of FcγRI (green) and SIRPα (red) at the surface of macrophages seeded as in A and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares are shown enlarged with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C–E) Nanocluster areas (C), density (D), and percentage of localizations in nanoclusters (E) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from two independent experiments. ns, not significant; *, P < 0.05; ****, P < 0.0001; two-tailed t test assuming unequal variance. (F) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRI in cells seeded as in A. Data are from a minimum of 30 cells from two independent experiments. Bars represent mean ± SD. (G) NND analysis from data shown in F. Each symbol represents median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ****, P < 0.0001; two-tailed t test assuming unequal variance. (H) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥10,000 clusters from a minimum of 10 cells per condition) from cells seeded onto control nonactivating (light gray) or hIgG-loaded activating (light red) SLBs.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Incubation, Staining, Labeling, Fluorescence, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Ligation of SIRPα impairs the reorganization of surface FcγRI. (A) Human macrophages were incubated for 24 h in wells coated with PLL, 20 µg/ml of hCD47, or with increasing concentrations of hCD47 in the presence of 10 µg/ml of hIgG, as indicated. M-CSF release was assessed by ELISA. Bars represent mean ± SD from three donors. Each color represents one individual donor. (B) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on slides coated with hCD47 or hCD47 plus hIgG and stained with fluorescently labeled specific antibody. Bars, 10 µm. (C) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with hCD47 (top) or hCD47 plus hIgG (bottom) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (D and G) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα (D) and for FcγRI and pSHP-1 Y536 (G) in cells seeded onto slides coated with PLL (light gray), hCD47 (light red), hCD47 plus hIgG (dark red), or hIgG (dark gray) for 10 (D) or 5 min (G). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (E and H) NND analysis from data shown in D and G, respectively. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (F and I) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). Graphs compare colocalization between FcγRI and SIRPα (F) and FCγRI and pSHP-1 Y536 (I). a.u., arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Ligation, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Segregation and reorganization of FcγRI is dependent on the actin cytoskeleton and formins, but not myosin II. (A) TIRF image of FcγRI (white; bars, 20 µm) and dSTORM images (bars, 5 µm) of FcγRI (green) and SIRPα (red) at the surface of human macrophages pretreated with 1 µM latrunculin A, 0.5 µM jasplakinolide, 10 µM blebbistatin or 10 µM SMIFH2. Cells were then seeded onto slides coated with PLL (nonactivated) or hIgG for 10 min, and stained with anti-FcγRI-AF488 and anti-SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column). Bars, 1 µm. (B) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα in cells pretreated with drugs as indicated and seeded onto slides coated with PLL (gray) or hIgG (latrunculin A [Lat A], dark gray; jasplakinolide [Jasp], red; SMIFH2, green; or blebbistatin [Bleb], blue) for 10 min. Data are from a minimum of 30 cells per condition from three independent donors. Bars represent mean ± SD. (C) NND analysis from data shown in B. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ****, P < 0.0001; two-tailed t test assuming unequal variance. (D) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). (E–G) Nanocluster areas (E), density (F), and percentage of localizations in nanoclusters (G) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions after pretreatment of cells with blebbistatin or DMSO control. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from three independent donors. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; two-tailed t test assuming unequal variance. NN, nearest neighbor.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Staining, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Src-family kinase signaling, but not Syk or PI3K signaling, is indispensable for reorganization of macrophage surfaces. (A) Immunoblots of phosphorylated AKT in nonactivated (PLL) or hIgG-activated human macrophages pretreated with vehicle (DMSO), as a control, 10 µM PP2 (left), 100 µM piceatannol (PCT; middle), or 1 µM wortmannin (Wort; right). Blots represent two independent experiments. (B) TIRF image of FcγRI (white; bars, 20 µm) and dSTORM images (bars, 5 µm) of FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated with vehicle (DMSO), PP2, PCT, or Wort, pretreated as in A. Cells were then seeded onto slides coated with PLL (nonactivated) or hIgG for 10 min and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column). Bars, 1 µm. (C) CBC histograms for FcγRI and SIRPα in cells pretreated as in A and seeded onto slides coated with PLL (gray) or hIgG (DMSO, dark gray; PP2, red; PCT, green; and Wort, blue) for 10 min, as indicated. Data are from a minimum of 30 cells from three independent donors. Bars show mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (E) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition).

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Western Blot, Incubation, Staining

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: VP treatment enhances apical AQP2 expression and its colocalization with ezrin. (A) VP treatment enhances apical AQP2 expression and its colocalization with ezrin in cultured renal epithelial cells. AQP2-MDCK cells were stained with antibodies against ezrin (green) and AQP2 (red) in the presence (VP) and absence (Control) of VP treatment (AVP 20 nM for 20 min). The larger panels represent confocal sections through the subapical regions of the cells above the nucleus. The smaller horizontal strips at the bottom of each panel are z-sections through the entire cell for direct comparison of the respective staining intensities of the apical and basolateral membranes, and the cytosol. Upper panels show that in the absence of VP stimulation, ezrin staining localized to the cytosol and basolateral region, while AQP2 staining was mainly detected in the subapical region. Lower panels show that after VP treatment, the ezrin signal was redistributed toward the apical and sub-apical regions and partially colocalized with the similarly apically redistributed AQP2. Scale bar: 10 μm. (B) Super-resolution Airyscan confocal microscopy imaging revealed that AQP2 and ezrin partially colocalize on the apical membrane in VP-treated MDCK cells. Left panels show no apparent colocalization of ezrin and AQP2, in the absence of VP stimulation. Right panels are cells treated with VP. Scale bar: 5 μm. (C) AQP2 and ezrin are co-expressed in principal cells of the Brattleboro rat collecting duct, and co-accumulate on the plasma membrane after vasopressin treatment. Without VP treatment (Control), ezrin was located in the cytosol and basal region, while AQP2 was detected mainly in the sub-apical region of the principal cells of the collecting ducts. After 7 days of VP treatment (VP), ezrin (red in the merge panel) colocalized with AQP2 (green in the merge panel) on the plasma membrane of the principal cells. Scale bar: 20 μm.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Expressing, Cell Culture, Staining, Confocal Microscopy, Imaging

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Interaction of AQP2 with ezrin is detected in co-IP experiments. (A) List of ezrin peptides detected by mass spectrometry from the AQP2 co-IP complex. (B,C) By using an anti-ezrin antibody for co-IP, we were able to detect AQP2 in the co-IP complex from stable AQP2-expressing LLC-PK1 cell lysates and mouse kidney (B). Similarly, ezrin signal was detected in the co-IP complex using anti-AQP2 antibody (C). WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Expressing, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 interacts with ezrin via the ezrin N-terminal FERM-containing domain. (A) Recombinant His-tagged ezrin full-length (FL, amino acids 1–586), N-terminus (NT, amino acids 1–308), and C-terminus (CT, amino acids 285–586) were expressed in E. coli and purified to homogeneity as revealed by SDS-PAGE together with purified recombinant AQP2 C-terminus (AQP2CT). Schematic representation of each recombinant protein is shown in B. (C) The purified His-tagged ezrin full-length protein (FL) and the N-terminal FERM-containing recombinant protein (but not the C-terminal protein) were able to pull down AQP2 from both LLC-AQP2 cell lysates and mouse kidney lysates. Lanes 1–3, beads alone pulled down with PBS (lane 1), LLC-AQP2 cell lysate (lane 2) and kidney lysate (lane 3); lanes 4–6, ezrin FL pulled down with PBS (lane 4), LLC-AQP2 cell lysate (lane 5) and kidney lysate (lane 6); lanes 7–9, ezrin NT pulled down with PBS (lane 7), LLC-AQP2 cell lysate (lane 8) and kidney lysate (lane 9); lanes 10–12, ezrin CT pulled down with PBS (lane 10), LLC-AQP2 cell lysate (lane 11) and kidney lysate (lane 12). WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Recombinant, Purification, SDS Page, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 C-terminus directly interacts with ezrin N-terminal FERM-containing domain. The direct interaction of AQP2 and ezrin is revealed by pulldown experiments using purified recombinant ezrin and AQP2 proteins. Only the purified ezrin full-length (FL) and the N-terminus FERM domain-containing (NT) recombinant protein were able to pull down the purified AQP2 C-terminus. The ezrin C-terminal domain (CT) did not pull down the AQP2 C-terminal domain. WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Purification, Recombinant, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Downregulating ezrin causes membrane accumulation of AQP2. (A) Immunoblotting revealed a strong knockdown of endogenous ezrin in LLC-AQP2 cells by ezrin shRNA lentivirus. There was an ∼80% reduction of endogenous ezrin in ezrin shRNA knockdown LLC-AQP2 cells (graph shows the quantification of ezrin band intensity relative to actin). Results are mean±s.e.m. (n≥3). ***P<0.001 (one-way ANOVA). (B) Immunofluorescence staining of AQP2 in control LLC-AQP2 cells and cells infected with ezrin shRNA lentivirus. After knocking down ezrin in LLC-AQP2 cells, AQP2 was found to increasingly accumulate on the cell surface under baseline conditions (without any stimulation). VP-treated LLC-AQP2 cells were used for comparison. Scale bar: 10 μm. (C) Surface biotinylation experiment revealed a significantly increased accumulation of AQP2 signal on cell surface after knocking down ezrin expression in cells. Transferrin receptor 1 (TFR-1) was used as an internal control for stable membrane proteins. WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Western Blot, shRNA, Immunofluorescence, Staining, Infection, Expressing

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Phosphorylation of key residues in AQP2 was not altered in ezrin knockdown cells. (A) Representative western blot showing that there is no alteration of the levels of total AQP2 and AQP2 phosphorylated at residues 256 or 261 in ezrin shRNA lentivirus-infected LLC-AQP2 cells unlike in cells treated with VP. Immunoblot using anti-β-actin antibody was used as control. Quantification of western blotting results for AQP2 phosphorylated on S256 (p256), S261 (p261) and total AQP2. (B) Intracellular cAMP measurement in LLC-PK1 cells. The intracellular cAMP concentration was significantly increased by 30 min VP treatment (20 nM LVP) without clonal variation. After ezrin knockdown no significant difference in cAMP concentration was observed compared to control. Results in A and B are mean±s.e.m. (n≥3). **P<0.01, ***P<0.001 (one-way ANOVA).

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Western Blot, shRNA, Infection, Concentration Assay

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Downregulating ezrin reduces clathrin-mediated endocytosis and causes concomitant membrane accumulation of AQP2 without affecting the overall exocytosis. (A) Ezrin knockdown does not affect the overall exocytosis in LLC-AQP2-ssYFP cells. LLC-AQP2 cells were stably transfected with ssYFP. The fluorescence signal in the extracellular medium was measured in LLC-AQP2-ssYFP cells with and without treatment with VP or ezrin shRNA lentivirus, respectively. The fluorescence intensity in the medium reflected the exocytotic activity of LLC-AQP2-ssYFP cells, and therefore the rate of exocytosis. No significant increase was seen in the overall exocytosis in LLC-AQP2-ssYFP cells treated with ezrin shRNA lentivirus compared to control. In contrast, a significant increase in exocytosis was observed in VP-treated LLC-AQP2-ssYFP cells, which is consistent with our previous reports (Nunes et al., 2008). (B) Endocytosis assay using Rhodamine-conjugated transferrin showed that Alexa Fluor 568-labeled transferrin accumulated on the apical membrane following ezrin knockdown (upper panel), and a simultaneous acute membrane accumulation of AQP2 (lower panel). Scale bar: 10 μm. (C) A bar graph showing that ezrin knockdown affects clathrin-mediated endocytosis. A block of endocytosis with MβCD was used as a positive control. Results in A and C are mean±s.e.m. (n≥3). **P<0.01, ***P<0.001 (one-way ANOVA).

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Stable Transfection, Transfection, Fluorescence, shRNA, Activity Assay, Endocytosis Assay, Labeling, Blocking Assay, Positive Control

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 internalization was reduced after ezrin knockdown in a cold block experiment. (A) Dynamic distribution of AQP2 after a 20°C cold block was revealed by immunofluorescence staining of LLC-AQP2 cells. Cells were incubated at 20°C for 30, 60 and 120 min to block AQP2 release from the trans-Golgi network. AQP2 formed a ‘perinuclear patch’ in cells at 20°C. In the absence of protein synthesis (blocked by cycloheximide), the speed of formation and immunostaining intensity of the perinuclear patch reflect the speed and extent of AQP2 internalization from the cell surface over time. Scale bar: 10 μm. (B) Quantification of the growth of the AQP2-positive perinuclear patch after 20°C cold block over time. The fluorescence intensity of AQP2 staining in the perinuclear patch was measured using Volocity software as described previously (Arthur et al., 2015). Results are mean±s.e.m. (n≥13 for each data point). Experiments were repeated at least three times.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Blocking Assay, Immunofluorescence, Staining, Incubation, Immunostaining, Fluorescence, Software

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) Cartoon of AC9-POPDC complex. ( B , C ) Proximity ligation assay was performed with HEK293 cells expressing pCDNA3 (background control), POPDC1-Myc alone, and YFP-AC9 in the presence of either POPDC1-Myc or POPDC2-Myc. The interaction between AC9 and Gβγ served as a positive control . ( B ) Images of PLA signal (red) and DAPI (blue). ( C ) Mean cellular fluorescence intensity was quantified by high content microscopy and shown as Box and Whisker plots. Kruskal-Wallis One Way ANOVA analysis was performed (n=7 experiments, P=0.003 between groups) with multiple comparisons by Bonferroni t-test (*P<0.05) ( D ) Quantification of COS7 cells expressing BiFC constructs for AC9, POPDC 1-3. The expressed proteins tagged with VN (top line) and VC (bottom line) are shown. Kruskal-Wallis One Way ANOVA analysis was performed (n=4 experiments, ***P<0.001 between groups) with multiple comparisons to control by Dunn’s method (**P<0.01, *P<0.05) ( E ) Representative live-cell images of indicated BiFC combinations in HEK293 cells (n>20 cells; scale bar is 10 um). Quantification of POPDC1-VN:AC9-VC is shown in and .

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Proximity Ligation Assay, Expressing, Positive Control, Fluorescence, Microscopy, Whisker Assay, Construct

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) Cell lysates from HEK293 cells expressing Flag-AC9 in the presence or absence of Myc-tagged POPDC1 or −2 were subjected to co-immunoprecipitation (Co-IP) with anti-MYC and assayed for AC activity with 300 nM Gαs-GTPγS. Kruskal-Wallis One Way ANOVA analysis on Ranks was performed (n=6 experiments, P=0.003 between all groups) with multiple comparisons to AC9 control by Dunn’s method (**P<0.01). ( B ) A portion of the lysates and Co-IP from ( A ) were subjected to western blot (WB) analysis with anti-MYC (POPDC) and anti-FLAG (AC9). Membranes isolated from Sf9 cells expressing Flag-AC9 served as a positive WB control. Note, POPDC protein runs as multiple bands (48-65 kDa) with altered sizes/patterns in different tissues due to changes in glycosylation patterns . Molecular weight markers are denoted as M. Quantitation of Flag-AC9 WB by One Way ANOVA, n=3-4 experiments, with comparisons by Tukey test, **P<0.01). ( C ) HEK293 cells expressing Flag-AC9 +/- POPDC1-Myc or POPDC2-Myc were subjected to Co-IP with anti-FLAG and subjected to WB analysis with anti-MYC and anti-FLAG. Quantitation of anti-MYC WB by One Way ANOVA, n=3 experiments, with comparisons by Tukey test, **P<0.01). ( D ) POPDC1 does not interact with control TM proteins. HEK293 cells expressing POPDC1-Myc +/- GFP-tagged AC9, EGFR, or LAMP1 were subjected to Co-IP with anti-MYC. Western blotting of lysates and Co-IPs for GFP (top) and Myc (bottom) are shown (n=3 experiments). ( E ) Schematic of POPDC1 truncations. ( F ) BiFC of AC9 and indicated POPDC1 truncations in COS-7 cells. Kruskal-Wallis One Way ANOVA analysis was performed (n=4 experiments, ***P<0.001 between groups) with multiple comparisons by Tukey test (**P=0.008). ( G ) Co-IP with anti-MYC in COS-7 cells expressing Flag-AC9 and indicated Myc-tagged POPDC1 truncations. Western blotting with anti-AC9 and anti-MYC (POPDC1) of Co-IP and lysates is shown. Kruskal-Wallis One Way ANOVA analysis was performed (n=3-5 experiments, P=0.003), with multiple comparisons by Dunn’s method (*P<0.05).

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Activity Assay, Western Blot, Isolation, Molecular Weight, Quantitation Assay

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) Co-localization of CFP-TREK-1 with the BiFC signal from AC9-VN:AC9-VC homodimer (top) and POPDC1-VN:AC9-VC complex (bottom) in HEK293 cells. Scale bar is 10 um. ( B ) The lifetime distribution of Cerulean-tagged proteins +/- the indicated YFP-tagged proteins expressed in HEK293 cells are displayed as box and whisker plots with all outliers shown. Mann-Whitney Rank Sum Test was performed (***P<0.001; n=cell number indicated on each bar). ( C ) Co-IP of Myc-tagged POPDC1 (anti-MYC) pulls down both Flag-AC9 and GFP-tagged TREK-1 (n=3 experiments). ( D ) Co-IP of TREK-1 (anti-GFP) pulls down Flag-AC9 and endogenous POPDC1. AKAP79 is not pulled down in complex (n=3 experiments). Quantification of Flag-AC9 WB (IP/total expression in lysate) is shown to the right of ( C ) and ( D ).

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Whisker Assay, MANN-WHITNEY, Co-Immunoprecipitation Assay, Expressing

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) BiFC signal between the indicated VN- and VC-tagged proteins expressed in HEK293 cells (POPDC1, P1). Kruskal-Wallis One-Way ANOVA on Ranks (n=5 experiments, P<0.001 between groups) with multiple comparison to VN and VC controls by Student-Newman-Keuls tests (*P<0.05). ( B ) Percent decrease of BiFC signal in ( A ) with ISO treatment (10 μM, 10 min at 37°C). Box and whisker plots are shown. Paired t-test was performed for each condition (vehicle versus ISO) using raw data prior to calculation of percent of vehicle control (n=6; *P<0.05; **P<0.01). ( C ) ISO dose-response curves for BiFC interactions between POPDC1:AC9, TREK-1:AC9, and TREK-1 with a catalytically inactive AC9 (TREK-1:AC9d). Data were analyzed by Two-way ANOVA with multiple comparisons by Holm Sidak method (n=3-4; *P<0.05). ( D ) TREK-1:AC9 BiFC signal is absent in COS-7 cells lacking detectable POPDC1 (TREK-1:TREK-1 BiFC is shown as a positive control). One-way ANOVA with multiple comparisons by Holm Sidak method (n=3 experiments; ***P<0.001); ( E ) Overexpression of POPDC1 truncation of Popeye domain (POPDC1-Δ172), but not WT POPDC1, abolishes ISO reduction of TREK-1:AC9 BiFC signal. One-way ANOVA with multiple comparisons by Holm Sidak method (n=3-4 experiments; ***P<0.001 compared to control). ( F ) Model of cAMP effects on AC9-POPDC1-TREK-1 complex formation.

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Whisker Assay, Positive Control, Over Expression

Journal: bioRxiv

Article Title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

doi: 10.1101/2021.12.21.473719

Figure Lengend Snippet: ( A ) IP-AC assay with IgG versus anti-TREK-1 in heart homogenates from WT and Adcy9 -/- mice; AC activity stimulated with 300 nM Gαs-GTPγS. IP-AC with anti-AKAP150 is shown as a positive control. Two-way ANOVA (overall P<0.001, n=3 mice per genotype) with multiple comparisons by Holm-Sidak test; # P<0.001 for indicated comparison or to respective IgG controls. IgG versus anti-TREK-1 in Adcy9 -/- was analyzed by Student’s t-test (**P=0.009, n=3). ( B ) IP-AC assay with IgG versus anti-TREK in WT and Popdc1 -/- mouse heart homogenates; AC activity stimulated as in (A). Statistics as in A, n=4 mice per genotype, #P<0.001. Paired t-test within Popdc1 -/ - , P=0.123. ( C ) Total AC activity in heart homogenates from WT or Popdc1 -/- ( P1 -/- ) mice stimulated with 300 nM Gαs-GTPγS or 100 μM calcium and 300 nM calmodulin. Analyzed by Student’s t-test (n=4, n.s.) ( D ) Cell lysates from HEK293 cells expressing AC1 or AC8 in the presence or absence of Myc-tagged POPDC1 were subjected to co-IP with anti-MYC and assayed for AC activity with 100 μM calcium and 300 nM calmodulin. Student’s t-test (n=3 experiments, **P=0.0011 and *P=0.012 for AC1 and AC8, respectively). (E) IP-AC assay with IgG versus anti-TREK-1 in WT and Popdc1 -/- mouse heart homogenates; AC activity stimulated 100 μM calcium and 300 nM calmodulin. Student’s t-test (*P=0.03; **P=0.005, n=4 mice per genotype).

Article Snippet: Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti-FLAG M2 agarose affinity gel (Sigma-Aldrich), mouse anti-DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti-MYC (purified by National Cell Culture from the ATCC hybridoma CRL-1729 for MYC 1-9E10.2), mouse anti-A.v. monoclonal antibody for green fluorescent protein (JL-8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti-GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti-β-actin (C4, Santa Cruz Biotechnology), anti-POPDC1 (early studies used Santa Cruz, goat #sc-49889 but now discontinued; Sigma-Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology).

Techniques: Activity Assay, Positive Control, Expressing, Co-Immunoprecipitation Assay

Journal: Cell metabolism

Article Title: Targeting Peripheral CB 1 Receptors Reduces Ethanol Intake via a Gut-Brain Axis

doi: 10.1016/j.cmet.2019.04.012

Figure Lengend Snippet: (A) Time course and rate of increase in oleate-driven respiratory activity in MGN3–1 cells. Cells were treated with 10 or 100 nM JD5037, 2.5 M of FCCP (positive control) or vehicle in the presence of the fluorescent extracellular O2 consumption reagent. Oxidative respiration was recorded every 90 sec and the slope of the initial linear increase was calculated. Points and bars are means ± SEM from n = 11–15 experiments, as indicated. *P < 0.05 compared to vehicle. (B) Verification of Cnr1 knockdown by rtPCR in cells used in panel C. Cellular uptake of the constructs was verified by fluorescent microscopy (20x magnification) and the degree of knockdown was determined by rt-PCR, *P < 0.05, n = 4. (C) Oleate-driven oxidative activity in MGN3–1 cells with shRNA-mediated knockdown of Cnr1 expression and their mock-transfected controls, n = 3–8 experiments, as indicated, *P < 0.05.

Article Snippet: Scramble shRNA with GFP Adenovirus (Ad-scramble-shRNA) , Vector Biolabs , 1122.

Techniques: Activity Assay, Positive Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Construct, Microscopy, shRNA, Expressing, Transfection

Journal: Pharmaceutics

Article Title: Safety Evaluation of Nanotechnology Products

doi: 10.3390/pharmaceutics13101615

Figure Lengend Snippet: A zebrafish model for liposome biodistribution. ( a ) Schematic of liposome injection and quantification in zebrafish. Fluorescently labeled liposomes (1 mM total lipids containing 1 mol% Rhod-PE) were injected into the duct of Cuvier at 54 hpf. Confocal microscopy was performed in a defined region (boxed) caudal to the yolk extension at 1, 8, 24, and 48 h after injection. ( b ) Whole-embryo view of liposome distribution in kdrl:GFP transgenic embryos, 1 hpi with three different liposome formulations (AmBisome, EndoTAG-1, and Myocet). ( c ) High-resolution imaging allows the quantification of liposomes in the circulation (measured in the lumen of the dorsal aorta (white box)) and liposome association with different blood vessel types. CHT-EC: caudal hematopoietic tissue endothelial cells, DLAV: dorsal longitudinal anastomotic vessel. ISV: intersegmental vessel. ( d ) Tissue=level view of liposome distribution in kdrl:gfp transgenic embryos, 1 h and 8 h after injection, with three different liposome formulations and a single confocal section through the dorsal aorta (DA) at 1 h after injection. ( e ) Quantification of liposome levels in circulation based on mean rhodamine fluorescence intensity in the lumen of the dorsal aorta at 1, 8, 24, and 48 h after injection (error bars: standard deviation.) n = 6 individually injected embryos per formulation per time point (in two experiments). ( f ) Quantification of liposome levels associated with venous vs. arterial endothelial cells based on rhodamine fluorescence intensity, associated with caudal vein (CV) vs. DA at 8 h after injection. ( g ) Quantification of extravascular liposome levels based on rhodamine fluorescence intensity outside of the vasculature between the DLAV and DA at 8 h after injection. ( h ) Quantification of liposome levels associated with the vessel wall based on rhodamine fluorescence intensity, associated with all endothelial cells relative to rhodamine fluorescence intensity in circulation at 1 h after injection. ( f − h ) Bar height represents median values, dots represent individual data points, and brackets indicate significantly different values (*: p < 0.05, **: p < 0.01, ***: p < 0.001), based on Kruskal−Wallis and Dunn’s tests with the Bonferroni correction for multiple testing. n = 12 individually injected embryos per group (in 2 experiments). ( i ) Whole-embryo view of liposome distribution in kdrl:GFP transgenic embryos, 1 h after injection with DOPG and DSPC liposomes. Liposome accumulation for both formulations was observed in the primitive head sinus (PHS), common cardinal vein (CCV), posterior cardinal vein (PCV), and caudal vein (CV). ( j ) Tissue-level view of liposome distribution in kdrl:GFP transgenic embryos, 1 h after injection with DOPG and DSPC liposomes at 102 hpf. Liposome accumulation was observed in the entire caudal vein (CV), but only on the dorsal side of the PCV (dPCV, arrows). Reproduced with permission from , American Chemical Society, 2018.

Article Snippet: They intravenously injected three types of liposomes, including Myocet (neutral), EndoTAG-1 (positively charged), and Ambisome (negatively charged) into the duct of Cuvier of zebrafish embryos.

Techniques: Injection, Labeling, Confocal Microscopy, Transgenic Assay, Imaging, Fluorescence, Standard Deviation

Journal: bioRxiv

Article Title: Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

doi: 10.1101/2022.07.17.500346

Figure Lengend Snippet: A) Percentage of SARS-CoV-2-infected cells detected in Caco-2 cell lines from different sources infected with different SARS-CoV-2 isolates at a multiplicity of infection (MOI) 0.01 as determined by immunostaining for the viral spike (S protein) 48h post infection. B) Cytopathogenic effect (CPE) formation in SARS-CoV-2 (MOI 0.01)-infected Caco-2 cell lines from different sources as determined 48h post infection. C) Susceptibility of Caco-2-F03 cells to a broad range of SARS-CoV-2 isolates after different times of cultivation. Cells had been frozen at passage 14 and were now resuscitated and cultivated for a further 30 passages. SARS-CoV-2 susceptibility was determined by immunostaining for S 48h after SARS-CoV-2 (MOI 0.01) infection 3 and 30 passages post resuscitation. D) ACE2 and TMPRSS2 levels in Caco-2-F03, Caco-2A, and single-cell derived clones from Caco-2A. E) Susceptibility of Caco-2A clones to selected SARS-CoV-2 isolates as indicated by immunostaining for S and CPE formation in SARS-CoV-2 (MOI 0.01)-infected cells 48h post-infection. F) Correlation of S staining and CPE formation with cellular ACE2 levels.

Article Snippet: Finally, caspase 3/7 activity reflected the effect of remdesivir on SARS-CoV-2 replication in CMS ( ).

Techniques: Infection, Immunostaining, Derivative Assay, Clone Assay, Staining

Journal: bioRxiv

Article Title: Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

doi: 10.1101/2022.07.17.500346

Figure Lengend Snippet: A) Caspase 3/7, caspase 8, and caspase 9 activity in Caco-2-F03 cells infected with a range of different SARS-CoV-2 isolates (MOI 0.01), as determined by Caspase-Glo assay assay (Promega) 48h post infection. Higher signal-to-basal (S/B) ratios and Z’ scores indicate higher assay robustness. B) Caspase 3/7 activity as determined by Caspase-Glo assay, C) SARS-CoV-2 Spike (S) protein staining, and D) virus titers as indicated by genomic RNA copy numbers determined by qPCR in Caco-2-F03 cells infected with a wide range of uncharacterized SARS-CoV-2 isolates (MOI 0.01) 48h post infection. E) Representative images indicating CPE formation in G614 (MOI 0.01)-infected Caco-2-F03 and Vero cells 48h post infection as indicated by phase contrast microscopy and immunofluoresce staining for the viral S protein in combination with DAPI-stained nuclei. F) Quantification of cellular S protein levels in Caco-2-F03 cells infected with G614 (MOI 0.01) 48h post infection by immunostaining. G) Only caspase 3/7 activity but not viability assays (MTT, CellTiter-Glo measuring ATP production) reflects G614 (MOI 0.01) replication 48h post infection in Vero cells, which do not display a virus-induced CPE. G614 (MOI 0.01)-infected Caco-2-F03 cells served as a control that displays a CPE. P values were calculated by one-way ANOVA. H) Caspase 3/7 activity in Caco-2-F03 cells infected with MERS-CoV, SARS-CoV, and HCoV-229E (MOI 0.01) as determined 48h post infection including S/B ratios and Z’ scores.

Article Snippet: Finally, caspase 3/7 activity reflected the effect of remdesivir on SARS-CoV-2 replication in CMS ( ).

Techniques: Activity Assay, Infection, Caspase-Glo Assay, Staining, Microscopy, Immunostaining

Journal: bioRxiv

Article Title: Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

doi: 10.1101/2022.07.17.500346

Figure Lengend Snippet: A) Dose-response curves and concentrations that inhibit virus infection by 50% (IC50) of antiviral agents as determined by caspase 3/7 activity and immunostaining for the coronavirus S protein in G614 (MOI 0.01)-infected Caco-2-F03 cells 24h post infection. B) Effects of the approved anti-SARS-CoV-2 drug remdesivir on cellular levels of the viral NP protein in G614 (MOI 1)-infected air liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells 120h post infection. C) Effects of remdesivir on caspase 3/7 activity and virus titers (genomic RNA copy numbers determined by PCR) in G614 (MOI 1)-infected ALI HBE cultures 120h post infection. D) Effects of remdesivir on caspase 3/7 activity and virus titers in G614 (MOI 1)-infected primary human cardiomyocytes (CMS) 48h post infection. E) Correlation of the neutralization capacity of sera derived from seven donors two weeks after their second dose of the mRNA-1273 vaccine determined by caspase 3/7 activity or cytopathogenic effect (CPE) scoring in D614, Alpha and Delta-infected Caco-2-F03 cells 48h post infection. F) Determination of neutralization titers by caspase 3/7 activity or CPE scoring using sera derived from seven donors two weeks after their second dose of the mRNA-1273 vaccine in Caco-2-F03 cells infected with D614, Alpha, and Delta isolates 72h post infection. P values were calculated using paired t-test.

Article Snippet: Finally, caspase 3/7 activity reflected the effect of remdesivir on SARS-CoV-2 replication in CMS ( ).

Techniques: Infection, Activity Assay, Immunostaining, Neutralization, Derivative Assay

Journal: bioRxiv

Article Title: Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

doi: 10.1101/2022.07.17.500346

Figure Lengend Snippet: A) Drug dose response curves and concentrations that reduce cellular levels of the SARS-CoV-2 S protein by 50% (IC50) in Caco-2-F03 and Calu-3 cells as determined by immunostaining 24h (Caco-2-F03) or 48h (Calu-3) post infection with the SARS-CoV-2 strain FFM3 or its remdesivir-adapted substrain FFM3rREM at MOI 0.01. B) Drug concentrations that reduce caspase 3/7 activity in FFM3 and FFM3rREM (MOI 0.01)-infected Caco-2-F03 cells 48h post infection. C) Sequence variants in FFM3rREM compared to FFM3. D) The polymerase complex with nsp7 and nsp8 and a template-primer RNA (cyan and deep teal) and remdesivir (magenta) bound. Gly671Ser is shown in red (as serine). E) The SARS-CoV-2 polymerase Gly671Ser sequence variant. Residue 671 is shown in red as serine, which would be able to form a hydrogen bond with Thr402 which would not be present as Gly671. All p values were calculated by two-way ANOVA.

Article Snippet: Finally, caspase 3/7 activity reflected the effect of remdesivir on SARS-CoV-2 replication in CMS ( ).

Techniques: Immunostaining, Infection, Activity Assay, Sequencing, Variant Assay

Journal: bioRxiv

Article Title: Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

doi: 10.1101/2022.07.17.500346

Figure Lengend Snippet: A) Overview of the proof-of-concept screen for anti-SARS-CoV-2 compounds using the Kinase inhibitor library L-1200 (Selleckchem, Germany) containing 1796 compounds (Selleckchem, Germany) in Delta (MOI 0.01)-infected Caco-2-F03 cells using caspase 3/7 activity as read-out 48h post infection. For the screen, every compound was tested at a concentration of 10 and 1 µM. 21 selected hits were then confirmed by determining drug-response curves. B) Quality controls, Z’scores served as quality controls (left). Only plates with a Źscore ≥ 0.5 were further analyzed. Remdesivir (10 µM) was used as positive control on each plate and produced consistent results (right). C) Number of hits at different inhibition cut-offs. D) Visualization of the distribution of hits according to their targets. Targets for which inhibitors were selected for confirmation are indicated. E) Heatmaps of the anti-SARS-CoV-2 activity of 21 hits by the determination of dose-response in Delta and Omicron (MOI 0.01)-infected Caco-2-F03 cells using immunostaining for the viral S protein as read-out 24h post infection.

Article Snippet: Finally, caspase 3/7 activity reflected the effect of remdesivir on SARS-CoV-2 replication in CMS ( ).

Techniques: Infection, Activity Assay, Concentration Assay, Positive Control, Produced, Inhibition, Immunostaining

Journal: bioRxiv

Article Title: Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

doi: 10.1101/2022.07.17.500346

Figure Lengend Snippet: A) Scheme of the testing of NCT-503 for anti-SARS-CoV-2 activity in air liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells. Effect of NCT-503 on (B) caspase 3/7 activity, (C) virus titers (determined as genomic RNA copy numbers by qPCR), (D) transepithelial electrical resistance (TEER), and (E) LDH release in ALI HBE cultures infected with Delta (MOI 1) 120h post infection. F) Anti-SARS-CoV-2 effects of NCT-503 in combination with 2-Deoxy-D-glucose (2DG). Illustration of how NCT-503 and 2DG can exert combined effects on a common metabolic pathway. G) Representative fluorescence images indicating the number of Delta and Omicron (MOI 0.01)-infected cells in NCT503 and/ or 2DG-treated Caco-2-F03 cultures 24h post infection. H) and I) Weighted combination indices (CIwt) determined by the method of Chou and Talalay indicating a strong synergism of NCT-503 and 2DG.

Article Snippet: Finally, caspase 3/7 activity reflected the effect of remdesivir on SARS-CoV-2 replication in CMS ( ).

Techniques: Activity Assay, Infection, Fluorescence

Journal:

Article Title: Imaging transcriptional regulation of p53-dependent genes with positron emission tomography in vivo

doi: 10.1073/pnas.161091198

Figure Lengend Snippet: Validation of Cis-p53/TKGFP reporter system in cell cultures. (A) Structure of the DXS53TGN retroviral vector bearing the Cis-p53/TKGFP reporter system. This vector has a mutation in the 3′LTR that renders the silencing of its promoter activity after duplication as 5′LTR during integration. The expression of the TKGFP gene is regulated by an artificial promoter containing multiple tandem repeats of a p53-specific DNA-binding motif. Constitutive expression of the neomycin-resistance gene (Neo) is driven by the simian virus 40 early immediate promoter, allowing for the selection of stably transduced cells. Fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis of a transduced U87p53/TKGFP cell population in the noninduced (control) state (B and C), and 24 h after a 2-h treatment with N,N′-bis(2-chloroethyl)-N-nitrosourea (BCNU) at 40 μg/ml (D and E). (F) Immunoblot analysis for total p53, activated p53 (Ser15 phosphorylated), p21, and TKGFP protein levels in U87p53/TKGFP cells in the noninduced state (0) and after treatment with different doses of etoposide (5–40 μg/ml), 40 μg/ml BCNU, or 400 mJ of UV radiation. (G) Reverse transcription (RT)-PCR analysis for p21 and TKGFP mRNA levels performed in the same samples as shown in F. The levels of phospho-p53, total p53, p21, and TKGFP proteins increase after etoposide treatment in a dose-dependent manner; similar increases were observed for BCNU and UV treatments (data not shown). (H) TKGFP expression in different cell populations as measured by the radiotracer assay. The FIAU/thymidine (TdR) ratio is low in wild-type U87 cells (negative control) and in noninduced U87p53/TKGFP cells. In contrast, BCNU-treated U87p53/TKGFP cells had a significantly higher FIAU/TdR accumulation ratio (higher TKGFP expression), which was within the range observed in RG2TKGFP cells that constitutively express TKGFP.

Article Snippet: The U87 and SaOS-2 cell lines were obtained from The American Type Culture Collection.

Techniques: Biomarker Discovery, Retroviral, Plasmid Preparation, Mutagenesis, Activity Assay, Expressing, Binding Assay, Virus, Selection, Stable Transfection, Fluorescence, Microscopy, FACS, Control, Western Blot, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal:

Article Title: Imaging transcriptional regulation of p53-dependent genes with positron emission tomography in vivo

doi: 10.1073/pnas.161091198

Figure Lengend Snippet: PET imaging of endogenous p53 activation. Transaxial PET images through the shoulder (A and C) and pelvis (B and D) of two rats are shown; the images are color-coded to the same radioactivity scale (% dose/g). A nontreated animal is shown on the left, and a BCNU-treated animal is shown on the right. Both animals had three s.c. tumor xenografts: U87p53TKGFP (test) in the right shoulder, U87 wild-type (negative control) in the left shoulder, and RG2TKGFP (positive control) in the left thigh. The nontreated animal on the left shows localization of radioactivity only in the positive control tumor (RG2TKGFP); the test (U87p53TKGFP) and negative control (U87wt) tumors are at background levels. The BCNU-treated animal on the right shows significant radioactivity localization in the test tumor (right shoulder) and in the positive control (left thigh), but no radioactivity above background in the negative control (left shoulder).

Article Snippet: The U87 and SaOS-2 cell lines were obtained from The American Type Culture Collection.

Techniques: Imaging, Activation Assay, Radioactivity, Negative Control, Positive Control

Journal: BMC Neuroscience

Article Title: Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Expression and subcellular localization of SLC10A4 in SH-SY5Y and CAD cells. a Relative SLC10A4 gene expression in SH-SY5Y cells after differentiation with TGF-ß1 + RA or BMP-2 + RA. Values represent mean ± SD of triplicate measurements. b Immunofluorescence analysis of the subcellular expression of the SLC10A4 protein in SH-SY5Y cells. Cells were either untreated (UD), or were differentiated with TGF-ß1 + RA or BMP-2 + RA over 4 days prior to immunolabeling. The SLC10A4 protein was detected with the anti-Slc10a4 1338 C antibody (1:1,000) and the Cy3-labelled anti-rabbit secondary antibody (1:800, red fluorescence) and nuclei were stained with DAPI (blue fluorescence). In all cases, the SLC10A4 protein showed a vesicle-like expression pattern within the perikarya and along the neurite-like cellular protrusions. c Even when a fluorescence-tagged SLC10A4-RFP construct was transiently transfected into SH-SY5Y cells, the SLC10A4-RFP protein showed a clear vesicular sorting pattern. d Relative Slc10a4 gene expression analysis in differentiated (Diff) and undifferentiated (UD) CAD cells. The values represent mean ± SD of triplicate measurements. e Endogenous expression of the SLC10A4 protein in CAD cells, cultivated in FCS containing medium (UD) or FCS-free medium (Diff). The SLC10A4 protein was detected with the anti-Slc10a4 1338 C antibody (1:500) and the Cy3-labelled secondary antibody (1:800, red fluorescence). For control, the primary anti-Slc10a4 antibody was omitted (control) or the antibody was pre-incubated with the immunizing peptide (peptide blocking). f Immunofluorescence detection of the SLC10A4 protein was performed with different SLC10A4-directed antibodies (green fluorescence): self-generated polyclonal rabbit anti-Slc10a4 1338 C antibody, rabbit anti-SLC10A4 Sigma Prestige antibody, rabbit anti-Slc10a4 Abnova antibody, and rabbit anti-SLC10A4 Abgent antibody. Membrane protein enriched fractions of the CAD cells were also subjected to Western Blot analysis with the same antibodies and revealed specific bands for the SLC10A4 protein at an apparent molecular weight of 30–32 kDa.

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques: Expressing, Gene Expression, Immunofluorescence, Immunolabeling, Fluorescence, Staining, Construct, Transfection, Control, Incubation, Blocking Assay, Generated, Membrane, Western Blot, Molecular Weight

Journal: BMC Neuroscience

Article Title: Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Transport measurements in transiently transfected HEK293 cells. HEK293 cells were transiently transfected with the indicated carriers SLC10A4, DAT, CHT1, or SERT, respectively. The uptake of 5 µM [ 3 H]dopamine, 5 µM [ 3 H]norepinephrine, 5 µM [ 3 H]choline, or 5 µM [ 3 H]serotonin was measured over the given time periods in the presence and absence of Na + (for CHT1 Na + was replaced by Li + ). Transport via DAT, CHT1, and SERT was blocked by the specific inhibitors nomifensine (10 µM), hemicholinium-3 (HC-3, 1 µM), and citalopram (2 µM), respectively. Values represent mean ± SD of representative experiments, each with quadruplicate determinations (n = 4). *Significantly different from control with p < 0.001. # Significantly different from positive uptake, p < 0.001.

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques: Transfection, Control

Journal: BMC Neuroscience

Article Title: Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Transport measurements in digitonin permeabilized SLC10A4-HEK293 and VMAT2-HEK293 cells. Prior to transport measurements, stably transfected human SLC10A4-HEK293 cells and human VMAT2-HEK293 cells were pre-incubated with 15 µM digitonin for 10 min for permeabilization. Then the uptake of 400 nM [ 3 H]serotonin, [ 3 H]norepinephrine, or [ 3 H]dopamine was measured over 10 min in the presence and absence of 5 mM ATP in the transport buffer. In addition to ATP, 2 µM of the potent VMAT2 inhibitor tetrabenazine (TBZ) or 5 µM of the proton ionophore carbonylcyanide-p-trifluoromethoxyphenylhydrazon (FCCP) were added to the transport buffer as indicated. After 10 min, the cells were washed with ice-cold PBS, lysed, and subjected to scintillation counting. The values represent mean ± SD of one representative experiment (for dopamine, n = 4) or two independent experiments (for serotonin and norepinephrine, n = 8). *Significantly different from control with p < 0.05. # Significantly different from positive uptake, p < 0.05.

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques: Stable Transfection, Transfection, Incubation, Control

Journal: BMC Neuroscience

Article Title: Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Transport measurements in Xenopus laevis oocytes. Xenopus laevis oocytes were injected with cRNA coding for human SLC10A4, SERT, DAT, or NTCP as well as mouse Oct1. Uptake of [ 3 H]serotonin, [ 3 H]histamine, [ 3 H]PREGS, [ 3 H]dopamine, [ 3 H]DHEAS, or [ 3 H]taurocholic acid, each at 1 µM, was measured over a time period of 10–60 min as indicated in the presence of sodium chloride in the transport buffer. SERT, Oct1, and DAT served as controls for the transport of [ 3 H]serotonin, [ 3 H]histamine, and [ 3 H]dopamine, respectively. NTCP was the reference carrier for PREGS, DHEAS and taurocholic acid. Afterwards, the oocytes were washed with ice-cold transport buffer, lysed and subjected to scintillation counting. The values represent mean ± SD of one representative experiment with n = 10 oocytes each. *Significantly different from control with p < 0.001.

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques: Injection, Control

Journal: BMC Neuroscience

Article Title: Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Transport measurements in stably transfected SLC10A4-HEK293 and NTCP-HEK293 cells after thrombin treatment. For the transport measurements, one part of the cells was pre-incubated with 1 U/200 µl thrombin over 3 h (+Thrombin), before the uptake of [ 3 H]DHEAS, [ 3 H]taurocholic acid, [ 3 H]PREGS, or [ 3 H]lithocholic acid (each at 300 nM) was measured over a time period of 10 min at 37°C. The cells were washed with ice-cold PBS, lysed, and subjected to scintillation counting. The values represent mean ± SD of two independent experiments each with triplicate determinations. *Significantly different from control with p < 0.001; n.s. not significantly different.

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques: Stable Transfection, Transfection, Incubation, Control

Journal: BMC Neuroscience

Article Title: Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Localization and transport function of SLC10A4/NTCP chimeras in CAD cells. a The shown SLC10A4/NTCP chimeric constructs were used. All chimeras were generated based on the full length sequences of SLC10A4 (grey marked transmembrane domains and loops with continuous lines) and NTCP (white transmembrane domains and loops as dotted lines), both with c-terminal V5-tag. Potential glycosylation sites were marked by “Y”. b All constructs were transiently transfected in CAD cells and cellular localization was analyzed by immunofluorescence microscopy using rabbit anti-V5 antibody and donkey Cy3-labelled anti-rabbit secondary antibody. Nuclei were stained with DAPI. Whereas SLC10A4 showed a clear vesicle-like expression pattern, the immunofluorescence signals for NTCP, NtSLC10A4-NTCP, SLC10A4-CtNTCP, and NtNTCP-SLC10A4-CtNTCP were clearly directed to the plasma membrane. When the 75 N-terminal amino acids were deleted in SLC10A4, the 75ΔSLC10A4 protein retained its vesicle-like intracellular expression comparable with full-length SLC10A4. c The SLC10A4/NTCP chimeras were also used for transport studies after transient transfection into CAD cells with [ 3 H]taurocholic acid and [ 3 H]serotonin, each at 5 µM. These measurements were performed by incubating the cells for 60 min at 37°C in 250 µl cell medium with 50 µl sodium transport buffer containing the radiolabeled and non-radiolabeled compounds. NTCP and SERT were used as a positive control, and empty-vector transfected cells served as the negative control. After the uptake phase, cells were washed with ice-cold PBS, lysed, and subjected to scintillation counting. Data represent mean ± SD of representative experiments each with quadruplicate determinations. *Significantly different from control with p < 0.01.

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques: Construct, Generated, Glycoproteomics, Transfection, Immunofluorescence, Microscopy, Staining, Expressing, Clinical Proteomics, Membrane, Positive Control, Plasmid Preparation, Negative Control, Control

Journal: BMC Neuroscience

Article Title: Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Transport studies in HEK293 cells

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques:

Journal: BMC Neuroscience

Article Title: Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes

doi: 10.1186/s12868-015-0174-2

Figure Lengend Snippet: Primers used for full-length carrier cloning

Article Snippet: PCR amplification was achieved with the TaqMan Gene Expression Assays Hs00293728 for human SLC10A4 and Mm00557788 for mouse Slc10a4 (Applied Biosystems).

Techniques:

Journal: Microorganisms

Article Title: Saccharomyces cerevisiae TAD1 Mutant Strain As Potential New Antimicrobial Agent: Studies on Its Antibacterial Activity and Mechanism of Action

doi: 10.3390/microorganisms13122848

Figure Lengend Snippet: Growth curve measurement. ( A ) Loss of TAD1 does not impair S. cerevisiae growth. Growth curves of parental strain BY4743, S. boulardii , and S. cerevisiae TAD1-KO cultivated in YPD at 30 °C, 200 rpm for 24 h. The superimposable curves indicate that TAD1 deletion is growth-neutral under standard laboratory conditions. ( B – D ) S. cerevisiae TAD1-KO elicits potent metabolite-mediated growth suppression. Growth curves of E. coli , S. aureus , and S. typhi exposed to 90% ( v / v ) S. cerevisiae TAD1-KO , BY4743, or nutrient controls at 37 °C, 200 rpm. S. cerevisiae TAD1-KO CFS significantly delayed lag-to-log transition, reduced µmax, and lowered stationary-phase density. Equivalent growth in sterile water and YPD controls excludes nutrient limitation, confirming inhibition by secreted bioactive metabolites. Data are means ± SD (n = 3); ** p < 0.01; *** p < 0.001; ns denotes no statistically significant difference.

Article Snippet: Even at 25% ( v / v ), CFS reduced the CV-stainable biomass below the untreated control, while 90% CFS suppressed biofilm formation to ≤16% for all tested strains: E. coli ATCC 25922 (10.5%), E. coli mcr-1 (11.1%), S. aureus ATCC 29213 (9.0%), MRSA (10.8%), and S. typhi SL1344 (15.9%).

Techniques: Sterility, Inhibition

Journal: Microorganisms

Article Title: Saccharomyces cerevisiae TAD1 Mutant Strain As Potential New Antimicrobial Agent: Studies on Its Antibacterial Activity and Mechanism of Action

doi: 10.3390/microorganisms13122848

Figure Lengend Snippet: S. cerevisiae TAD1-KO CFS inhibits biofilm formation and downregulates biofilm-associated genes: ( A ) Quantitative CV assay showing dose-dependent suppression of biofilms in E. coli ATCC25922, E. coli ( mcr-1 ), S. aureus ATCC29213, MRSA, and S. typhi SL1344 after 24 h exposure to 25–90% ( v / v ) S. cerevisiae TAD1-KO CFS. ( B ) 96-well plate images confirming progressive biofilm disruption with increasing CFS concentration. ( C ) Light-microscopy images confirming progressive biofilm disruption with increasing CFS concentration. ( D ) qRT-PCR demonstrating significant downregulation of key biofilm genes ( fimH , fliC , csgA , csgD ) in E. coli treated with 50% S. cerevisiae TAD1-KO CFS versus untreated control. Data are means ± SD (n = 3); * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Even at 25% ( v / v ), CFS reduced the CV-stainable biomass below the untreated control, while 90% CFS suppressed biofilm formation to ≤16% for all tested strains: E. coli ATCC 25922 (10.5%), E. coli mcr-1 (11.1%), S. aureus ATCC 29213 (9.0%), MRSA (10.8%), and S. typhi SL1344 (15.9%).

Techniques: Disruption, Concentration Assay, Light Microscopy, Quantitative RT-PCR, Control

Journal: Microorganisms

Article Title: Saccharomyces cerevisiae TAD1 Mutant Strain As Potential New Antimicrobial Agent: Studies on Its Antibacterial Activity and Mechanism of Action

doi: 10.3390/microorganisms13122848

Figure Lengend Snippet: S. cerevisiae TAD1-KO CFS impairs adhesion, hydrophobicity, and EPS production: ( A ) Adhesion to glass after 24 h, ( B ) cell-surface hydrophobicity, and ( C ) EPS yield of E. coli , S. aureus , and S. typhi following 24 h exposure to 50% ( v / v ) S. cerevisiae TAD1-KO CFS. All parameters were significantly reduced versus untreated controls, confirming that CFS targets the early physical determinants of biofilm formation. Data are means ± SD (n = 3); * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Even at 25% ( v / v ), CFS reduced the CV-stainable biomass below the untreated control, while 90% CFS suppressed biofilm formation to ≤16% for all tested strains: E. coli ATCC 25922 (10.5%), E. coli mcr-1 (11.1%), S. aureus ATCC 29213 (9.0%), MRSA (10.8%), and S. typhi SL1344 (15.9%).

Techniques: Cell Surface Hydrophobicity

Journal: Microorganisms

Article Title: Saccharomyces cerevisiae TAD1 Mutant Strain As Potential New Antimicrobial Agent: Studies on Its Antibacterial Activity and Mechanism of Action

doi: 10.3390/microorganisms13122848

Figure Lengend Snippet: Time–kill kinetics and ultrastructural damage induced by S. cerevisiae TAD1-KO CFS. ( A ) Viable counts of E. coli exposed to 90% S. cerevisiae TAD1-KO CFS, 90% BY4743 CFS, or LB at 37 °C; bactericidal effect could be achieved within 4 h, with maximum bactericidal activity at 8 h. ( B ) Representative TEM micrographs after 6 h treatment with 50% S. cerevisiae TAD1-KO CFS: E. coli and S. aureus exhibit swelling, membrane rupture, cytoplasmic leakage, and plasmolysis compared with intact untreated cells, confirming metabolite-mediated envelope disruption. The black scale in the image represents 2 µm and 500 nm. Data are means ± SD (n = 3); *** p < 0.001.

Article Snippet: Even at 25% ( v / v ), CFS reduced the CV-stainable biomass below the untreated control, while 90% CFS suppressed biofilm formation to ≤16% for all tested strains: E. coli ATCC 25922 (10.5%), E. coli mcr-1 (11.1%), S. aureus ATCC 29213 (9.0%), MRSA (10.8%), and S. typhi SL1344 (15.9%).

Techniques: Activity Assay, Membrane, Disruption

Journal: Microorganisms

Article Title: Saccharomyces cerevisiae TAD1 Mutant Strain As Potential New Antimicrobial Agent: Studies on Its Antibacterial Activity and Mechanism of Action

doi: 10.3390/microorganisms13122848

Figure Lengend Snippet: S. cerevisiae TAD1-KO CFS sequentially disrupts envelope integrity and elicits intracellular ROS. ( A ) Time-dependent leakage of periplasmic AKP (OD 520 ) after 50% S. cerevisiae TAD1-KO CFS exposure; significant increase from 4 h onward. ( B ) NPN fluorescence assay: 50% S. cerevisiae TAD1-KO CFS raises outer-membrane permeability to the level of 1 mg mL −1 EDTA-2Na (positive control). ( C ) ONPG hydrolysis (OD 415 ), indicating inner-membrane permeabilization; slightly higher but statistically different from control (* p < 0.05). ( D ) DCFH-DA fluorescence: 90% S. cerevisiae TAD1-KO CFS triggers robust intracellular ROS accumulation (*** p < 0.001), contributing to rapid bacterial killing. ( E , F ) ROS scavengers do not impair S. cerevisiae TAD1-KO CFS bactericidal efficacy. E. coli (1 × 10 7 CFU mL −1 ) were cultured with 90% S. cerevisiae TAD1-KO CFS alone or supplemented with 1 mmol L −1 or 2 mmol L −1 VC, thiourea, or both. Viable counts (CFU mL −1 ) were determined at 0, 4 and 8 h (37 °C, 200 rpm). No statistically significant difference between groups ( p > 0.05) indicates that ROS contributes minimally to CFS-mediated killing; the lethal effect is primarily attributable to direct envelope disruption. Data are means ± SD (n = 3); * p < 0.05; ** p < 0.01; *** p < 0.001; ns denotes no statistically significant difference.

Article Snippet: Even at 25% ( v / v ), CFS reduced the CV-stainable biomass below the untreated control, while 90% CFS suppressed biofilm formation to ≤16% for all tested strains: E. coli ATCC 25922 (10.5%), E. coli mcr-1 (11.1%), S. aureus ATCC 29213 (9.0%), MRSA (10.8%), and S. typhi SL1344 (15.9%).

Techniques: Fluorescence, Membrane, Permeability, Positive Control, Control, Cell Culture, Disruption

Journal: Microorganisms

Article Title: Saccharomyces cerevisiae TAD1 Mutant Strain As Potential New Antimicrobial Agent: Studies on Its Antibacterial Activity and Mechanism of Action

doi: 10.3390/microorganisms13122848

Figure Lengend Snippet: S. cerevisiae TAD1-KO tolerates simulated gastrointestinal stress and displays superior aggregation traits. ( A ) Survival after 3 h in simulated gastric fluid (pH 3.0, pepsin 3 mg mL −1 ); S. cerevisiae TAD1-KO retained 86.0% viability. ( B ) Survival after 4 h in intestinal fluid (pH 8.0, trypsin 1 mg mL −1 , 0.3% bile); 80.7% cells remained viable. ( C ) Auto-aggregation at 5 h; S. cerevisiae TAD1-KO exceeded BY4743 by 23%. ( D ) Co-aggregation increments with E. coli , S. aureus , and S. typhi (28%, 17%, and 30% higher than BY4743, respectively). Data are means ± SD (n = 3); ** p < 0.01; *** p < 0.001; ns denotes no statistically significant difference.

Article Snippet: Even at 25% ( v / v ), CFS reduced the CV-stainable biomass below the untreated control, while 90% CFS suppressed biofilm formation to ≤16% for all tested strains: E. coli ATCC 25922 (10.5%), E. coli mcr-1 (11.1%), S. aureus ATCC 29213 (9.0%), MRSA (10.8%), and S. typhi SL1344 (15.9%).

Techniques:

Journal: Microorganisms

Article Title: Saccharomyces cerevisiae TAD1 Mutant Strain As Potential New Antimicrobial Agent: Studies on Its Antibacterial Activity and Mechanism of Action

doi: 10.3390/microorganisms13122848

Figure Lengend Snippet: G. mellonella toxicity and therapeutic efficacy of S. cerevisiae TAD1-KO . ( A ) Survival curves after a single injection of live yeast, heat-killed yeast, or CFS (n = 5 per group); no larval mortality within 5 days confirms low toxicity. ( B , C ) Models of infection with E. coli and S. aureus : live S. cerevisiae TAD1-KO and its CFS significantly extended survival (*** p < 0.001 vs. PBS), whereas heat-inactivated yeast had no effect, demonstrating that protection requires metabolically active cells. ( D ) Survival rates of G. mellonella in each group on the first day of the E. coli infection model and the S. aureus infection model. ( E , F ) On the fifth day post-infection, homogenize G. mellonella and calculate the bacterial load within each group. ( G ) After homogenization, each group was spread onto LB agar plates for counting. ** p < 0.01; *** p < 0.001; ns denotes no statistically significant difference.

Article Snippet: Even at 25% ( v / v ), CFS reduced the CV-stainable biomass below the untreated control, while 90% CFS suppressed biofilm formation to ≤16% for all tested strains: E. coli ATCC 25922 (10.5%), E. coli mcr-1 (11.1%), S. aureus ATCC 29213 (9.0%), MRSA (10.8%), and S. typhi SL1344 (15.9%).

Techniques: Drug discovery, Injection, Infection, Metabolic Labelling, Homogenization

Journal: Microorganisms

Article Title: Saccharomyces cerevisiae TAD1 Mutant Strain As Potential New Antimicrobial Agent: Studies on Its Antibacterial Activity and Mechanism of Action

doi: 10.3390/microorganisms13122848

Figure Lengend Snippet: ( A – C ) Direct co-culture reveals enhanced pathogen suppression by live S. cerevisiae TAD1-KO . Viable counts (CFU mL −1 ) of E. coli , S. aureus , and S. typhi after 16 h contact with live S. cerevisiae TAD1-KO or BY4743 at 37 °C, 200 rpm. Inhibition rates: 89.5%, 55.1%, and 52.2% for S. cerevisiae TAD1-KO versus ≤ 20% for BY4743, confirming the contribution of TAD1 deletion to antimicrobial competence. Data are means ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001; ns denotes no statistically significant difference.

Article Snippet: Even at 25% ( v / v ), CFS reduced the CV-stainable biomass below the untreated control, while 90% CFS suppressed biofilm formation to ≤16% for all tested strains: E. coli ATCC 25922 (10.5%), E. coli mcr-1 (11.1%), S. aureus ATCC 29213 (9.0%), MRSA (10.8%), and S. typhi SL1344 (15.9%).

Techniques: Co-Culture Assay, Inhibition

Journal: Microorganisms

Article Title: Saccharomyces cerevisiae TAD1 Mutant Strain As Potential New Antimicrobial Agent: Studies on Its Antibacterial Activity and Mechanism of Action

doi: 10.3390/microorganisms13122848

Figure Lengend Snippet: Transwell assay confirms metabolite-mediated antibacterial activity of S. cerevisiae TAD1-KO . ( A , B ) Viable counts of E. coli and S. aureus after 16 h at 37 °C. Inhibition rates: mixed culture 81.7%, separated culture 83.7%; no significant difference between regimes ( p > 0.05, n = 3), demonstrating that diffusible metabolites, not direct contact, drive antibacterial activity. Data are means ± SD; ** p < 0.01; *** p < 0.001; ns denotes no statistically significant difference.

Article Snippet: Even at 25% ( v / v ), CFS reduced the CV-stainable biomass below the untreated control, while 90% CFS suppressed biofilm formation to ≤16% for all tested strains: E. coli ATCC 25922 (10.5%), E. coli mcr-1 (11.1%), S. aureus ATCC 29213 (9.0%), MRSA (10.8%), and S. typhi SL1344 (15.9%).

Techniques: Transwell Assay, Activity Assay, Inhibition

Journal: Microorganisms

Article Title: Saccharomyces cerevisiae TAD1 Mutant Strain As Potential New Antimicrobial Agent: Studies on Its Antibacterial Activity and Mechanism of Action

doi: 10.3390/microorganisms13122848

Figure Lengend Snippet: Antimicrobial activity of S. cerevisiae TAD1-KO is pH-dependent. Inhibitory efficacy against E. coli and S. aureus (OD 600 , 24 h) declined sharply as the native CFS (pH 4.0 ± 0.37) was stepwise neutralized to pH 7.0, demonstrating that the bactericidal effect is mediated predominantly by acidic metabolites identified in the metabolomic analysis. Data are means ± SD; *** p < 0.001; ns denotes no statistically significant difference.

Article Snippet: Even at 25% ( v / v ), CFS reduced the CV-stainable biomass below the untreated control, while 90% CFS suppressed biofilm formation to ≤16% for all tested strains: E. coli ATCC 25922 (10.5%), E. coli mcr-1 (11.1%), S. aureus ATCC 29213 (9.0%), MRSA (10.8%), and S. typhi SL1344 (15.9%).

Techniques: Activity Assay

Journal: Microorganisms

Article Title: Saccharomyces cerevisiae TAD1 Mutant Strain As Potential New Antimicrobial Agent: Studies on Its Antibacterial Activity and Mechanism of Action

doi: 10.3390/microorganisms13122848

Figure Lengend Snippet: Mechanism diagram of S. cerevisiae TAD1-KO against E. coli . S. cerevisiae TAD1-KO CFS secretes large quantities of organic acids, causing a reduction in the expression of biofilm-associated genes in E. coli (blue arrow); CFS disrupts the normal cellular structure of E. coli , leading to increased leakage of AKP (blue arrow) and NPN (pink arrow) into the extracellular space, alongside elevated intracellular ONP levels (yellow arrow). Concurrently, organic acids promote intracellular ROS accumulation (red arrow), inducing oxidative stress damage within the cell.

Article Snippet: Even at 25% ( v / v ), CFS reduced the CV-stainable biomass below the untreated control, while 90% CFS suppressed biofilm formation to ≤16% for all tested strains: E. coli ATCC 25922 (10.5%), E. coli mcr-1 (11.1%), S. aureus ATCC 29213 (9.0%), MRSA (10.8%), and S. typhi SL1344 (15.9%).

Techniques: Expressing

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