Journal: Medicina

Article Title: Secretory Carcinoma of the Breast with Apocrine Differentiation—A Peculiar Entity

doi: 10.3390/medicina60060924

Figure Lengend Snippet: ( A ) Ob20×: S100 is diffusely positive in the secretory component (green circle) and negative in the apocrine component. ( B ) Ob20×: Polyclonal CEA is diffusely positive in the secretory component (green diamond) and negative in the apocrine component. ( C ) Ob20×: CK5/6 is diffusely positive in the secretory component (green star) and negative in the apocrine component.

Article Snippet: The immunohistochemical panel included the following: ER, EP1 clone, Ready to Use (RTU), Dako PR, PgR636 clone, RTU, Dako AR, Ar441 clone, Concentrated, Dilution 1:50, Dako Ki67, MIB-1 clone, RTU, Dako Her2, c-erbB-2 oncoprotein, Concentrated, Dilution 1:200, Dako S-100, 4C4.9, Concentrated, Dilution 1:150, Zeta Gata-3, L50-823, Concentrated, Dilution 1:100, Zeta CEA, Polyclonal RPab, Concentrated, Dilution 1:250, Biosb CK 5/6, D5/16 B4 clone RTU, Dako.

Techniques:

Journal: Cell reports

Article Title: Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs.

doi: 10.1016/j.celrep.2023.112221

Figure Lengend Snippet: Figure 1. LRRK2 interacts with VAMP4, VAMP7, and VAMP8 (A, C, E, and G) HEK293T cells were co-transfected with mCherry-tagged LRRK2-WT, LRRK2-G2019S, or LRRK2-R1441C mutant constructs and GFP-tagged v-SNARES or GFP as mock, as indicated. Forty-eight h after transfection, lysates were subjected to co-immunoprecipitation using GFP antibody and processed for SDS-PAGE and western blot analysis. Inputs represent 3% of the cell extract used for immunoprecipitation. Membranes were cut to allow independent incubations and immunoblotted with LRRK2 antibody in the upper part and GFP antibody in the bottom part. (B, D, F, and H) Quantification of co-immunoprecipitation (co-immunoprecipitated/input signal ratio; see STAR Methods for details) was estimated by densi- tometry analysis. (I) In vitro pull-down was performed with indicated GST-tagged proteins and purified WT LRRK2 protein. Input (3% of total reaction) and pull-down were sub- mitted to SDS-PAGE and immunoblotted with LRRK2 antibody. Amount of GST proteins present in each reaction is detected using Ponceau staining. (J) Quantification of pull-down (pulled-down LRRK2/Ponceau signal). In each panel, mean (colored triangles) and SEM from 3 independent experiments are displayed. One-way ANOVA with Tukey’s multiple-comparison test is labeled on graphs. *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti CD63 BD Biosciences Cat# 551458; RRID:AB_394205 Anti GAPDH Sigma-Aldrich Cat# G9545; RRID:AB_796208 Anti GFP serum N/A rpAb TG32 Anti GFP purified Roche Roche Cat# 11814460001; RRID:AB_390913 Anti GM130 BD Biosciences Cat# 610823; RRID:AB_398142 Anti HSC70 Santa Cruz Biotechnologies Cat# sc-1059; RRID:AB_2120291 Anti LAMP1 Sigma-Aldrich Cat# L1418; RRID:AB_477157 Anti LRRK2 purified Abcam Cat # ab133474; RRID:AB_2713963 Anti LRRK2 serum Antibodies Incorporated Cat# 73–253; RRID:AB_10671178 Anti TGN46 Bio-rad Cat# AHP500GT; RRID:AB_2203291 Anti TSG101 Genetex Cat# GTX70255; RRID:AB_373239 Anti VAMP2 N/A mAB 69.1 Anti VAMP4 N/A rpAb TG19B Anti VAMP7 N/A rpAb TG50 Anti VGF Santa Cruz Biotechnology Cat# sc-365397; RRID:AB_10846955 Deposited data Analyzed Proteomic Dataset from PC12 cells This manuscript ProteomeXchange Consortium via PRIDE, ID = PXD028321 https://www.ebi.ac.uk/pride/archive/ projects/PXD028321 Proteomics results with legends are summarized in Table S1 This manuscript N/A Experimental models: Cell lines PC12 ATCC Cat# CRL-1721.1 HEK293T Evelyne Bloch-Gallego, Institut Cochin N/A Oligonucleotides Guide RNA to KO VAMP 2 CACCgTGGCAGCGGTAGCCGACCTAAAAC TAGGTCGGCTACCGCTGCCAc This manuscript N/A Guide RNA to KO VAMP 4 CACCgTTTAGGTGGCGCTTGAACTTAAACA AGTTCAAGCGCCACCTAAAc This manuscript N/A See Table S2 for primers used for cloning This manuscript N/A Recombinant DNA See Table S2 for cloning details (initial plasmids, primers, cloning methods used) This manuscript N/A RFP-VAMP4 This manuscript N/A mCherry-LRRK2 WT This manuscript N/A mCherry-LRRK2 G2019S This manuscript N/A mCherry-LRRK2 R1441C This manuscript N/A RUSH-VGF-mCherry This manuscript N/A (Continued on next page) Cell Reports 42, 112221, March 28, 2023 17

Techniques: Transfection, Mutagenesis, Construct, Immunoprecipitation, SDS Page, Western Blot, In Vitro, Staining, Comparison, Labeling

Journal: Cell reports

Article Title: Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs.

doi: 10.1016/j.celrep.2023.112221

Figure Lengend Snippet: Figure 3. Validation of VAMP4 and VAMP7 KO secretomes by western blot (A, C, and E) WT, VAMP2 KO, VAMP4 KO, and VAMP7 KO PC12 were differentiated with NGF for one week (A and C). (E) HEK293T cells were transfected with indicated constructs. Equal amounts of cell lysate proteins (15 mg) and volumes of secreted fractions (‘‘total release,’’ ‘‘15K pellet,’’ and ‘‘200K pellet’’) cor- responding to equal cell lysate protein concentration were processed for SDS-PAGE and western blot analysis. Membranes were probed with indicated anti- bodies. (B, D, and F) Cell content (cell lysates/GAPDH), fractional release for total and 15K/200K pellet (secreted fraction/cell lysate) of pro-VGF was estimated by densitometry analysis of the corresponding bands from three independent experiments. Mean (colored triangles) and SEM of three independent experiments are displayed. One-way ANOVA with Tukey’s multiple-comparison test is labeled on graphs. *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti CD63 BD Biosciences Cat# 551458; RRID:AB_394205 Anti GAPDH Sigma-Aldrich Cat# G9545; RRID:AB_796208 Anti GFP serum N/A rpAb TG32 Anti GFP purified Roche Roche Cat# 11814460001; RRID:AB_390913 Anti GM130 BD Biosciences Cat# 610823; RRID:AB_398142 Anti HSC70 Santa Cruz Biotechnologies Cat# sc-1059; RRID:AB_2120291 Anti LAMP1 Sigma-Aldrich Cat# L1418; RRID:AB_477157 Anti LRRK2 purified Abcam Cat # ab133474; RRID:AB_2713963 Anti LRRK2 serum Antibodies Incorporated Cat# 73–253; RRID:AB_10671178 Anti TGN46 Bio-rad Cat# AHP500GT; RRID:AB_2203291 Anti TSG101 Genetex Cat# GTX70255; RRID:AB_373239 Anti VAMP2 N/A mAB 69.1 Anti VAMP4 N/A rpAb TG19B Anti VAMP7 N/A rpAb TG50 Anti VGF Santa Cruz Biotechnology Cat# sc-365397; RRID:AB_10846955 Deposited data Analyzed Proteomic Dataset from PC12 cells This manuscript ProteomeXchange Consortium via PRIDE, ID = PXD028321 https://www.ebi.ac.uk/pride/archive/ projects/PXD028321 Proteomics results with legends are summarized in Table S1 This manuscript N/A Experimental models: Cell lines PC12 ATCC Cat# CRL-1721.1 HEK293T Evelyne Bloch-Gallego, Institut Cochin N/A Oligonucleotides Guide RNA to KO VAMP 2 CACCgTGGCAGCGGTAGCCGACCTAAAAC TAGGTCGGCTACCGCTGCCAc This manuscript N/A Guide RNA to KO VAMP 4 CACCgTTTAGGTGGCGCTTGAACTTAAACA AGTTCAAGCGCCACCTAAAc This manuscript N/A See Table S2 for primers used for cloning This manuscript N/A Recombinant DNA See Table S2 for cloning details (initial plasmids, primers, cloning methods used) This manuscript N/A RFP-VAMP4 This manuscript N/A mCherry-LRRK2 WT This manuscript N/A mCherry-LRRK2 G2019S This manuscript N/A mCherry-LRRK2 R1441C This manuscript N/A RUSH-VGF-mCherry This manuscript N/A (Continued on next page) Cell Reports 42, 112221, March 28, 2023 17

Techniques: Biomarker Discovery, Western Blot, Transfection, Construct, Protein Concentration, SDS Page, Comparison, Labeling

Journal: Cell reports

Article Title: Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs.

doi: 10.1016/j.celrep.2023.112221

Figure Lengend Snippet: Figure 4. A pool of VGF localizes in VAMP4, VAMP7, and LAMP1 compartments (A) WT PC12 cells were differentiated with NGF for one week and processed for immunocytochemistry. The cells were stained for endogenous VAMP4 (left, red) or VAMP7 (right, red) and VGF (green). Magnifications of dash-delimited regions are displayed in the bottom row. Projections of confocal microscopy optical section are shown. Arrowheads indicate red-green co-stained structures. Scale bar, 10 mm. (B) WT, VAMP4 KO, and VAMP7 KO PC12 were differentiated with NGF for 1 week and processed for immunocytochemistry. Whole projections of confocal microscopy optical section are shown. Cells were stained for LAMP1 (red) and VGF (green). Arrowheads indicate red-green co-stained structures. Scale bar, 10 mm. (C) Quantification of VGF signal detected in LAMP1+ vesicles in cell body area (% of total VGF signal in cell body). One-way ANOVA with Tukey’s multiple- comparison test is displayed on graph. ns, not significant.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti CD63 BD Biosciences Cat# 551458; RRID:AB_394205 Anti GAPDH Sigma-Aldrich Cat# G9545; RRID:AB_796208 Anti GFP serum N/A rpAb TG32 Anti GFP purified Roche Roche Cat# 11814460001; RRID:AB_390913 Anti GM130 BD Biosciences Cat# 610823; RRID:AB_398142 Anti HSC70 Santa Cruz Biotechnologies Cat# sc-1059; RRID:AB_2120291 Anti LAMP1 Sigma-Aldrich Cat# L1418; RRID:AB_477157 Anti LRRK2 purified Abcam Cat # ab133474; RRID:AB_2713963 Anti LRRK2 serum Antibodies Incorporated Cat# 73–253; RRID:AB_10671178 Anti TGN46 Bio-rad Cat# AHP500GT; RRID:AB_2203291 Anti TSG101 Genetex Cat# GTX70255; RRID:AB_373239 Anti VAMP2 N/A mAB 69.1 Anti VAMP4 N/A rpAb TG19B Anti VAMP7 N/A rpAb TG50 Anti VGF Santa Cruz Biotechnology Cat# sc-365397; RRID:AB_10846955 Deposited data Analyzed Proteomic Dataset from PC12 cells This manuscript ProteomeXchange Consortium via PRIDE, ID = PXD028321 https://www.ebi.ac.uk/pride/archive/ projects/PXD028321 Proteomics results with legends are summarized in Table S1 This manuscript N/A Experimental models: Cell lines PC12 ATCC Cat# CRL-1721.1 HEK293T Evelyne Bloch-Gallego, Institut Cochin N/A Oligonucleotides Guide RNA to KO VAMP 2 CACCgTGGCAGCGGTAGCCGACCTAAAAC TAGGTCGGCTACCGCTGCCAc This manuscript N/A Guide RNA to KO VAMP 4 CACCgTTTAGGTGGCGCTTGAACTTAAACA AGTTCAAGCGCCACCTAAAc This manuscript N/A See Table S2 for primers used for cloning This manuscript N/A Recombinant DNA See Table S2 for cloning details (initial plasmids, primers, cloning methods used) This manuscript N/A RFP-VAMP4 This manuscript N/A mCherry-LRRK2 WT This manuscript N/A mCherry-LRRK2 G2019S This manuscript N/A mCherry-LRRK2 R1441C This manuscript N/A RUSH-VGF-mCherry This manuscript N/A (Continued on next page) Cell Reports 42, 112221, March 28, 2023 17

Techniques: Immunocytochemistry, Staining, Confocal Microscopy, Comparison

Journal: Cell reports

Article Title: Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs.

doi: 10.1016/j.celrep.2023.112221

Figure Lengend Snippet: Figure 5. VGF follows ER to PM route via VAMP4 and VAMP7 compartments (A) Schematic representation of RUSH-VGF-mCherry trafficking upon biotin addition. The RUSH system is based on the expression of two fusion proteins: (1) an ER retention hook (Lys-Asp-Glu-Leu; KDEL) fused to core streptavidin and (2) a cargo protein fused to SBP (streptavidin-binding protein) and mCherry. Because of the high-affinity SBP/streptavidin interaction, the VGF cargo is retained in the ER. Addition of biotin to the cell medium triggers a synchronous release of the cargo from the ER and its transport across the GA to the cell surface, which can be monitored via mCherry tag. (B) Still images from Video S1. Trafficking of RUSH-VGF-mCherry co-expressed in HEK293T cells with GA marker (N-acetylglucosaminetransferase [NAGT]) upon biotin addition: t = 0 min (addition of biotin), at the ER; t = 15 min at the GA; t = 45 min, partially still at the GA and post-Golgi vesicles; t = 80 min, mainly targeted at the PM. (C) HEK293T transfected with RUSH-VGF-mCherry were submitted to biotin chase for the indicated times and lysed. Equal amounts of cell lysate proteins were processed for SDS-PAGE and mCherry immunoblot (short and long ECL exposures are shown). Note the presence of multiples bands below pro-VGF (80 kDa) that correspond to C-terminally mCherry-tagged cleaved VGF peptides. (D) RUSH-VGF-mCherry trafficking in HEK293T cells. After 15 min (top) and 60 min (bottom) of biotin incubation, cells were immunostained for mCherry (red), TGN marker TGN46 (green), and VAMP4 (blue) 15 min after biotin addition (top panels) and for CD63 (green) and VAMP7 (blue) 60 min after biotin addition (bottom panels). Projections of confocal microscopy optical sections of less than 1 mm are shown (black and white and three color micrographs). Whole Z projections of confocal microscopy optical sections are shown for each condition in the right panels as indicated. Bottom rows show magnifications of the colored boxed areas depicting co-stained structures further indicated by arrows. Scale bar, 10 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti CD63 BD Biosciences Cat# 551458; RRID:AB_394205 Anti GAPDH Sigma-Aldrich Cat# G9545; RRID:AB_796208 Anti GFP serum N/A rpAb TG32 Anti GFP purified Roche Roche Cat# 11814460001; RRID:AB_390913 Anti GM130 BD Biosciences Cat# 610823; RRID:AB_398142 Anti HSC70 Santa Cruz Biotechnologies Cat# sc-1059; RRID:AB_2120291 Anti LAMP1 Sigma-Aldrich Cat# L1418; RRID:AB_477157 Anti LRRK2 purified Abcam Cat # ab133474; RRID:AB_2713963 Anti LRRK2 serum Antibodies Incorporated Cat# 73–253; RRID:AB_10671178 Anti TGN46 Bio-rad Cat# AHP500GT; RRID:AB_2203291 Anti TSG101 Genetex Cat# GTX70255; RRID:AB_373239 Anti VAMP2 N/A mAB 69.1 Anti VAMP4 N/A rpAb TG19B Anti VAMP7 N/A rpAb TG50 Anti VGF Santa Cruz Biotechnology Cat# sc-365397; RRID:AB_10846955 Deposited data Analyzed Proteomic Dataset from PC12 cells This manuscript ProteomeXchange Consortium via PRIDE, ID = PXD028321 https://www.ebi.ac.uk/pride/archive/ projects/PXD028321 Proteomics results with legends are summarized in Table S1 This manuscript N/A Experimental models: Cell lines PC12 ATCC Cat# CRL-1721.1 HEK293T Evelyne Bloch-Gallego, Institut Cochin N/A Oligonucleotides Guide RNA to KO VAMP 2 CACCgTGGCAGCGGTAGCCGACCTAAAAC TAGGTCGGCTACCGCTGCCAc This manuscript N/A Guide RNA to KO VAMP 4 CACCgTTTAGGTGGCGCTTGAACTTAAACA AGTTCAAGCGCCACCTAAAc This manuscript N/A See Table S2 for primers used for cloning This manuscript N/A Recombinant DNA See Table S2 for cloning details (initial plasmids, primers, cloning methods used) This manuscript N/A RFP-VAMP4 This manuscript N/A mCherry-LRRK2 WT This manuscript N/A mCherry-LRRK2 G2019S This manuscript N/A mCherry-LRRK2 R1441C This manuscript N/A RUSH-VGF-mCherry This manuscript N/A (Continued on next page) Cell Reports 42, 112221, March 28, 2023 17

Techniques: Expressing, Binding Assay, Marker, Transfection, SDS Page, Western Blot, Incubation, Confocal Microscopy, Staining

Journal: Cell reports

Article Title: Secretion of VGF relies on the interplay between LRRK2 and post-Golgi v-SNAREs.

doi: 10.1016/j.celrep.2023.112221

Figure Lengend Snippet: Figure 7. Defect of VGF transport to neurites in VAMP7-LD and LRRK2-expressing primary neurons and hypothetical model (A) DIV6 primary hippocampal neurons were transfected with CMV-VGF and RFP, RFP-VAMP7-LD, mCherry-LRRK2 WT, or mCherry-LRRK2 R1441C, fixed at DIV8, and stained with mCherry and VGF antibodies. Wide-field fluorescent images are shown. Pound sign and asterisk correspond to RFP and RFP+ cell soma, respectively. (B) Quantification of peripheral (excluded from soma) over total VGF signal ratio. Horizontal lines show mean ± SEM of 3 color-coded independent experiments, while circles and triangles represent values of individual cells and mean of each experiment, respectively. One-way ANOVA with Tukey’s multiple-comparison test was performed from each triplicate mean values. **p < 0.01 and ***p < 0.001. Scale bar, 50 mm. (C) Hypothetical model of the routes of pro-VGF trafficking unraveled in this study: (1) VAMP2-dependent regulated secretion of VGF peptides, (2) VAMP7- dependent late endosomal secretion of pro-VGF, and (3) ATG5-dependent autophagic degradation of pro-VGF. LRRK2 interaction with VAMP4 and VAMP7 would function as a checkpoint for the secretory pathway in the cell center.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti CD63 BD Biosciences Cat# 551458; RRID:AB_394205 Anti GAPDH Sigma-Aldrich Cat# G9545; RRID:AB_796208 Anti GFP serum N/A rpAb TG32 Anti GFP purified Roche Roche Cat# 11814460001; RRID:AB_390913 Anti GM130 BD Biosciences Cat# 610823; RRID:AB_398142 Anti HSC70 Santa Cruz Biotechnologies Cat# sc-1059; RRID:AB_2120291 Anti LAMP1 Sigma-Aldrich Cat# L1418; RRID:AB_477157 Anti LRRK2 purified Abcam Cat # ab133474; RRID:AB_2713963 Anti LRRK2 serum Antibodies Incorporated Cat# 73–253; RRID:AB_10671178 Anti TGN46 Bio-rad Cat# AHP500GT; RRID:AB_2203291 Anti TSG101 Genetex Cat# GTX70255; RRID:AB_373239 Anti VAMP2 N/A mAB 69.1 Anti VAMP4 N/A rpAb TG19B Anti VAMP7 N/A rpAb TG50 Anti VGF Santa Cruz Biotechnology Cat# sc-365397; RRID:AB_10846955 Deposited data Analyzed Proteomic Dataset from PC12 cells This manuscript ProteomeXchange Consortium via PRIDE, ID = PXD028321 https://www.ebi.ac.uk/pride/archive/ projects/PXD028321 Proteomics results with legends are summarized in Table S1 This manuscript N/A Experimental models: Cell lines PC12 ATCC Cat# CRL-1721.1 HEK293T Evelyne Bloch-Gallego, Institut Cochin N/A Oligonucleotides Guide RNA to KO VAMP 2 CACCgTGGCAGCGGTAGCCGACCTAAAAC TAGGTCGGCTACCGCTGCCAc This manuscript N/A Guide RNA to KO VAMP 4 CACCgTTTAGGTGGCGCTTGAACTTAAACA AGTTCAAGCGCCACCTAAAc This manuscript N/A See Table S2 for primers used for cloning This manuscript N/A Recombinant DNA See Table S2 for cloning details (initial plasmids, primers, cloning methods used) This manuscript N/A RFP-VAMP4 This manuscript N/A mCherry-LRRK2 WT This manuscript N/A mCherry-LRRK2 G2019S This manuscript N/A mCherry-LRRK2 R1441C This manuscript N/A RUSH-VGF-mCherry This manuscript N/A (Continued on next page) Cell Reports 42, 112221, March 28, 2023 17

Techniques: Expressing, Transfection, Staining, Comparison

Journal: PLoS ONE

Article Title: The MyD88+ Phenotype Is an Adverse Prognostic Factor in Epithelial Ovarian Cancer

doi: 10.1371/journal.pone.0100816

Figure Lengend Snippet: MyD88 positive tumours (n = 12) had significantly reduced progression-free survival (A) and overall survival (B) (p = 0.018 and p = 0.008, respectively).

Article Snippet: TLR4 and MyD88 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) at initial concentrations of 200 µg/mL: rabbit polyclonal antibody (RpAb) to TLR4 (clone H-80); RpAb to MyD88 (clone HFL-296).

Techniques:

Journal: PLoS ONE

Article Title: The MyD88+ Phenotype Is an Adverse Prognostic Factor in Epithelial Ovarian Cancer

doi: 10.1371/journal.pone.0100816

Figure Lengend Snippet: Distribution of TLR4 & MyD88 protein expression in all patient samples.

Article Snippet: TLR4 and MyD88 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) at initial concentrations of 200 µg/mL: rabbit polyclonal antibody (RpAb) to TLR4 (clone H-80); RpAb to MyD88 (clone HFL-296).

Techniques: Expressing

Journal: PLoS ONE

Article Title: The MyD88+ Phenotype Is an Adverse Prognostic Factor in Epithelial Ovarian Cancer

doi: 10.1371/journal.pone.0100816

Figure Lengend Snippet: Characterisation of TLR4 & MyD88 expression in ovarian cancer.

Article Snippet: TLR4 and MyD88 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) at initial concentrations of 200 µg/mL: rabbit polyclonal antibody (RpAb) to TLR4 (clone H-80); RpAb to MyD88 (clone HFL-296).

Techniques: Expressing

Journal: PLoS ONE

Article Title: The MyD88+ Phenotype Is an Adverse Prognostic Factor in Epithelial Ovarian Cancer

doi: 10.1371/journal.pone.0100816

Figure Lengend Snippet: TLR4 (A) or MyD88 (B) negative cases had significantly better PFS (15 & 18 months longer; p<0.05).

Article Snippet: TLR4 and MyD88 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) at initial concentrations of 200 µg/mL: rabbit polyclonal antibody (RpAb) to TLR4 (clone H-80); RpAb to MyD88 (clone HFL-296).

Techniques:

Journal: PLoS ONE

Article Title: The MyD88+ Phenotype Is an Adverse Prognostic Factor in Epithelial Ovarian Cancer

doi: 10.1371/journal.pone.0100816

Figure Lengend Snippet: Survival was longer in MyD88 (B) negative cases (by 19 months; p<0.05). The difference in survival associated with TLR4 (A) is not significant (p>0.5).

Article Snippet: TLR4 and MyD88 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) at initial concentrations of 200 µg/mL: rabbit polyclonal antibody (RpAb) to TLR4 (clone H-80); RpAb to MyD88 (clone HFL-296).

Techniques:

Journal: PLoS ONE

Article Title: The MyD88+ Phenotype Is an Adverse Prognostic Factor in Epithelial Ovarian Cancer

doi: 10.1371/journal.pone.0100816

Figure Lengend Snippet: Scatter plots showing relative microRNA expression with standard deviation (fold changes calculated via the 2 −ΔΔCt method). 20 EOC cases (serous carcinomas) grouped as MyD88+ or MyD88- based on protein expression; data shown relative to each group. Average expression of miR-21 & miR-146a increased in MyD88 negative EOC (p<0.05).

Article Snippet: TLR4 and MyD88 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) at initial concentrations of 200 µg/mL: rabbit polyclonal antibody (RpAb) to TLR4 (clone H-80); RpAb to MyD88 (clone HFL-296).

Techniques: Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: The MyD88+ Phenotype Is an Adverse Prognostic Factor in Epithelial Ovarian Cancer

doi: 10.1371/journal.pone.0100816

Figure Lengend Snippet: Scatter plots showing relative gene and microRNA expression with standard deviation (fold changes calculated via the 2 −ΔΔCt method). 22 EOC cases (serous carcinomas) grouped as MyD88+ or MyD88- based on protein expression; data shown relative to each group. A & B, TLR4 & MyD88 mRNA statistically unchanged between MyD88 positive & MyD88 negative groups (p>0.05); C & D, levels of both miR-21 & miR-146a up-regulated in MyD88 negative EOC (p<0.05).

Article Snippet: TLR4 and MyD88 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) at initial concentrations of 200 µg/mL: rabbit polyclonal antibody (RpAb) to TLR4 (clone H-80); RpAb to MyD88 (clone HFL-296).

Techniques: Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: The MyD88+ Phenotype Is an Adverse Prognostic Factor in Epithelial Ovarian Cancer

doi: 10.1371/journal.pone.0100816

Figure Lengend Snippet: SKOV-3 cells were left untransfected (Unt), transfected with negative control siRNA (siNeg), MyD88 targeting siRNA (siMyD88) or TLR4 targeting siRNA (siTLR4). The transfected cells were incubated for 72 hrs before either harvesting for mRNA analysis (A), for protein analysis (B) or treatment with paclitaxel (C). (A) MyD88 and TLR4 mRNA expression levels were evaluated by TaqMan RT-PCR. MyD88 and TLR4 mRNA expression was normalised to that of an endogenous control, B2M, and calibrated to that of untreated cells to establish the relative percentage of mRNA expression (n = 3, mean +SD). (B) MyD88 and TLR4 mRNA expression levels were evaluated by western blot analysis. GAPDH was used as a loading control. (C) Transfected cells were either left untreated, treated with DMSO (vehicle control) or 3.5 nM of paclitaxel (IC25). 48 hrs post treatment, cell viability was assessed by means of the CCK-8 assay. % cell viability rate was calculated by comparing the absorbance values for the vehicle control to the corresponding paclitaxel treated samples. Results are expressed as mean +SD, n = 3; *p<0.05, **p<0.01 (un-paired Student's t-test).

Article Snippet: TLR4 and MyD88 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) at initial concentrations of 200 µg/mL: rabbit polyclonal antibody (RpAb) to TLR4 (clone H-80); RpAb to MyD88 (clone HFL-296).

Techniques: Transfection, Negative Control, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, CCK-8 Assay

Journal: PLoS ONE

Article Title: The MyD88+ Phenotype Is an Adverse Prognostic Factor in Epithelial Ovarian Cancer

doi: 10.1371/journal.pone.0100816

Figure Lengend Snippet: A , qPCR data: MyD88 expression is down-regulated upon NTera2 differentiation, with minimal changes in TLR4; in contrast 2102Ep cells avoid differentiation and maintain both TLR4 & MyD88 expression (fold changes shown are proportional to internal control gene GAPDH and calculated via the 2 −ΔΔCt method). B , TLR4 and MyD88 protein expression in NTera2 cells: TLR4 staining was unchanged following differentiation (left panel); MyD88 staining in undifferentiated NTera2 cells showed significantly reduced staining after differentiation (right panel) (all 40x). C , TLR4 and MyD88 expression in 2102Ep cells: minimal changes in TLR4 (left panel) or MyD88 (right panel) were observed following RA-treatment (all at 40x).

Article Snippet: TLR4 and MyD88 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) at initial concentrations of 200 µg/mL: rabbit polyclonal antibody (RpAb) to TLR4 (clone H-80); RpAb to MyD88 (clone HFL-296).

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: The MyD88+ Phenotype Is an Adverse Prognostic Factor in Epithelial Ovarian Cancer

doi: 10.1371/journal.pone.0100816

Figure Lengend Snippet: Quantification of immunohistochemical staining of TLR4 and MyD88 (0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining).

Article Snippet: TLR4 and MyD88 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) at initial concentrations of 200 µg/mL: rabbit polyclonal antibody (RpAb) to TLR4 (clone H-80); RpAb to MyD88 (clone HFL-296).

Techniques: Immunohistochemical staining, Staining

Journal: Acta Neuropathologica Communications

Article Title: The Arctic AβPP mutation leads to Alzheimer’s disease pathology with highly variable topographic deposition of differentially truncated Aβ

doi: 10.1186/2051-5960-1-60

Figure Lengend Snippet: The list of antibodies used in immunohistochemistry and immunoprecipitation experiments

Article Snippet: rpAb cathepsin D , Lysosomes , Dako (production discontinued).

Techniques: Immunohistochemistry, Immunoprecipitation

Journal: Acta Neuropathologica Communications

Article Title: The Arctic AβPP mutation leads to Alzheimer’s disease pathology with highly variable topographic deposition of differentially truncated Aβ

doi: 10.1186/2051-5960-1-60

Figure Lengend Snippet: The list of antibodies used in immunohistochemistry and immunoprecipitation experiments

Article Snippet: rpAb 42 , Aβx-42, neoepitope , Biosource/Invitrogen.

Techniques: Immunohistochemistry, Immunoprecipitation