Journal: Journal of Pathology and Translational Medicine

Article Title: Adrenal Cortical Neoplasm with Uncertain Malignant Potential Arising in the Heterotopic Adrenal Cortex in the Liver of a Patient with Beckwith-Wiedemann Syndrome

doi: 10.4132/jptm.2018.11.13

Figure Lengend Snippet: Detailed information and results of immunohistochemistry

Article Snippet: Chromogranin , - , - , - , 1:1,600 , M0869 , Dako, Glostrup, Denmark.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Interaction of Calcium-dependent Activator Protein for Secretion 1 (CAPS1) with the Class II ADP-ribosylation Factor Small GTPases Is Required for Dense-core Vesicle Trafficking in the trans -Golgi Network *

doi: 10.1074/jbc.M110.137414

Figure Lengend Snippet: CAPS1 knockdown induces chromogranin accumulation in the Golgi complex. A, KD of CAPS1 expression in PC12 cells by siRNA. siRNA specific for CAPS1 was electroporated into PC12 cells, and the culture medium was concentrated and electrophoresed. Top, three bands were greatly reduced in the culture medium of the CAPS1 KD cells and were identified as chromogranin A (ChgA), chromogranin B (ChgB), and secretogranin II (SgII) by LC-MS/MS. Bottom, reduction of CAPS1 expression in PC12 cells by siRNA KD was assayed by immunoblotting of cell lysates with anti-CAPS1 antibody. B, high KCl-induced chromogranin A and secretogranin II release from CAPS1 KD PC12 cells. PC12 cells were transfected with either control siRNA or CAPS1 siRNA together with a ChgA-HA or SgII-HA expression plasmid. The culture media, treated with 5 mm KCl (5K) or 50 mm KCl (50K) (see “Experimental Procedures”), were collected and analyzed by immunoblotting with anti-HA antibody. C, statistical analyses of the effect of CAPS1 KD on ChgA-HA release from PC12 cells. PC12 cells were transfected with ChgA-HA together with the control (white bar) or CAPS1 siRNA (black bar). The amounts of ChgA-HA released into the culture media with 5 or 50 mm KCl stimulation were analyzed by Western blotting, followed by densitometric analysis (n = 6). There was no significant difference in the amount of ChgA-HA in cell lysates between the control and CAPS1 KD groups (n = 6). The signal intensities of the extracellular ChgA-HA bands of the culture media were normalized against those of the intracellular ChgA-HA bands of the cell lysates. AU, arbitrary unit. Error bars, S.E. **, p < 0.01 by Student's t test. D, subcellular localization of endogenous chromogranin A in NGF-differentiated PC12 cells without (left) and with (right) CAPS1 KD; immunostaining for chromogranin A. Scale bars, 20 μm. E, subcellular localization of ChgA-HA expressed in NGF-differentiated PC12 cells without (left) and with (right) CAPS1 KD; immunostaining for HA. Scale bars, 20 μm. F, subcellular localization of C-terminal HA-tagged TrkB expressed in NGF-differentiated PC12 cells without (left) and with (right) CAPS1 KD; immunostaining for HA. Scale bars, 20 μm.

Article Snippet: The following primary antibodies were used for immunocyto- or immunohistochemistry: mouse monoclonal anti-chromogranin A (1:5,000 dilution; catalog no. 611844, BD Biosciences), mouse monoclonal anti-syntaxin 6 (1:500 dilution; catalog no. 610635, BD Biosciences), mouse monoclonal anti-FLAG (1:250 dilution; catalog no. F1804, Sigma), and rat monoclonal anti-HA (1:250 dilution; catalog no. 1867423, Roche Applied Science).

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Western Blot, Transfection, Plasmid Preparation, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: Interaction of Calcium-dependent Activator Protein for Secretion 1 (CAPS1) with the Class II ADP-ribosylation Factor Small GTPases Is Required for Dense-core Vesicle Trafficking in the trans -Golgi Network *

doi: 10.1074/jbc.M110.137414

Figure Lengend Snippet: Subcellular distribution and release of chromogranin is disrupted by either ARF4/5 siRNA knockdown or expression of CAPS1-binding-deficient ARF5. A–D, HA-tagged chromogranin (ChgA-HA) expressed in PC12 cells showed a Golgi-accumulated pattern following KD of ARF4/5 expression. Shown are immunocytochemical staining patterns of control (A), ARF4 KD (B), ARF5 KD (C), and ARF4/5 double KD (D) cells with anti-HA antibody. ARF4 and ARF5 siRNAs efficiently reduced the expression levels of ARF4 and ARF5 proteins, respectively (supplemental Fig. S3). Scale bar, 10 μm. E–G, ARF5(3,4A) and ARF5(3,4,6A) had a disrupted N-terminal CAPS1 binding site and showed a decrease in regulated release of ChgA-HA from PC12 cells. ChgA-HA and one of the ARF5 constructs (wild-type ARF5, ARF5(3,4A), or ARF5(3,4,5A)) were transfected into PC12 cells. Culture medium after treatment with 5 mm KCl (5K) or 50 mm KCl (50K) was examined by Western blot analysis with anti-HA antibody (E), and the cell lysates were analyzed by Western blotting (WB) with anti-HA antibody or anti-FLAG antibody (F). G, statistical analyses of the effect of ARF5 and CAPS1 binding-deficient ARF5 on ChgA-HA release from PC12 cells. PC12 cells were transfected with ChgA-HA together with wild-type ARF5, CAPS1 binding-deficient ARF5(3,4A), or ARF5(3,4,6A). The amount of ChgA-HA released into the culture media with 5 mm KCl (white bar) or 50 mm KCl (black bar) stimulation was analyzed by Western blotting followed by densitometric analysis (n = 4). The signal intensities of the extracellular ChgA-HA bands from the culture media were normalized against those of the intracellular ChgA-HA bands from the cell lysates. AU, arbitrary unit. Error bars, S.E. **, p < 0.01 by Student's t test. H–J, CAPS1 binding-deficient ARF5(3,4,6A) coexpressed in PC12 cells induces accumulation of ChgA-HA in the Golgi. Immunocytochemical staining with anti-HA antibody indicates that ChgA-HA is localized in the tips of processes and around the nuclei in control cells (H) and cells coexpressing wild-type ARF5 (I) but is accumulated in the Golgi in cells coexpressing ARF5(3,4,6A) (J). Scale bar, 25 μm. K and L, CAPS1 binding-deficient ARF5(3,4,6A) coexpressed in primary cultured hippocampal neurons (DIV8) induces accumulation of ChgA-HA in the Golgi. Immunocytochemistry with anti-HA antibody shows that expressed ChgA-HA is localized in neurites and in soma of cells coexpressing wild-type ARF5 (K), whereas it is accumulated in the Golgi in cells coexpressing ARF5(3,4,6A) (L). Scale bar, 50 μm.

Article Snippet: The following primary antibodies were used for immunocyto- or immunohistochemistry: mouse monoclonal anti-chromogranin A (1:5,000 dilution; catalog no. 611844, BD Biosciences), mouse monoclonal anti-syntaxin 6 (1:500 dilution; catalog no. 610635, BD Biosciences), mouse monoclonal anti-FLAG (1:250 dilution; catalog no. F1804, Sigma), and rat monoclonal anti-HA (1:250 dilution; catalog no. 1867423, Roche Applied Science).

Techniques: Expressing, Binding Assay, Staining, Construct, Transfection, Western Blot, Cell Culture, Immunocytochemistry

Journal: mBio

Article Title: Citrobacter rodentium Infection Induces Persistent Molecular Changes and Interferon Gamma-Dependent Major Histocompatibility Complex Class II Expression in the Colonic Epithelium

doi: 10.1128/mbio.03233-21

Figure Lengend Snippet: Changes to the cellular composition of the crypt. (A, C, G, and K) Temporal measurements of CCH (A), the crypt PCNA + zone (C), goblet cell density (G), and CHGB-positive cell density (K). Each point represents the mean for an individual mouse. *, P < 0.0.5; ****, P < 0.0001 (by one-way ANOVA with Dunnett’s posttest for multiple comparisons to the control group [uninfected {UI} mice]). (B and L) Representative distal colonic sections showing temporal changes in the crypt PCNA + zone (B) and CHGB-positive cell density (L). Bars = 50 μm (B) or 100 μm (L). (D and F) Average Log 2 FC values of selected significantly changed (FDR of <0.05 by one-way ANOVA) proteins involved in cell proliferation and cIEC metabolic changes (D) and cIEC markers (F). DSC, deep secretory cell. In panel F, “−ve” indicates that proteins are negative regulators of terminal differentiation into the specified cell type. (E, I, and J) qRT-PCR of the indicated mRNAs isolated from cIECs. Each point represents an individual mouse. *, P < 0.05; **, P < 0.01 (by one-way ANOVA with Dunnett’s posttest for multiple comparisons to the control group [uninfected mice]). For panels G and J, square data points were identified as outliers and not included in the statistical analysis. (H) z scores of selected protein abundances across the entire C. rodentium infection time course (4 to 48 DPI). Means ± standard errors of the means (SEM) are shown.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti‐ C. rodentium antibody (1:50) (Statens Serum Institute, Copenhagen, Denmark), mouse anti-PCNA antibody (1:500) (catalog number ab29; Abcam), and rabbit anti-CHGB antibody (1:50) (catalog number 14968-1-AP; Proteintech).

Techniques: Control, Quantitative RT-PCR, Isolation, Infection

Journal: bioRxiv

Article Title: The expression of YAP1 is increased in high-grade prostatic adenocarcinoma but is reduced in neuroendocrine prostate cancer

doi: 10.1101/832360

Figure Lengend Snippet: Microscopic examination of the prostate of TRAMP and LADY mice revealed that the expression of YAP1 increased in PIN but decreased in NEPC. ( A ) Wild-type prostate demonstrating positive staining of YAP1 in the occasional basal epithelia but negative staining in luminal epithelia. ( B&C ) Serial sections of a PIN tumor of TRAMP demonstrating diffuse positive staining of YAP1 and the rare FOXA2-positive cells in PIN. ( D to F ) Serial sections of a NEPC tumor of TRAMP demonstrating diffuse positive staining of YAP1 in PIN components but negative in NEPC cells. NEPC markers chromogranin A (CHGA) and FOXA2 were stained positive in NEPC cells but negative in PIN. ( G & H ) Serial sections of a 12T-10 NEPC tumor demonstrating negativity of YAP1 in NEPC cells in contrast to the positive staining in focal PIN. Positive staining of FOXA2 was seen in the NEPC cells. ( I ) NE10 tumor showed negative YAP1 staining. ( J to O ) The expression of YAP1 in NEPC metastases. Sections of the liver (J to L) and lung (M to O) with metastasis of NEPC demonstrated negative YAP1 staining in metastatic NEPC. In contrast, FOXA2 and CHGA were positive in NEPC metastases.

Article Snippet: Primary antibodies of YAP1 and Chromogranin A (CHGA) were purchased from Santa Cruz Biotechnology (Dallas, TX), Synaptophysin (SYP) (BD biosciences, San Jose, CA), p63 and FOXA2 (Abcam, Cambridge, MA).

Techniques: Expressing, Staining, Negative Staining

Journal: bioRxiv

Article Title: The expression of YAP1 is increased in high-grade prostatic adenocarcinoma but is reduced in neuroendocrine prostate cancer

doi: 10.1101/832360

Figure Lengend Snippet: The expression of YAP1 in human prostatic tissues. ( A ) Benign prostatic tissue demonstrated positive YAP1 staining in the stroma cells and basal cells. ( B ) Low-grade prostate adenocarcinoma. YAP1 expression was present in stromal cells and occasional basal cells but was absent in adenocarcinoma cells. ( C ) High-grade prostate adenocarcinoma. YAP1 was expressed in prostate adenocarcinomas with a Gleason score of 8 or higher. ( D to F ) Serial sections of a NEPC with focal adenocarcinoma. YAP1 protein was expressed in high-grade adenocarcinoma components, but the expression was lost in NEPC cells. CHGA and FOXA2 were diffusely expressed in NEPC but not in adenocarcinoma cells. ( G to I ) Serial sections of a small cell carcinoma demonstrating negative YAP1 and positive synaptophysin (SYP) staining in NEPC cells.

Article Snippet: Primary antibodies of YAP1 and Chromogranin A (CHGA) were purchased from Santa Cruz Biotechnology (Dallas, TX), Synaptophysin (SYP) (BD biosciences, San Jose, CA), p63 and FOXA2 (Abcam, Cambridge, MA).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: The expression of YAP1 is increased in high-grade prostatic adenocarcinoma but is reduced in neuroendocrine prostate cancer

doi: 10.1101/832360

Figure Lengend Snippet: The expression of YAP1 in LuCaP PDXs. The expression levels of YAP1, TAZ, AR, and NEPC markers (FOXA2, SOX2, CHGA, and SYP) were extracted from RNAseq data derived from 46 LuCaP PDXs including (from left to right) AR + /NE − (group 1, n=35), AR low /NE − (group 2, n=1), AR − /NE − (group 3, n=1), AR + /NE + (group 4, n=1), and AR − /NE + tumors (group 5, n=8). YAP1 expression was lost in all the NE positive LuCaP PDXs. Whereas, YAP1 was expressed in all but one prostate adenocarcinoma cases.

Article Snippet: Primary antibodies of YAP1 and Chromogranin A (CHGA) were purchased from Santa Cruz Biotechnology (Dallas, TX), Synaptophysin (SYP) (BD biosciences, San Jose, CA), p63 and FOXA2 (Abcam, Cambridge, MA).

Techniques: Expressing, Derivative Assay