Journal: bioRxiv

Article Title: Parallel signaling through IRE1α and PERK regulates pancreatic neuroendocrine tumor growth and survival

doi: 10.1101/522102

Figure Lengend Snippet: A , In response to the accumulation of misfolded proteins in the ER, IRE1α and PERK homodimerize and signal an adaptive stress response through splicing of Xbp1 and phosphorylation of eIF2α, respectively. However, under sustained ER stress, these pathways promote apoptosis through RIDD and upregulation of pro-apoptotic CHOP. B-C , Representative ( B ) H&E and ( C ) BiP/GRP78 IHC on normal pancreas and primary human PanNET. Star indicates islet of Langerhans (scale bars, 50 μm). D-E , ( D ) Percent XBP1 splicing and ( E ) relative ATF4 mRNA expression from normal human pancreas and four primary human PanNETs. Technical replicate error bars shown in E. F , PanNET xenograft experimental setup. INS-1 cells (control vs. transgenic variant) injected s.c. in bilateral flanks of NSG mice. Tumors become palpable by ∼10 d; mice are sacrificed at 4 weeks post-injection. G-K , IHC of human PanNETs and INS-1 mouse xenografts stained with the indicated antibodies (CgA=chromogranin A, SPH=synaptophysin; scale bars, 50 μm). L , INS-1 cells were grown in tissue culture ( in vitro ) or as xenografts ( in vivo ) in NSG mice for 2 or 3 weeks. Three unique replicates of each condition were harvested and analyzed by immunoblotting with the indicated antibodies. Quantified in Fig. S1B and C.

Article Snippet: Antibodies used for immunohistochemistry: BiP [C50B12] (Cell Signaling Technology #3177, 1:200), CD31 (CST #77699 1:100), Chromogranin A [polyclonal] (Cell Marque, 1:4), Cleaved Caspase-3 (CST #9661 1:200), Insulin (DAKO A0564, 1:200), IRE1α (CST #3294, 1:100), Ki67 (Ventana #790-4286, Undiluted), Myc (Sigma M4439, 1:5000), Synaptophysin [LK2H10 clone] (Cell Marque, 1:100).

Techniques: Expressing, Transgenic Assay, Variant Assay, Injection, Staining, In Vitro, In Vivo, Western Blot

Journal: Biological Research

Article Title: The tankyrase inhibitor G007-LK inhibits small intestine LGR5 + stem cell proliferation without altering tissue morphology

doi: 10.1186/s40659-017-0151-6

Figure Lengend Snippet: Morphology and cellular composition of duodenal tissue. ( a – c ) Haematoxylin and eosin (H&E)-stained sections from control ( a ), short-term gavage, 50 mg G007-LK/kg body mass ( b ), and long-term enriched chow, 100 mg G007-LK/kg chow ( c ) treated mice. d , e Periodic acid-Schiff (PAS)-stained cells (indicated with black arrows) were identified in duodenal tissue samples from control ( d ) and G007-LK ( e ) treated (gavage 50 mg G007-LK/kg body mass) mice (inset shows enlargements of boxed areas in main panel). f Quantification of the average number of PAS-positive cells per villus in duodenal tissue sections from control and G007-LK-treated mice (mean ± SEM, n = 3). g , h IHC of duodenal tissue sections, stained with an antibody against Lysozyme, from control ( g ) and G007-LK treated, gavage 10 mg G007-LK/kg body mass ( h ) mice (inset shows enlargements of boxed areas in main panel). ( i , j ) IHC of duodenal tissue sections, stained with an antibody against Chromogranin A (positive cells indicated with black arrows), from control ( i ) and G007-LK-treated, long term enriched chow, 100 mg G007-LK/kg chow ( j ) mice (inset shows enlargements of boxed areas in main panel)

Article Snippet: IHC staining was performed as previously described [ ], with primary antibodies against β-catenin, green fluorescent protein (GFP), Lysozyme and Chromogranin A (rabbit monoclonal anti-β-catenin, Abcam ab32572; rabbit monoclonal anti-GFP, Cell Signaling Technology #2956; rabbit monoclonal anti-Lysozyme, Abcam ab108508; rabbit polyclonal anti-Chromogranin A, Abcam ab15160).

Techniques: Staining

Journal: Nature Communications

Article Title: Stress-induced plasticity of a CRH/GABA projection disrupts reward behaviors in mice

doi: 10.1038/s41467-023-36780-x

Figure Lengend Snippet: a Schematic of experimental design for electrophysiology recordings in the whole-cell patch-clamp configuration. Horizontal brain slices containing BLA and NAc from CRH-ires-CRE mice that were injected with Cre-dependent ChR2-EYFP in BLA. b Representative traces of optically evoked IPSCs (oIPSCs). These were blocked in the presence of GABA A receptor antagonist picrotoxin. c A neurobiotin (brown) filled neuron from which oIPSCs were recorded. Note spines (arrows) suggesting the cell is a medium spiny neuron (MSN). Arrowheads denote ChR2-expressing boutons from BLA-origin CRH + axons (CRH; blue) on the soma. d oIPSCs amplitudes pre and post picrotoxin ( n = 7 neurons, 5 mice). e Time-course plot of normalized oIPSCs amplitudes throughout the recording and following application of picrotoxin. Black triangles in e denote trace recordings in b and timepoint analysis in d and e . f Representative trace of a NAc cell showing no response from optically evoked EPSC (oEPSC) at −70 mV, obtained after verifying oIPSCs at 0 mV in the presence of picrotoxin. g Representative trace showing spontaneous EPSCs (sEPSCs) were still present; gray box shows magnified view recording. In d and e , bars represent mean. In e circles represent mean ± SEM. Two-sided paired t -tests in d and e . d oIPSCs (current) pre vs. post PTX: P = 0.0338. e oIPSCs (normalized) pre vs. post PTX: P = 0.0004. 3 V = third ventricle, Hip Hippocampus, LV lateral ventricle. Source data are provided as a Source Data file.

Article Snippet: Sections were rinsed in PBS-T and then processed for CRH (1:20,000, PBL#rC68) or ChR2-EYFP (rabbit anti-GFP in 1:2000, Cat # 2555 S, Lot # 2, Cell Signaling, US) immunostaining.

Techniques: Patch Clamp, Injection, Expressing

Journal: Nature Communications

Article Title: Stress-induced plasticity of a CRH/GABA projection disrupts reward behaviors in mice

doi: 10.1038/s41467-023-36780-x

Figure Lengend Snippet: a , b Coordinate locations of a , DREADD injection and CNO infusion and b opsin injection and fiber placement. c schematic of the reward tasks. d , e Chemogenetically stimulating the CRH/GABA BLA → NAc projection with microinfusion of CNO in the medial NAc shell suppressed d , palatable food consumption ( n = 10 male mice; 12 female mice) and e preference for a sex-cue ( n = 12 male mice; 12 female mice) in males, but not females. f , g Inhibiting the CRH/GABA BLA → NAc projection in TR male mice did not increase f , palatable food consumption ( n = 11 mice) or g , preference for a sex-cue ( n = 8 mice). h , i stimulating the CRH/GABA ChR2-expressing BLA → NAc projection decreased h palatable food consumption ( n = 9 mice) and i , approach time to a sex-cue ( n = 11 mice). In d – i bars represent mean. Two-way ANOVA with repeated measures followed by post hoc tests ( d , e ). d hM3Dq BLA → NAc: Sex x Treatment— F = 5.691, DFn = 1, DFd = 40, P = 0.0219; post hoc with Tukey’s multiple comparison (Veh vs. CNO: Male— P = 0.0016; Female— P = 0.9366). e hM3Dq BLA → NAc: Treatment— F = 6.448, DFn = 1, DFd = 44, P = 0.0147; post hoc with Sidak’s multiple comparison (Veh vs. CNO: Male— P = 0.0081; Female— P = 0.8241). Two-sided paired t -tests ( f – i ). f hM4Di BLA → NAc: P = 0.1036; g hM4Di BLA → NAc: P = 0.1139. h ChR2 BLA → NAc: P = 0.0064; i ChR2 BLA → NAc: P = 0.0034. PF palatable food. Gray = vehicle/light off, teal = CNO, blue = light on. Source data are provided as a Source Data file.

Article Snippet: Sections were rinsed in PBS-T and then processed for CRH (1:20,000, PBL#rC68) or ChR2-EYFP (rabbit anti-GFP in 1:2000, Cat # 2555 S, Lot # 2, Cell Signaling, US) immunostaining.

Techniques: Injection, Expressing, Comparison