Journal: PLoS Pathogens

Article Title: Global Functional Analyses of Cellular Responses to Pore-Forming Toxins

doi: 10.1371/journal.ppat.1001314

Figure Lengend Snippet: ( A ) Western blot of mammalian c-JUN in HaCaT cells treated with 125 ng/mL SLO for time indicated. Abbreviations: S: constitutively active version of SLO, N: non-lytic mutant N402 SLO; Co: no-SLO control. ( B ) HaCaT cells were treated with SLO and biotinylated AP-1 decoy-oligonucleotide (right panel) or biotinylated control oligonucleotide (left panel). Oligonucleotide is stained red (SA-Alexa594), and DNA is stained blue (4',6-diamidino-2-phenylindole/DAPI). The images presented are a single representative from three independent assays. Cells treated with AP-1 decoy show a greater proportion of intoxicated cells with condensed chromosomes, characteristic of becoming pyknotic ( e.g. , white arrowhead). The proportion of pyknotic cells was quantified as described in the section; the bar graph shows mean values from three independent experiments (m = mismatched oligonucleotide; AP-1 = AP-1 decoy; error bars: standard error of the mean, P value was determined with paired, one-tailed student's t-test). ( C ) A model for interconnected regulation of defense to PFT. The KGB-1 JNK-like MAPK regulates both p38 MAPK–dependent (e.g., ttm-1 , ttm-2 , UPR) and p38 MAPK-independent ( e.g. , jun-1 , kin-18 ) PFT-induced protection genes. There are also PFT protection genes that, to date, have not been linked to either MAPK pathway.

Article Snippet: For the AP-1 decoy experiment, HaCaT cells were treated in Ca 2+- free medium with 500 ng/mL SLO and 3 μM biotinylated AP-1 decoy oligodeoxynucleotide (ODN), or mismatched AP-1 decoy ODN for 30 min, (Genedetect, Auckland, New Zealand).

Techniques: Western Blot, Mutagenesis, Control, Staining, One-tailed Test

Journal: Cell Stress & Chaperones

Article Title: An unfolded protein response is the initial cellular response to the expression of mutant matrilin-3 in a mouse model of multiple epiphyseal dysplasia

doi: 10.1007/s12192-010-0193-y

Figure Lengend Snippet: Histological and immunohistochemical analysis of wild-type and mutant xiphoid and rib cartilage. H&E staining of wild-type (wt) and mutant (m/m) a rib and b xiphoid cartilage growth plates from 5- and 21-day-old mice. Cells in the proliferative zone form ordered columns along the vertical axis of the GP ( black boxes ). In the m/m mice by 5 days of age, the chondrocytes ( red circles ) of the proliferative zone are rounder and more randomly orientated compared to the wt ( black box ). Scale bar is 100 µm. IHC with an anti-matrilin-3 antibody on c rib and d xiphoid growth plates from 5-, 14- and 21-day-old mice. In wild-type mice (wt), matrilin-3 staining was seen in the cartilage ECM at all ages. In mutant mice (m/m), matrilin-3 staining was predominantly within the chondrocytes with only minimal staining present in the ECM. Intracellular staining was observed from 5 days of age and was still present by 21 days of age

Article Snippet: Antibodies used were rabbit anti-matrilin-3 (R&D), rat anti-BrdU (Abcam) and goat anti-Grp94 (Santa Cruz).

Techniques: Immunohistochemical staining, Mutagenesis, Staining

Journal: Cell Stress & Chaperones

Article Title: An unfolded protein response is the initial cellular response to the expression of mutant matrilin-3 in a mouse model of multiple epiphyseal dysplasia

doi: 10.1007/s12192-010-0193-y

Figure Lengend Snippet: The effect of SPB treatment on the phenotype of mutant mice and the relative levels of matrilin-3 retention, chondrocyte proliferation and apoptosis. a Bone length measurements of treated and untreated mutant male mice were performed on radiographs taken at 9 weeks of age showed no significant differences. Measurements were made of the inner canthal distance ( ICD ), femur ( F ), pelvis ( P ) and tibia (T). ( n > 23 mice per group; nested ANOVA). b The body weights of male and female mutant mice showed no significant differences between untreated and treated groups. ( n > 23 mice; nested ANOVA). The relative levels of apoptosis in c the hypertrophic zone (HZ) and d at the VIF of the growth plate was calculated by counting the number of TUNEL-positive chondrocytes in the HZ or at the VIF compared to the total number of DAPI-stained chondrocytes in the growth plate. Although there appeared to be relatively less TUNEL-positive cells in the HZ and more apoptosis at the VIF in the treated group, these differences were not statistically significant. ( n > 20 sections per group; nested ANOVA). e Twenty-one-day-old mice were administered with 0.01 ml/g of the nucleotide analogue BrdU 2 h prior to sacrifice. IHC was performed on tibia sections using an anti-BrdU antibody. The relative levels of chondrocyte proliferation was determined by counting the number of BrdU-labelled nuclei compared to the total number of chondrocytes in the proliferative zone of the growth plate. There were no significant differences in the proportion of proliferating cells between the treated and untreated groups. ( n > 20 sections per group; nested ANOVA). f IHC using anti-matrilin-3 antibody on tibia growth plates from 21-day-old mutant mice either treated or untreated with SPB. In both groups, there is extensive intracellular retention of mutant matrilin-3 ( scale bar is 100 µm)

Article Snippet: Antibodies used were rabbit anti-matrilin-3 (R&D), rat anti-BrdU (Abcam) and goat anti-Grp94 (Santa Cruz).

Techniques: Mutagenesis, TUNEL Assay, Staining

Journal: BMJ Open Diabetes Research & Care

Article Title: Mouse model of metformin-induced diarrhea

doi: 10.1136/bmjdrc-2019-000898

Figure Lengend Snippet: Effects of wood creosote on diabetic obese mice treated with metformin. Wood creosote does not affect efficacy of metformin. Weight change (A) and blood glucose levels (fed condition) (B) were examined during the experiment in . Data are shown as means±SEM. No significant differences between the metformin group and the metformin with wood creosote (M+C) group were observed. Mice were fasted for 3 hours after the final administration of drugs. (C) One g/kg glucose was administrated via oral gavage. One to 2 µL blood was collected via tail vein at indicated time points, and blood glucose was measured by using GlucoSensor. (D) At day 4 of , mice were fasted for 2 hours and then injected intraperitoneally with 36 µg/kg insulin. Blood glucose levels are shown as % initial. At day 5 in , blood was collected and measured serum bile acid levels (E) and alanine aminotransferase (F) were measured. (G) Western blots were carried out using liver protein (50 µg) to detect levels of phospho-AMPK and phoshopho-AKT. AKT, Protein Kinase B; AMPK, AMP-activated kinase.

Article Snippet: Antibodies are as follows: Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1E6D9, Proteintech, Rosemont, Illinois, USA), anti-AMPK (ABV10739, ABGENT, San Diego, California, USA), anti-phospho AMPK (pT172) (40H9, Cell Signaling Technology, Danvers, Massachusetts, USA), anti-total Protein Kinase B (AKT) (200–401 N98, Rockland, Limerick, Pennsylvania, USA), and anti-phosho AKT (pS473) (D9E, Cell Signaling Technology).

Techniques: Injection, Western Blot