Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Sulforaphane promotes ER stress, autophagy, and cell death: implications for cataract surgery

doi: 10.1007/s00109-016-1502-4

Figure Lengend Snippet: MAPK signaling involvement in SFN-induced autophagy in FHL124 human lens epithelial cells. The effects of 100 μM SFN on p38, JNK, and ERK phosphorylation in conjunction with LC3-II levels detected by Western blot methods ( a , b ); representative blots showing products at ∼44/42 kDa (phospho and total ERK1/2), 46 kDa (phospho and total JNK1), 38 kDa (phospho and total p38), and 45 kDa (β-actin) ( a ) and pooled quantitative data ( b ) are presented. Data are presented as mean ± SEM ( n = 4). Asterisk indicates a significant difference between treated and nontreated control groups ( p ≤ 0.05; ANOVA with Dunnett’s post hoc test). The influence of inhibiting ERK phosphorylation (using 5 μM U0126) on SFN induced LC3-II expression ( c , d ) and autophagic vesicles ( e , f ). Western blots detected products for LC3-I and LC3-II at ∼18 and 16 kDa; phospho and total ERK1/2 were detected at ∼44/42 kDa; a band corresponding to β-actin was detected at 45 kDa. Data are presented as mean ± SEM ( n = 3). Asterisk indicates a significant difference from all other groups ( p ≤ 0.05; ANOVA with Tukey’s post hoc test)

Article Snippet: The membrane was blocked with PBS containing 5% nonfat dry milk and 0.1% Tween-20, hybridized with primary antibody (anti-LC3, (Sigma-Aldrich, Poole, Dorset); anti-ERK, anti-JNK, anti-p38, anti-β-actin (Cell Signaling Technology, Beverly, MA, USA), anti-EIF-2α, anti-BiP/GRP78 (BioSource International, Rockville, MD); anti-IRE1, anti-ATF6 (Abcam, Cambridge, UK)) followed by incubation with secondary antibody (Amersham Biosciences, Bucks, UK).

Techniques: Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Adiponectin confers protection from acute colitis and restricts a B cell immune response

doi: 10.1074/jbc.M115.712646

Figure Lengend Snippet: APN-KO colitic mice exhibit increased stress signaling, an altered APN receptor profile, and APN co-localizes with AdipoR1. A, Western blots of proliferative and cellular stress markers in APN-KO DSS colons versus APN-KO controls. Densitometry analysis of: B, p-p38 MAPK demonstrates an increase in APN-KO DSS mice compared with APN-KO controls (p < 0.01); C, p-ERK1/2 showing an increase in APN-KO DSS mice versus APN-KO controls (p < 0.05); D, PI3K showing a reduction in APN-KO DSS colonic protein versus APN-KO controls (p < 0.01); E, p-Akt showing a reduction in APN-KO DSS-treated mice compared with APN-KO controls (p < 0.001); F, AdipoR1 showing an increase in APN-KO DSS versus APN-KO control (p < 0.001); and G, AdipoR2 showing a reduction in APN-KO DSS compared with APN-KO controls (p < 0.05). H, immunofluorescence ×60 image of the colon showing APN (green), AdipoR1 (red), and DAPI (blue). Scale bar, 25 μm. White arrows indicate co-localization of APN and AdipoR1. *, p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were then blocked in TBST (0.1% Tween 20) with 5% skim milk for 1 h, and subsequently incubated in primary antibody against: adiponectin (Affinity Bioreagent; catalog number PA1–84881), Caspase-3 (Santa Cruz; catalog number sc-7148), AdipoR1 (Santa Cruz; catalog number sc-46748); and from Cell Signaling: Akt (catalog number 9272), p-Akt (catalog number 9271), ERK1/2 (catalog number 9102), p-ERK1/2 (catalog number 9101), PI3k (catalog number 4249), p-STAT3 (catalog number 9131), p38 MAPK (catalog number 9212), p-p38 (catalog number 9212) and p53 (catalog number 9282), and β-actin (Sigma; catalog number A2228) in 4% skim milk solution overnight at 4 °C.

Techniques: Western Blot, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Adiponectin confers protection from acute colitis and restricts a B cell immune response

doi: 10.1074/jbc.M115.712646

Figure Lengend Snippet: APN is important in mediating epithelial homeostasis in an in vitro model of DSS colitis. A, Western blots showing cellular stress markers following DSS treatment, and the restoration of balance in HCT116 colonic epithelial cells when APN is present with DSS. Densitometry analysis showing: B, AdipoR1, with an increase in DSS-treated cells compared with APN treatment (p < 0.05), and DSS + APN treatment (p < 0.01); C, p53, with an increase in DSS treatment compared with controls (p < 0.05); D, Bcl-2, with a significant reduction in DSS-treated cells compared with controls (p < 0.001), APN (p < 0.001), and DSS + APN-treated cells (p < 0.01); E, p-Akt, with a reduction in DSS treated cells compared with controls (p < 0.001) and APN- treated cells (p < 0.05); F, p-ERK1/2, with a significant increase in DSS treatment compared with controls (p < 0.01); and G, p-p38 MAPK, with an increase in DSS treatment compared with controls (p < 0.01). H, immunofluorescent images showing DAPI (blue) and caspases-3 and -7 (green) in HCT116 cells, where APN reduces apoptosis in the presence of DSS. Analysis of caspase-3 and -7 staining showing an increase in DSS treatment versus controls, and APN-treated cells (p < 0.01 for both groups), and a significant decrease in DSS + APN-treated cells, compared with DSS alone (p < 0.01). I, immunofluorescent images showing DAPI (blue) and Ki67 (green) in HCT 116 cells, where APN promotes proliferation in the presence of DSS. Analysis of Ki67 staining showing a reduction after DSS treatment versus controls and APN-treated cells (p < 0.01 for both groups), and a significant restoration in DSS + APN-treated cells, compared with DSS alone (p < 0.05). Scale bar, 25 μm. J, BrdU incorporation assays confirmed reduced proliferation after DSS treatment versus controls and APN-treated cells (p < 0.001 and 0.01, respectively), and that the addition of APN to DSS-treated cells rescued proliferation, compared with DSS alone (p < 0.05). *, p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were then blocked in TBST (0.1% Tween 20) with 5% skim milk for 1 h, and subsequently incubated in primary antibody against: adiponectin (Affinity Bioreagent; catalog number PA1–84881), Caspase-3 (Santa Cruz; catalog number sc-7148), AdipoR1 (Santa Cruz; catalog number sc-46748); and from Cell Signaling: Akt (catalog number 9272), p-Akt (catalog number 9271), ERK1/2 (catalog number 9102), p-ERK1/2 (catalog number 9101), PI3k (catalog number 4249), p-STAT3 (catalog number 9131), p38 MAPK (catalog number 9212), p-p38 (catalog number 9212) and p53 (catalog number 9282), and β-actin (Sigma; catalog number A2228) in 4% skim milk solution overnight at 4 °C.

Techniques: In Vitro, Western Blot, Staining, BrdU Incorporation Assay

Journal: The Journal of Biological Chemistry

Article Title: Adiponectin confers protection from acute colitis and restricts a B cell immune response

doi: 10.1074/jbc.M115.712646

Figure Lengend Snippet: APN mediates its homeostatic/restorative effects in vitro via interactions with its receptor, AdipoR1. A, Western blot confirming AdipoR1 knockdown via siRNA in HCT116 colonic epithelial cells. B, densitometry analysis of AdipoR1 following knockdown, showing a significant reduction in the siRNA-treated cells compared with scrambled conditions and controls (p < 0.05 for both groups). C, qRT-PCR analysis confirming knockdown of the AdipoR1 gene in the knockdown groups versus scrambled (p < 0.001). D, Western blots showing the lack of restorative effects of APN following AdipoR1 knockdown in HCT116 cells. Densitometry analysis following siRNA-mediated AdipoR1 knockdown in HCT116 colonic epithelial cells, showing p53 (E), with significant increases in DSS treatment compared with control and APN-treated cells (p < 0.05 for both), as well as in DSS + APN treatment compared with controls (p < 0.01) and APN-treated cells (p < 0.05); F, Bcl-2, with a significant reduction in DSS-treated cells compared with control and APN groups (p < 0.01 for both), as well as a reduction in DSS + APN-treated cells compared with controls and APN-treated cells (p < 0.01 and 0.05, respectively); G, p-Akt, with a significant reduction in DSS-treated cells compared with controls (p < 0.05) and APN-treated cells (p < 0.01), as well as DSS + APN-treated cells compared with controls and APN treatment alone (p < 0.05 for both groups); H, p-ERK1/2 with significant increases in DSS-treated cells compared with controls and APN-treated cells (p < 0.01 and 0.05, respectively), as well as DSS + APN-treated cells compared with controls and APN-treated groups (p < 0.01 for both comparisons); and I, p-p38 MAPK, with a significant increase in DSS-treated cells compared with control and APN treatment (p < 0.001 for both groups), as well as in DSS + APN treatment compared with controls and APN treatment alone (p < 0.001 for both groups). J, immunofluoresence images of DAPI (blue) and Ki67 (green) in HCT116 cells with AdipoR1 ablation, showing a reduction in proliferation with DSS treatment, and a lack of restoration with DSS + APN treatment. Analysis of immunofluorescence showing a reduction of Ki67 with DSS treatment compared with controls and APN treatment alone (p < 0.001 for both groups), as well as with DSS + APN conditions compared with controls and APN treatment alone (p < 0.001 for both groups). K, BrdU analyses confirmed reduced proliferation with DSS and DSS + APN treatment after AdipoR1 siRNA treatment. Proliferation with DSS treatment was reduced compared with controls and APN treatment alone (p < 0.001 for both groups), as well as with DSS + APN compared with controls and APN treatment alone (p < 0.001 and 0.05, respectively). L, immunofluorescence of DAPI (blue) and caspase-3 and -7 (green) illustrating an increase in apoptosis following DSS treatment in HCT 116 cells with AdipoR1 ablation, and the absence of a reduction in apoptosis following APN treatment in the presence of DSS. Analysis of immunofluorescence showing an increase in caspases-3 and -7 in DSS treatment compared with controls and APN-treated cells (p < 0.01 for both groups), as well as in DSS + APN groups compared with controls and APN-treated cells (p < 0.05 for both groups). Scale bar, 25 μm. *, p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were then blocked in TBST (0.1% Tween 20) with 5% skim milk for 1 h, and subsequently incubated in primary antibody against: adiponectin (Affinity Bioreagent; catalog number PA1–84881), Caspase-3 (Santa Cruz; catalog number sc-7148), AdipoR1 (Santa Cruz; catalog number sc-46748); and from Cell Signaling: Akt (catalog number 9272), p-Akt (catalog number 9271), ERK1/2 (catalog number 9102), p-ERK1/2 (catalog number 9101), PI3k (catalog number 4249), p-STAT3 (catalog number 9131), p38 MAPK (catalog number 9212), p-p38 (catalog number 9212) and p53 (catalog number 9282), and β-actin (Sigma; catalog number A2228) in 4% skim milk solution overnight at 4 °C.

Techniques: In Vitro, Western Blot, Quantitative RT-PCR, Immunofluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully immunocompetent mouse model

doi: 10.1186/s13046-018-0907-z

Figure Lengend Snippet: JPH203 decreases mTORC1 activity, but not ERK or AKT Western blot analyses of mTORC1, ERK and AKT activity in thyroid cancer cell lines after JPH203 treatment. P-p70 S6 kinase, p-ERK and p-AKT levels were assayed in ( a ) PTC cell lines and ( b ) ATC cell lines and ( c ) LNCaP as negative control. Cells were plated in triplicates using normal growth medium, after overnight incubation cells were washed and 0.125X EAA was added. Cells were treated with 10 μM JPH203 or DMSO as control and lysed after 24 h

Article Snippet: Primary antibody used (purchased from Cell signaling unless specified): P-p70S6K (cat. no. 2708), p70S6K (cat. no. 2708) P-S6 S240/244 (cat. no. 5364), P-S6 S235/236 (cat. no. 4858), S6 (cat. no. 2217), P-ERK (cat. no. 9107), Tot-ERK (cat. no. 4370), P-AKT S473 (cat. no. 4060), pan-AKT (cat. no. 2920), β-actin (Sigma-Aldrich, cat. no. A5316) and LAT1 (TransGenic Inc., cat. no. KE026).

Techniques: Activity Assay, Western Blot, Negative Control, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Muscle Proteomic Profile before and after Enzyme Replacement Therapy in Late-Onset Pompe Disease

doi: 10.3390/ijms22062850

Figure Lengend Snippet: Lysosomal autophagy and apoptosis. Representative immunoblot images (cropped images) and histograms (mean ± SD) showing protein abundance of: ( A ) microtubule associated protein 1 light chain 3 beta (LC3B/MAP1LC3B), ubiquitin-binding protein p62/sequestosome 1 (p62/SQSTM1), sintaxin 17 (STX17), synaptosomal-associated protein 29 kDa (SNAP29), endobrevin (VAMP8), Golgi reassembly-stacking protein 2 (GORASP2), lysosome-associated membrane protein 2 (LAMP2), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) in CTR, pre- and post-ERT LOPD; and ( B ) protein kinase AMP-activated catalytic subunit alpha 1 (AMPK/PRKKA1), p38 mitogen-activated protein kinases 11 (p38β/MAPK11), and fork head box O3 (FoxO3) in CTR, LOPD PRE and LOPD POST. The data were normalized against the total amount of loaded proteins stained with Sypro Ruby. Statistical analysis was performed by ANOVA and Tukey’s test (* p < 0.05). Blot images have been cropped for a better visualization, full-length images are available in . O.D. = optical density.

Article Snippet: The membrane was blocked with 5% bovine serum albumin for 1 h at room temperature and incubated, overnight at 4 °C, with rabbit, mouse, or goat polyclonal primary antibodies (Cell Signaling Technology, Danvers, MA, USA, except when differently stated) as follows: anti-microtubule associated protein 1 light chain 3 beta (LC3B/MAP1LC3B, 2775), 1:1000; anti-ubiquitin-binding protein p62/sequestosome 1 (p62/SQSTM1, P0067; Sigma–Aldrich, St. Louis, MO, USA), 1:1000; anti-sintaxin 17 (STX17, HPA001204; Sigma–Aldrich, St. Louis, MO, USA), 1:1000; anti-synaptosomal-associated protein 29 kDa (SNAP29, sc-135564; Santa Cruz Biotechnology, Dallas, TX, USA), 1:500; anti-endobrevin (VAMP8, sc-166820; Santa Cruz Biotechnology, Dallas, TX, USA), 1:500; anti-golgi reassembly-stacking protein 2 (GORASP2, HPA035275; Sigma–Aldrich, St. Louis, MO, USA), 1:1000; anti-lysosome-associated membrane protein 2 (LAMP2, 49067), 1:1000; anti-BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3, B7931; Sigma–Aldrich, St. Louis, MO, USA), 1:1000; anti-protein kinase AMP-activated catalytic subunit alpha 1 (AMPK/PRKAA1, 2532), 1:1000; anti-phosphoAMPK (2531), 1:1000; anti-p38 mitogen-activated protein kinases 11 (p38β/MAPK11, 8690), 1:1000; anti-phosphop38β (9215), 1:1000; anti-forkhead box O3 (FoxO3, 2497), 1:1000; anti-phosphoFoxO3 (9464), 1:1000; anti-multi transcriptional factor C/EBP-homologous protein (CHOP/DDIT3, 2895), 1:1000; anti-GLUL (6640; Santa Cruz Biotechnology, Dallas, TX, USA), 1:500.

Techniques: Western Blot, Binding Assay, Staining