Journal: Journal of Clinical Investigation

Article Title: Collagen VII plays a dual role in wound healing

doi: 10.1172/jci68127

Figure Lengend Snippet: Figure 5 Mechanisms of delayed wound closure in Col7a1-hypomorphic keratinocytes. (A) In in vitro assays, wound closure by keratinocytes was assessed at 8, 20, and 24 hours. At 20 and 24 hours, Col7a1-hypomorphic keratinocytes closed wounds at a reduced rate. n = 6; values rep- resent mean ± SEM. *P < 0.05. (B–D) Western blotting of wild-type and Col7a1-hypomorphic keratinocyte protein lysates revealed increased expression of integrin β4 subunit (B) and increased phosphorylation of Ser473 AKT (C) and JNK2 (D) in cultured Col7a1-hypomorphic keratino- cytes. Densitometric quantification of keratinocyte isolations is also shown. n ≥ 3; values represent mean ± SD. *P < 0.05; **P < 0.01. (E) Western blots on cell lysates from tamoxifen-induced Col7a1 knockout mouse keratinocytes. Keratinocytes from the same isolation were divided into 2 pools: one was treated with 4-OH tamoxifen to induce Col7a1 knockout, the other received only DMSO and served as control. The changes in Col7a1-hypomorphic keratinocytes were replicated in keratinocytes after forced COL7A1 loss. Loss of COL7A1 did not affect expression of lam- inin-332 (LM-β3/γ2; representing the laminin β3 and γ2 chains of laminin-332), but increased expression of integrin β4 and phosphorylation of Ser473 AKT and JNK2. Total AKT and GAPDH were used to ensure equal loading. (F) Altered Laminin-332 organization in Col7a1-hypomorphic keratinocytes. Primary keratinocytes of the indicated genotypes and treatments were cultured for 24 hours in the presence of ascorbate and stained for COL7A1 (red) and laminin-332 (green). Original magnification, ×400. Tam, tamoxifen.

Article Snippet: The following antibodies were used: mouse antiGAPDH (Millipore), rabbit anti–COL7A1 (Millipore), mouse anti–β-actin (Sigma-Aldrich), rabbit anti-tubulin (Abcam), rat anti–integrin α6 (GoH3) (Progen), rat anti–integrin β1 (BD Biosciences), rat anti–integrin β4 (BD Biosciences), rabbit anti–integrin β4 (Santa Cruz Biotechnology), mouse anti–active JNK1 and JNK2, rabbit anti-JNK (Abcam), rabbit anti–phospho–Tyr705 STAT3 (Cell Signaling Technology), mouse anti-AKT (Santa Cruz Biotechnology), rabbit anti–phospho–Ser473 AKT (Cell Signaling Technology), rat anti–Ki-67 (Dako), Cy3-conjugated mouse anti–α-SMA (Sigma-Aldrich), rat anti-CD11b (BD Biosciences), rabbit anti-mouse collagen I (Acris), rabbit anti–laminin-332 (gift from K. Yancey, University of Texas Southwestern, Dallas, Texas, USA), rabbit anti–pan-laminin (Progen), and rat anti–laminin α5 (gift from L. Sorokin, Muenster University, Muenster, Germany).

Techniques: In Vitro, Western Blot, Expressing, Phospho-proteomics, Cell Culture, Knock-Out, Isolation, Control, Staining

Journal: Oncology reports

Article Title: Inhibition of basal JNK activity by small interfering RNAs enhances cisplatin sensitivity and decreases DNA repair in T98G glioblastoma cells.

doi: 10.3892/or.2014.3570

Figure Lengend Snippet: Figure 1. Expression of JNK1 and JNK2 protein levels in T98G glioblastoma cell line in response to several stimuli. T98G cells were treated with FBS 0.5%, FBS 10%, TPA 30 nM, and TNF-α 20 ng/ml. Untreated T98G cells were used as control. β-actin was used as a loading control. One of three similar experiments is shown. Lane 1, untreated; lane 2, FBS 0.5%; lane 3, FBS 10%; lane 4, TPA (30 nM); lane 5, TNF-α (20 ng/ml).

Article Snippet: The cells were incubated with 60 nmol/l siRNAs (Santa Cruz) targeting JNK1 (sc-29380), JNK2 (sc-39101) or control ‘scrambled’ siRNA (sc-37007) and 6 μl siRNA transfection reagent, according to the manufacturer's recommendation (Santa Cruz) in 1 ml serum-free medium (MEM) at 37 ̊C for 6 h. After transfection, cells were cultured in complete medium for 42 h or until being used.

Techniques: Expressing, Control

Journal: Oncology reports

Article Title: Inhibition of basal JNK activity by small interfering RNAs enhances cisplatin sensitivity and decreases DNA repair in T98G glioblastoma cells.

doi: 10.3892/or.2014.3570

Figure Lengend Snippet: Figure 2. Inhibition of JNK1 and JNK2 protein synthesis by small interfering RNAs and sensitization of T98G cells to cisplatin. T98G glioblastoma cells were transfected with small interfering RNAs (siRNAs) against JNK1 and JNK2 genes, or with scrambled siRNA, in presence or absence of cisplatin (100 µM). Western analysis of T98G cells at 24 h after transfection with siRNAs against JNK1 and JNK2 alone or in combination with cisplatin (cul- tured for an additional 2 h), as shown in Materials and methods. One of three similar experiments is shown.

Article Snippet: The cells were incubated with 60 nmol/l siRNAs (Santa Cruz) targeting JNK1 (sc-29380), JNK2 (sc-39101) or control ‘scrambled’ siRNA (sc-37007) and 6 μl siRNA transfection reagent, according to the manufacturer's recommendation (Santa Cruz) in 1 ml serum-free medium (MEM) at 37 ̊C for 6 h. After transfection, cells were cultured in complete medium for 42 h or until being used.

Techniques: Inhibition, Transfection, Western Blot

Journal: Oncology reports

Article Title: Inhibition of basal JNK activity by small interfering RNAs enhances cisplatin sensitivity and decreases DNA repair in T98G glioblastoma cells.

doi: 10.3892/or.2014.3570

Figure Lengend Snippet: Figure 3. Soft agar assays of cells treated with siRNAs against JNK1 and JNK2, in the presence or absence of cisplatin (100 µM), compared with glioma parental cell lines (untreated cells) and those transfected with the scrambled siRNA. T98G cells treated with cisplatin and/or transfected with siRNAs against JNK1 or JNK2 displayed significantly fewer colonies compared with the parental and scrambled siRNA-transfected cells. A further reduction in colony formation was observed when T98G cells were treated with a combi- nation of cisplatin and JNK1-siRNA or JNK2-siRNA. Control and scrambled siRNA caused no inhibition of colony-forming ability of these cells on soft agar. Results are representative of three independent experiments.

Article Snippet: The cells were incubated with 60 nmol/l siRNAs (Santa Cruz) targeting JNK1 (sc-29380), JNK2 (sc-39101) or control ‘scrambled’ siRNA (sc-37007) and 6 μl siRNA transfection reagent, according to the manufacturer's recommendation (Santa Cruz) in 1 ml serum-free medium (MEM) at 37 ̊C for 6 h. After transfection, cells were cultured in complete medium for 42 h or until being used.

Techniques: Transfection, Control, Inhibition

Journal: Oncology reports

Article Title: Inhibition of basal JNK activity by small interfering RNAs enhances cisplatin sensitivity and decreases DNA repair in T98G glioblastoma cells.

doi: 10.3892/or.2014.3570

Figure Lengend Snippet: Figure 4. Inhibition of JNK1 and JNK2 by specific siRNAs inhibits cell survival and sensitizes T98G cells to cisplatin. T98G glioblastoma cells were transfected with scrambled siRNA and with siRNAs against JNK1 or JNK-2. Twenty four hours after transfection, cells were treated with cisplatin (100 µM) for an additional 2 h. (A) Whole cell proteins were extracted and IAP enzymatic activities in the T98G cells were analyzed. (B) After transfec- tion with JNK1- and JNK2-siRNAs or scrambled siRNA, T98G cells were treated with cisplatin (100 µM) for an additional 2 h. Apoptosis was analyzed by determination of DNA fragmentation by an ELISA method. Results are representative of three independent experiments.

Article Snippet: The cells were incubated with 60 nmol/l siRNAs (Santa Cruz) targeting JNK1 (sc-29380), JNK2 (sc-39101) or control ‘scrambled’ siRNA (sc-37007) and 6 μl siRNA transfection reagent, according to the manufacturer's recommendation (Santa Cruz) in 1 ml serum-free medium (MEM) at 37 ̊C for 6 h. After transfection, cells were cultured in complete medium for 42 h or until being used.

Techniques: Inhibition, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Oncology reports

Article Title: Inhibition of basal JNK activity by small interfering RNAs enhances cisplatin sensitivity and decreases DNA repair in T98G glioblastoma cells.

doi: 10.3892/or.2014.3570

Figure Lengend Snippet: Figure 5. RT-PCR analysis of (A) bcl-2 and (B) bax mRNA levels in T98G glioblastoma cells in response to treatments with scrambled siRNA, cisplatin, JNK1-siRNA and JNK2-siRNA and the combination treatment of cisplatin and JNK1-siRNA or JNK2-siRNA. Levels are represented as the fold change over the untreated controls. Results are representative of three independent experiments.

Article Snippet: The cells were incubated with 60 nmol/l siRNAs (Santa Cruz) targeting JNK1 (sc-29380), JNK2 (sc-39101) or control ‘scrambled’ siRNA (sc-37007) and 6 μl siRNA transfection reagent, according to the manufacturer's recommendation (Santa Cruz) in 1 ml serum-free medium (MEM) at 37 ̊C for 6 h. After transfection, cells were cultured in complete medium for 42 h or until being used.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a Quantitative analysis of relative mRNA levels of Jnk s in intact retinae or retinae 1 day (1 d), 1 week (1 w) or 2 weeks (2 w) after ONA ( n = 3 for each time point). b Representative western blots from at least three biological repeats showing JNK expression after ONA. Intact retina or retinae 1 d, 1 w, 2 w after ONA were harvested, lysed and immunoblotted using antibodies against JNK1, JNK2, JNK3, pJNK or GAPDH. The location of molecular weight markers is shown in kDa on the right. c Representative fluorescence microscope images show immunostaining of mouse retina flat-mounts from wild-type or JNK1, JNK2, or JNK3-null mice ( n = 6 for each genotype) 14 days after ONA using an anti-Tuj1 antibody. d The bar chart shows quantitative analysis of Tuj1 + RGC numbers 14 days after ONA. Scale bar, 50 μm. One-way ANOVA followed by post-hoc Bonferroni test: ** p < 0.01. Error bars represent SEM

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Western Blot, Expressing, Molecular Weight, Fluorescence, Microscopy, Immunostaining