Journal: Scientific Reports

Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

doi: 10.1038/srep11009

Figure Lengend Snippet: ( a ) Cells transfected with Flag-JWA (4 μg) or without transfection were incubated with EGCG (20–40 μM) for 24 h. JWA protein expression was assessed by Western blot analysis. β-actin expression served as a loading control. ( b ) NCI-H460 cells were treated with EGCG (20–40 μM) for 24 h and total cellular RNA was extracted. mRNA level of JWA was detected by real-time PCR. GAPDH was used as an internal control. ( c ) Western blot analysis of the protein level of JWA in EGCG-treated A549 cells for 24 h. β-actin expression served as a loading control. ( d ) After A549 cells were incubated with EGCG (20–40 μM) for 24 h, total RNAs were prepared and real-time PCR was applied to measure the JWA mRNA level. GAPDH was used as an internal control. ( e ) NCI-H460 cells were transfected with or without Flag-topoisomerase IIα plasmid (4 μg) and then treated with EGCG (20–40 μM) for 24 h. Protein from cell was subjected to western blot analysis. β-actin expression was served as a loading control. ( f ) Total RNAs from NCI-H460 cells incubated with EGCG (20–40 μM) for 24 h were extracted and subjected to real-time PCR using primer for topoisomerase IIα. GAPDH was used as an internal control. ( g ) A549 cells were treated with EGCG (20–40 μM) for 24 h and then protein from cell lysate was subjected to western blot analysis. β-actin expression was served as a loading control. ( h ) A549 cells were incubated in the absence or presence of EGCG (20–40 μM) for 24 h. Then cells were lysed for the detection expression of topoisomerase IIα mRNA by real-time PCR. GAPDH was used as an internal control. The A549 xenograft nude mice model was established and treated with EGCG or normal saline. ( i ) Tumor size was checked twice per week. ( j ) Protein obtained from tumor tissues was subjected to western blot. β-tubulin expression was served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.

Article Snippet: Cells (2 × 10 5 ) were transfected with predesigned human topoisomerase IIα siRNA or siRNA control (100 pM) (RiboBio, Guangzhou, China) in 35-mm culture plates using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif, USA).

Techniques: Transfection, Incubation, Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, Plasmid Preparation, Saline

Journal: Scientific Reports

Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

doi: 10.1038/srep11009

Figure Lengend Snippet: ( a ) and ( b ) Expression of topoisomerase IIα was elevated and that of topoisomerase IIα was reduced in lung cancer tissues compared with those in matched normal tissues. β-Tubulin expression was served as a loading control. JWA and topoisomerase IIα expressions in cancer tissues (T) and paired non-cancerous normal tissues (N) of lung cancer patients were analyzed by Western blotting. The level of each protein was normalized against β-tubulin. NCI-H460 cells were transiently transfected with Flag-JWA or Flag- topoisomerase IIα plasmid (1–4 μg). Whole-cell extracts were prepared 24 h after transfection and the expression of target proteins and mRNAs was examined. As analyzed by western blot assay, topoisomerase IIα levels were decreased after transfection with JWA plasmid ( c ) Also, overexpression of topoisomerase IIα does dependently inhibited JWA protein expression ( d ) β-actin was used for the protein loading control. Total RNAs were isolated from NCI-H460 cells transfected with Flag-JWA or Flag- topoisomerase IIα plasmid (4 μg) and subjected to real-time PCR. JWA transfection suppressed topoisomerase IIα mRNA expression ( e ) The level of JWA mRNA was down-regulated in topoisomerase IIα overexpressed NCI-H460 cells ( f ) GAPDH was used as an internal control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.

Article Snippet: Cells (2 × 10 5 ) were transfected with predesigned human topoisomerase IIα siRNA or siRNA control (100 pM) (RiboBio, Guangzhou, China) in 35-mm culture plates using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif, USA).

Techniques: Expressing, Control, Western Blot, Transfection, Plasmid Preparation, Over Expression, Isolation, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

doi: 10.1038/srep11009

Figure Lengend Snippet: ( a ) and ( b ) NCI-H460 cells were transiently transfected with Flag-JWA plasmid (2.5 μg) for 24 h, incubated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. Western blotting was carried out to confirm the level of topoisomerase IIα. ( c ) and (d) NCI-H460 cells were transiently transfected with Flag- topoisomerase IIα plasmid (2.5 μg) for 24 h and co-treated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. JWA protein was detected by anti-JWA antibody. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.

Article Snippet: Cells (2 × 10 5 ) were transfected with predesigned human topoisomerase IIα siRNA or siRNA control (100 pM) (RiboBio, Guangzhou, China) in 35-mm culture plates using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif, USA).

Techniques: Transfection, Plasmid Preparation, Incubation, Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

doi: 10.1038/srep11009

Figure Lengend Snippet: ( a ) Schematic showed the domains of full length and indicated fragments JWA protein. “Dark grey” represented the TMD of JWA. Amino acid sequences ranged from 1 to 60 for JWA-1, 1 to 90 for JWA-2, 1 to 140 for JWA-3 and 141–188 for JWA-4. ( b ) NCI-H460 cells were transiently transfected with full-length Flag-JWA plasmid and four different fragments of JWA plasmid respectively. Whole-cell extracts were prepared 24 h after transfection, and topoisomerase IIα expression was measured by western blotting. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.

Article Snippet: Cells (2 × 10 5 ) were transfected with predesigned human topoisomerase IIα siRNA or siRNA control (100 pM) (RiboBio, Guangzhou, China) in 35-mm culture plates using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot

Journal: Scientific Reports

Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

doi: 10.1038/srep11009

Figure Lengend Snippet: NCI-H460 cells were plated on 60 mm plates in DMEM (10% fetal bovine serum), which was changed to starving DMEM (without serum) for 24 h or 30 h. ( a ) Flow cytometric analysed the change of cell cycle. And the protein levels of JWA and topoisomerase IIα were examined by Western blotting. ( c ) and ( d ) Then NCI-H460 cells were treated with 200 nM nocodazole, respectively for 12 h, 18 h and 24 h. ( b ) Flow cytometric analysed the cell cycle. ( e ) and ( f )The expression of JWA and topoisomerase IIα were examined by Western blotting. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01 and ***p < 0.001.

Article Snippet: Cells (2 × 10 5 ) were transfected with predesigned human topoisomerase IIα siRNA or siRNA control (100 pM) (RiboBio, Guangzhou, China) in 35-mm culture plates using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif, USA).

Techniques: Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

doi: 10.1038/srep11009

Figure Lengend Snippet: ( a ) NCI-H460 cells were transfected with siRNA-topoisomerase IIα (100 pmol), shRNA-JWA, JWA and topoisomerase IIα plasmids (4 μg) as well as the control vector. Migration ability of the cells at various time points after transfection (24 h, 48 h) was assessed by scratch migration assay. ( b ) The JWA or topoisomerase IIα plasmid (4 μg) was transiently transfected into NCI-H460 cells. 24 hours later, target proteins in cell lysates were detected by immunoblotting using antibodies against MMP-2/9, N- cadherin, ZEB1, slug, snail and E-cadherin. β-actin expression served as a loading control. ( c ) and ( d ) NCI-H460 cells were transfected with Flag-JWA , Flag- topoisomerase IIα and Flag-vector plasmids, (4 μg) in the presence or absence of EGCG (40 μM) for 24 h. The cells were harvested and lysed for the detection expression of JWA or topoisomerase by Western blot analysis. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.

Article Snippet: Cells (2 × 10 5 ) were transfected with predesigned human topoisomerase IIα siRNA or siRNA control (100 pM) (RiboBio, Guangzhou, China) in 35-mm culture plates using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif, USA).

Techniques: Transfection, shRNA, Control, Plasmid Preparation, Migration, Western Blot, Expressing

Journal: Nature Communications

Article Title: Peroxiredoxin 6 mediates Gαi protein-coupled receptor inactivation by cJun kinase

doi: 10.1038/s41467-017-00791-2

Figure Lengend Snippet: PRDX6 is recruited to Gαi3 following norBNI treatment and regulates KOR/Gαi association. a Scatterplot of FLAG-Gαi3-interacting proteins, depicting the ratio of norBNI/vehicle in forward and reverse replicates. For experiment design, see Supplementary Fig. ; for individual proteins, see Supplementary Data . Proteins whose associations were changed by norBNI treatment in both replicates are indicated in red . b The increase in Gαi immunoprecipitated with myc after norBNI in mycKOR expressing cells was blocked by 10 µM MJ33 pretreatment (*, Student’s t -test, P < 0.05, n = 3–5). c NorBNI treatment (10 µM, 1 h) significantly increased phospho-JNK immunoreactivity in KORGFP expressing cells and was not blocked by 10 µM MJ33 pretreatment. (Significant effect of norBNI, ** P < 0.01, but not MJ33 or interaction; two-way ANOVA, n = 9). d – f MycKOR expressing cells were immunostained for KOR and PRDX6. Colocalization was quantified by the Pearson correlation coefficient between intensity of KOR and PRDX6 immunoreactivity across 7–10 cells for each replicate. Representative images are shown in Supplementary Fig. . Representative line plot profile analysis for a vehicle ( d ) and norBNI (10 µM, 1 h) ( e ) treated cell. Line plot is presented as the intensity normalized to the maximal intensity for that cell. f NorBNI treatment (significantly increased colocalization of KOR-IR and PRDX6-IR (*, paired t -test, P < 0.05, n = 8). No change after norBNI was observed when pretreated with 1 µM SP610025 (paired t -test, P = 0.6, n = 6). g NorBNI (10 µM, 1 h) significantly increased HA immunoreactivity in the membrane fraction of HEK293 cells stably expressing mycKOR and HA-PRDX6 (( P < 0.05) one sample t -test, n = 7). This increased was blocked by pretreatment with JNK-IN-8 (1 µM) (Student’s t -test, P < 0.05, n = 4). h Representative immunoblot for g . i HEK293 cells stably co-expressing mycKOR and HA-PRDX6 were treated with vehicle or norBNI (10 µM, 1 h) and lysed in PLA2 buffer. Whole cell, membrane, or cytosol fractions were collected and analyzed for PLA2 activity. NorBNI selectively increased PLA2 activity in the membrane fraction (& ( P < 0.05) one sample t -test, n = 8). This increase was blocked by pretreatment with JNK-IN-8 (1 µM) or MJ33 (10 µM) ( P < 0.05, one-way ANOVA; * P < 0.05 Holm–Sidak post hoc vs. vehicle + norBNI; n = 4-8). Error bars represent mean ± SEM

Article Snippet: MycKOR expressing HEK293 cells were transiently transfected with control siRNA (Fisher Scientific #4390843) or predesigned validated siRNA against PRDX6 from Fisher Scientific (combination of s18428 and s18429, Fisher Scientific #4427038 and #4390824).

Techniques: Immunoprecipitation, Expressing, Membrane, Stable Transfection, Western Blot, Activity Assay

Journal: Nature Communications

Article Title: Peroxiredoxin 6 mediates Gαi protein-coupled receptor inactivation by cJun kinase

doi: 10.1038/s41467-017-00791-2

Figure Lengend Snippet: NorBNI and morphine induce ROS production via JNK and PRDX6. MycKOR a , c or mycMOR b , d expressing HEK293 cells were pretreated with vehicle, AACOCF3 (10 µM), JNK-IN-8 (1 µM), SP610025 (1 µM), MJ33 (10 µM), or N-acetyl-cysteine (NAC, 10 µM) 30 min prior to being treated for 1 h with norBNI (10 µM), morphine (10 µM), fentanyl (10 µM) or naloxone (10 µM). a , b Cells were imaged for ROS using CellROX Green; scale bar represents 12.5 µm. c Quantification of ROS in mycKOR cells. NorBNI, but not naloxone, increased CellROX Green fluorescence; this was blocked by pretreatment with MJ33, JNK-IN-8, SP610025, or NAC, but not AACOCF3 (one-way ANOVA, P < 0.0001, n = 3-11; *, # P < 0.05, ** P < 0.01, Holm–Sidak post hoc analysis). d Quantification of ROS in mycMOR cells. Morphine, but not fentanyl or norBNI, increased CellROX Green fluorescence; this increase was blocked by SP610025 (one-way ANOVA, P < 0.001, n = 4–5; * P < 0.05, ** P < 0.01, Holm–Sidak post hoc analysis). e Schematic illustrating the experimental protocol used to generate the results shown in f , g , and in Supplementary Fig. . HEK293 cells stably co-expressing mycKOR and FLAG-GΑI3 were pretreated with vehicle, SP610025, or MJ33 and treated 5.5 h with norBNI prior to harvest. Cell membranes were extracted in the presence of N-ethylmaleimide (50 mM) to covalently bind unmodified cysteines, and immunoprecipitated with anti-myc to isolate mycKOR and mycKOR-associated FLAG-Gαi. Myc immunoprecipitates were treated with hydroxylamine to cleave palmitic acids and then incubated with BMCC-biotin to biotinylate previously palmitoylated cysteines. Palmitoylation was measured by probing with streptavidin and normalizing to anti-Gαi immunoreactivity. f NorBNI significantly reduced palmitoylation of Gαi coimmunoprecipitated with mycKOR, but not Gαi that was not KOR-associated ( & P < 0.05, one sample t -test with correction for multiple comparisons; n = 5–6); this change in palmitoylation was blocked by MJ33 pretreatment (* P < 0.05 Student’s t -test with Welch’s correction). g Representative immunoblots from f . Error bars represent mean ± SEM

Article Snippet: MycKOR expressing HEK293 cells were transiently transfected with control siRNA (Fisher Scientific #4390843) or predesigned validated siRNA against PRDX6 from Fisher Scientific (combination of s18428 and s18429, Fisher Scientific #4427038 and #4390824).

Techniques: Expressing, Fluorescence, Stable Transfection, Immunoprecipitation, Incubation, Western Blot

Journal: Nature Communications

Article Title: Peroxiredoxin 6 mediates Gαi protein-coupled receptor inactivation by cJun kinase

doi: 10.1038/s41467-017-00791-2

Figure Lengend Snippet: PRDX6 mediates the long-lasting effects of norBNI and acute morphine tolerance. a , b Mice were injected with saline, MJ33 (1.25 mg kg −1 ), AACOCF3 (10 mg kg −1 ), or NAC (100 mg kg −1 ) 1 h prior to norBNI (10 mg kg −1 ). Three days later, tail withdrawal latency from 52.5 ± 0.3 °C water bath was measured prior to and 30 min after U50,488 (10 mg kg −1 ). a The U50,488 stimulated increase in latency was blocked by norBNI following saline or AACOCF3 pretreatment, but not MJ33 (paired two-way ANOVA; significant effect of norBNI, U50,488, and interaction ( P < 0.01); * P < 0.01, ** P < 0.01, **** P < 0.0001, Holm–Sidak post hoc analysis of post vs. pre; # P < 0.05 and comparing post-U50,488 latency between groups # P < 0.05, ## P < 0.01, ### P < 0.001, n = 4–16). b NorBNI failed to block U50,488-stimulated analgesia when mice were pretreated with NAC (paired two-way ANOVA, significant effect of U50,488, norBNI, and interaction ( P < 0.05; n = 8); * P < 0.05, **** P < 0.0001 Holm–Sidak post hoc). c , d Mice were injected with saline, AACOCF3, or MJ33 2 h prior to the initial injection of morphine (10 mg kg −1 ) or fentanyl (0.3 mg kg −1 ). Tail withdrawal latency was measured prior to and at 30 min intervals following morphine or fentanyl. For area under the curve (AUC) values see Supplementary Fig. . c MJ33 pretreatment increased the analgesic response to the second morphine injection, compared to saline or AACOCF3 (paired two-way ANOVA; significant effect of time, subject matching, and interaction ( P < 0.05); *(vs. saline ( red ) or AACOCF3 ( black ), P < 0.05), # (vs. 30 min, P < 0.01, Holm–Sidak post hoc; n = 6–8). d MJ33 pretreatment had no effect on the analgesic response to a second fentanyl injection (paired two-way ANOVA on second injection; significant effect of time only ( P < 0.0001), n = 6–7; &, significant difference in AUC). e Mice were pretreated twice daily for 5 days with saline or MJ33 2 h prior to morphine (5 mg kg −1 ), and tail withdrawal latency was measured prior to or 30 min after morphine. Data are represented as increase in latency following morphine. MJ33 pretreatment increased the analgesic response to repeated morphine and delayed tolerance (paired two-way ANOVA, significant effect of MJ33, morphine treatment, and interaction ( P < 0.0001); vs. saline *( P < 0.05), ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs. first injection ### P < 0.001, #### P < 0.00001, Holm–Sidak post hoc; n = 11–14). Error bars represent mean ± SEM

Article Snippet: MycKOR expressing HEK293 cells were transiently transfected with control siRNA (Fisher Scientific #4390843) or predesigned validated siRNA against PRDX6 from Fisher Scientific (combination of s18428 and s18429, Fisher Scientific #4427038 and #4390824).

Techniques: Injection, Saline, Blocking Assay

Journal: Nature Communications

Article Title: Peroxiredoxin 6 mediates Gαi protein-coupled receptor inactivation by cJun kinase

doi: 10.1038/s41467-017-00791-2

Figure Lengend Snippet: D2 dopamine receptor tolerance is mediated by PRDX6-dependent ROS production. a HEK293 cells transiently expressing HA-D2DR(L) were pretreated with vehicle or MJ33 (10 µM) 30 min prior to being treated for 1 h with vehicle or quinpirole (100 nM). Cells were imaged for ROS using CellROX Green; scale bar represents 12.5 µm. b Quantification of ROS in a . Quinpirole increased CellROX Green fluorescence; this was blocked by pretreatment with MJ33 (two-way ANOVA, significant effect of quinpirole ( P < 0.01) and significant interaction ( P < 0.05), n = 4–5; * P < 0.05, ** P < 0.01, Holm–Sidak post hoc analysis of MJ33 vs. vehicle pretreatment). c , d Mice were injected with saline or MJ33 (1.25 mg kg −1 i.p.) 1 h prior to the initial injection of quinpirole (0.2 mg kg −1 i.p.) or saline. Two hr later, mice were injected with a quinpirole (0.2 mg kg −1 i.p.) or saline (baseline) placed in a novel cage, and locomotor activity was measured over 20 min. c Locomotion was measured as distance traveled (cm), in 5 min bins. Pretreatment significantly affected locomotion. (two-way ANOVA, significant effect of treatment ( P < 0.001), time ( P < 0.001), and significant interaction ( P < 0.01), n = 7–9). d Total locomotion over 20 min following quinpirole administration was quantified as a percent locomotion observed in saline treated mice. Quinpirole significantly reduced locomotor activity in all pretreatment groups, but mice pretreated with quinpirole moved significantly more than mice pretreated with saline or MJ33 + quinpirole (one-way ANOVA, P < 0.01; * P < 0.05, ** P < 0.01, *** P < 0.01, Holm–Sidak post hoc analysis). Error bars represent mean ± SEM

Article Snippet: MycKOR expressing HEK293 cells were transiently transfected with control siRNA (Fisher Scientific #4390843) or predesigned validated siRNA against PRDX6 from Fisher Scientific (combination of s18428 and s18429, Fisher Scientific #4427038 and #4390824).

Techniques: Expressing, Fluorescence, Injection, Saline, Activity Assay

Journal: Nature Communications

Article Title: Peroxiredoxin 6 mediates Gαi protein-coupled receptor inactivation by cJun kinase

doi: 10.1038/s41467-017-00791-2

Figure Lengend Snippet: PRDX6 mediates GPCR cross-tolerance. a MycKOR expressing or wild-type HEK293 cells transiently transfected with HA-D2DR(L) were pretreated with vehicle or MJ33 (10 µM) 30 min prior to being treated for 3-5 h with vehicle or norBNI (10 µM). Cells were then treated with 5 min with vehicle or quinpirole (100 nM) and cell lysates analyzed for phospho-ERK1/2 immunoreactivity. NorBNI reduced quinpirole-stimulated ERK1/2 phosphorylation; the norBNI inhibition of quinpirole response was blocked by MJ33 pretreatment and not observed in cells only expressing HA-D2DR(L). (Two-way ANOVA; significant interaction ( P < 0.05), significant effect of norBNI ( P < 0.01), and significant effect of KOR or MJ33 ( P < 0.05), n = 5–8; ** P < 0.01 Holm–Sidak post hoc analysis of norBNI vs. vehicle pretreatment; ## P < 0.01) Holm–Sidak post hoc analysis compared to KOR + D2DR without MJ33 pretreatment.). b Representative immunoblots for a . c EC50 values were calculated from concentration-response curves for quinpirole inhibition of dopamine release 5–7 h after vehicle, norBNI (10 mg kg −1 , i.p.), or MJ33 (1.25 mg kg −1 i.p.) prior to norBNI. NorBNI treatment resulted in a significant increase in quinpirole EC 50 , which was reduced by MJ33 pretreatment (one-way ANOVA, P < 0.0001; * P < 0.05, ** P < 0.01, **** P < 0.0001, Holm–Sidak post hoc). Error bars represent mean ± SEM

Article Snippet: MycKOR expressing HEK293 cells were transiently transfected with control siRNA (Fisher Scientific #4390843) or predesigned validated siRNA against PRDX6 from Fisher Scientific (combination of s18428 and s18429, Fisher Scientific #4427038 and #4390824).

Techniques: Expressing, Transfection, Phospho-proteomics, Inhibition, Western Blot, Concentration Assay

Journal: Nature Communications

Article Title: Peroxiredoxin 6 mediates Gαi protein-coupled receptor inactivation by cJun kinase

doi: 10.1038/s41467-017-00791-2

Figure Lengend Snippet: Model of GPCR inactivation by PRDX6-mediated oxidation and depalmitoylation. a Proposed model of JNK-mediated Gαi regulation by norBNI- and morphine-like drugs. JNK promotes membrane localization of PRDX6 and PLA2 activity, thereby locally activating NADPH oxidase (NOX) and stimulating the generation of ROS. The Gαi-PRDX6 interaction may help promote PRDX6 translocation and/or locally target PLA2 activity. Gαi oxidation disrupts Gαi palmitoylation, jamming the G-protein receptor complex in an inactive conformation. b Oxidation of cys-3 of Gαi blocks repalmitoylation of the residue. c Proposed model of GPCR cross-regulation by this mechanism. ROS generated as result JNK/PRDX6 activation by GPCR (i.e., KOR) diffuse locally, oxidizing nearby GPCR-Gαi complexes, function as paracrine or intracrine signals to silence inhibitory GPCRs

Article Snippet: MycKOR expressing HEK293 cells were transiently transfected with control siRNA (Fisher Scientific #4390843) or predesigned validated siRNA against PRDX6 from Fisher Scientific (combination of s18428 and s18429, Fisher Scientific #4427038 and #4390824).

Techniques: Membrane, Activity Assay, Translocation Assay, Residue, Generated, Activation Assay

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Interference efficiency of mGluR4 siRNA in bone marrow-derived dendritic cells (BMDC). Three mGluR4-targeted siRNAs were predesigned. Mouse BMDC were transfected with negative control siRNA (NC) or one of the three mGluR4 siRNAs (siRNA). Twenty-four hours later, the efficiency of silencing was detected by qRT-PCR ( A ) and western blotting analysis ( B ). C , Quantitative mGluR4/β-actin expression levels. Data are reported as means±SD. *P<0.05, ***P<0.001 vs NC (ANOVA).

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Derivative Assay, Transfection, Negative Control, Quantitative RT-PCR, Western Blot, Expressing

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Th17 cell differentiation induced by mGluR4 knockdown in bone marrow-derived dendritic cells (BMDC). The relative mRNA expressions of interleukin (IL)-6 ( A ) and IL-23 ( B ) were evaluated by qRT-PCR in immature (Control), mature (lipopolysaccharide, LPS), and mGluR4 siRNA transfected (siRNA) BMDC. CD4 + T cells were co-cultured with the BMDC for 24 h at the ratio of 1:1. The relative mRNA expressions of IL-17A ( C ) and RORγt ( D ) in the co-culture environment were determined by qRT-PCR. Data are reported as means±SD. ***P<0.001 vs Control (ANOVA).

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Cell Differentiation, Derivative Assay, Quantitative RT-PCR, Transfection, Cell Culture, Co-Culture Assay

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Effect of increased production of Th17-related cytokines on melanocyte function. B16 cells were treated with co-culture medium of CD4 + T cells and the negative control siRNA transduced dendritic cells (DC), mGluR4 siRNA transduced DC (siRNA), or only RIPM-1640 medium (Control). Twenty-four hours later, the mRNA expressions of MITF ( A ), TYR ( B ), and TRP-1 ( C ) were measured by qRT-PCR. D , Forty-eight hours later, B16 cells exposed to the different interventions were treated with 1 M NaOH and processed for absorbance (OD) at 405 nm. Data are reported as means±SD. ***P<0.001 vs Control (ANOVA).

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Co-Culture Assay, Negative Control, Quantitative RT-PCR

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Effect of increased production of Th17-related cytokines on morphology of B16 cells. B16 cells were exposed to the co-culture medium of CD4 + T cells and the negative control siRNA transduced dendritic cells (DC), mGluR4 siRNA transduced DC (siRNA), or only RIPM-1640 medium (Control). Twenty-four hours later, melanocyte morphology was observed under a microscope with 100 and 200× magnification (scale bars: upper panels 20 µm; lower panels 10 µm). Representative images are shown.

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Co-Culture Assay, Negative Control, Microscopy

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: mGluR4 over-expression in bone marrow-derived dentritic cells (BMDC). BMDC were transfected with empty vector (negative control, NC), plasmid expressing mGluR4 (mGluR4), or untreated (Control). Twenty-four hours later, the expression of mGluR4 was determined by qRT-PCR ( A ) and western blotting analysis ( B ). C , Quantitative mGluR4/β-actin expression levels. Data are reported as means±SD. **P<0.01, ***P<0.001 vs Control (ANOVA).

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Over Expression, Derivative Assay, Transfection, Plasmid Preparation, Negative Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Effect of mGluR4 over-expression on surface marker expression of costimulatory molecules CD80 and CD86 in dendritic cells (DC). DC were transfected with empty vector (negative control, NC), plasmid expressing mGluR4 (mGluR4), or untreated (Control). Twenty-four hours later, cells were collected and the percent of DC expressing CD80 ( A ) and CD86 ( B ) was quantified by flow cytometry. Data are reported as means±SD. *P<0.05, ***P< 0.001 vs Control (ANOVA).

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Over Expression, Marker, Expressing, Transfection, Plasmid Preparation, Negative Control, Flow Cytometry

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Effect of mGluR4 over-expression on Th17 differentiation and melanogenesis of B16 cells. Dendritic cells (DC) were transfected with empty vector (negative control, NC), plasmid expressing mGluR4 (mGluR4), or untreated (Control). Twenty-four hours later, the relative mRNA expressions of interleukin (IL)-6 ( A ) and IL-23 ( B ) were evaluated by qRT-PCR. CD4 + T cells were co-cultured with the above DC for 24 h at the ratio of 1:1. The relative mRNA expressions of IL-17A ( C ) and RORγt ( D ) in co-culture environment were determined by qRT-PCR. B16 cells were treated with co-culture medium of CD4 + T cells and empty vector transfected DC (DC), pcDNA3.1 mGluR4 transfected DC (mGluR4), or only RIPM-1640 medium (Control) for 24 h ( E ). mRNA expression of MITF of B16 cells was measured by qRT-PCR. Data are reported as means±SD. ***P<0.001 vs Control (ANOVA).

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Over Expression, Transfection, Plasmid Preparation, Negative Control, Expressing, Quantitative RT-PCR, Cell Culture, Co-Culture Assay

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

doi: 10.1590/1414-431X20209282

Figure Lengend Snippet: Effect of decreased production of Th17-related cytokines on morphology of B16 cells. B16 cells were exposed to the co-culture medium of CD4 + T cells and empty vector transfected dendritic cells (DC), pcDNA3.1 mGluR4 transfected DC (mGluR4), or only RIPM-1640 medium (Control). Twenty-four hours later, melanocyte morphology was observed under the microscope with 100 and 200× magnification (scale bars: upper panels 20 µm; lower panels 10 µm). Representative images are shown.

Article Snippet: For transient transfection experiments, three predesigned mGluR4-targeted siRNAs and negative control siRNA (NC) (GenePharma Co., China) were diluted with Opti-MEM (Gibco/BRL, Thermo fisher Scientific, USA) and transfected into cells using INTERFERin transfection reagent (Polyplus Transfection, France) according to the manufacturer's instructions.

Techniques: Co-Culture Assay, Plasmid Preparation, Transfection, Microscopy

Journal: Oncology Letters

Article Title: Hypoxia‑induced SREBP1‑mediated lipogenesis and autophagy promote cell survival via fatty acid oxidation in breast cancer cells

doi: 10.3892/ol.2025.14921

Figure Lengend Snippet: SREBP1, analyzed through SREBF1 mRNA expression, serves as a prognostic marker for survival outcomes among patients with triple negative breast cancer. The recurrence-free survival and distant metastasis-free survival rates of (A) ER+/PR-/HER2-(n=200 and 118, respectively) and (B) ER-/PR-/HER2-(n=392 and 306, respectively) patients with breast cancer were analyzed over 250 months using Kaplan-Meier plots to assess the impact of SREBF1 mRNA expression. SREBF1, sterol regulatory element-binding transcription factor 1; ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2; HR, hazard ratio.

Article Snippet: Gene knockdown experiments were performed by transfection with AccuTarget negative control siRNA (cat. no. SN-1011; Bioneer Corporation) and AccuTarget Genome-wide Predesigned siRNA targeting Human SREBF1 (Bioneer Corporation).

Techniques: Expressing, Marker, Binding Assay