Journal: Nature Communications

Article Title: Nucleus-translocated GCLM promotes chemoresistance in colorectal cancer through a moonlighting function

doi: 10.1038/s41467-024-55568-1

Figure Lengend Snippet: a IHC staining and scores of GCLM expression in primary CRC tumor (T) and adjacent normal tissues (N) (n = 406, CRC tissue specimens). Scale bar = 50 μm. The data are presented as a box-and-whisker graph (minimum–maximum), and the horizontal line across the box indicates the median ( a right). b Overall survival and disease-free survival assays of CRC patients based on the GCLM protein level in ( a ). c IHC staining of GCLM expression in CRC patients receiving FOLFOX or XELOX chemotherapy. The blue arrowheads indicate the GCLM expression in nucleus. The correlation between GCLM expression and the response of patients to the standard chemotherapy (right), which is presented in the form of the percentage of total samples. (n = 58, CRC tissue specimens). Scale bar = 50 μm. d Cell viability of CRC cells overexpressing control or rGCLM WT, which were treated with PBS, oxaliplatin (40 μM for HCT116, 80 μM for DLD1, 24 h) or BSO (150 μM, 24 h). e IB detection of total, cytoplasmic and nuclear GCLM expression in HCT116 cells treated with 40 μM oxaliplatin (Oxa), cisplatin (CDDP) and carboplatin (CBP), and 20 μM irinotecan (CPT-11) and 5-fluorouracil (5-FU) for 24 h. f IF staining showing the localization of GCLM in HCT116 cells with the treatment of PBS, oxaliplatin or cisplatin (40 μM, 24 h). Scale bar = 10 μm. Cell viability ( g ) and apoptotic cells ( h ) among HCT116 cells overexpressing control, nuclear GCLM (NLS) or C193/194 A mutant (NLS-CA) with oxaliplatin or cisplatin treatment (40 μM, 24 h). i Photographs and tumor volumes, weights analysis of CDX model after the implantation of HCT116 cells overexpressing control or nuclear GCLM, followed by intraperitoneal injections of PBS or oxaliplatin (5 mg/kg). IB experiments were repeated three times and n = 3 biologically independent experiments in ( d , g , h ) and n = 5 mice in ( i ). The data are presented as the mean ± S.D ( d , g , h , i ). The P values were calculated by two-tailed paired Student’s t test ( a ), Kaplan–Meier analysis (log-rank test) ( b ), two-sided chi-square test ( c ), one-way ANOVA ( h , i right), and two-way ANOVA ( d , g , i middle). PD progressive disease, SD stable disease, PR partial response, CR complete response, NLS nuclear localization signals.

Article Snippet: For Co-IP, the total cell lysate or cytoplasmic and nuclear fractions were incubated with anti-Flag, HA, GCLM, NKRF antibody or IgG and protein A/G beads (MedChemExpress, NJ, USA) overnight at 4 °C.

Techniques: Immunohistochemistry, Expressing, Whisker Assay, Control, Staining, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Nucleus-translocated GCLM promotes chemoresistance in colorectal cancer through a moonlighting function

doi: 10.1038/s41467-024-55568-1

Figure Lengend Snippet: a Coomassie blue staining showing the bound proteins of cytoplasmic and nuclear GCLM in 293 T cells overexpressing Flag-tagged GCLM under oxaliplatin treatment (40 μM, 24 h). Red box indicates the main proteins that differed between the cytoplasmic and nuclear fractions. b Co-IP analysis showing the level of NKRF bound by GCLM using anti-GCLM or IgG antibody. c GST pull-down analysis detecting the level of purified His-NKRF interacting with purified GST-GCLM. d Co-IP analysis demonstrating the level of NKRF bound by total, cytoplasmic and nuclear GCLM in 293 T cells overexpressing Flag-tagged GCLM. Co-IP ( e ) and Duolink ( f ) analyses showing the interaction (red dots in f ) of GCLM and NKRF in 293 T cells overexpressing Flag-tagged GCLM WT or nuclear GCLM ( + NLS). Scale bar = 10 μm. g Co-IP analysis showing the interaction of GCLM and NKRF in HCT116 cells treated with different concentrations of oxaliplatin or cisplatin for 24 h. h Co-IP analysis demonstrating the level of GCLM bound by WT or truncated NKRF mutants (M1-M5) in 293 T cells. Control or GCLM-knockdown HCT116 or 293 T cells overexpressed HA-tagged NKRF. Co-IP analysis showing the interaction of NKRF and p65/p50 ( i ) and the interaction of p65 and p50 ( j ) with oxaliplatin treatment (40 μM, 24 h). k HCT116 or 293 T cells were depleted GCLM and re-expressed nuclear GCLM (NLS) with control or NKRF inhibition in the presence of oxaliplatin (40 μM, 24 h). The transcriptional activity of NF-κB/p65 was detected. l Q-PCR analysis of the expression of NF-κB/p65-targeted genes (CCND1, XIAP, BCL2, BCL-xl, and iNOS) in HCT116 cells when depleting GCLM and re-expressiong nuclear GCLM in the presence of oxaliplatin (40 μM, 24 h). m Cell viability and apoptotic cells analysis in HCT116 cells when depleting GCLM and re-expressiong nuclear GCLM with control or p65 silencing with or without oxaliplatin treatment (40 μM, 24 h). IB experiments were repeated three times and n = 3 biologically independent experiments in ( k – m ). All the data are presented as the mean ± S.D. The P values were calculated by one-way ANOVA ( k , l ) and two-way ANOVA ( m ).

Article Snippet: For Co-IP, the total cell lysate or cytoplasmic and nuclear fractions were incubated with anti-Flag, HA, GCLM, NKRF antibody or IgG and protein A/G beads (MedChemExpress, NJ, USA) overnight at 4 °C.

Techniques: Staining, Co-Immunoprecipitation Assay, Purification, Control, Knockdown, Inhibition, Activity Assay, Expressing

Journal: Nature Communications

Article Title: Nucleus-translocated GCLM promotes chemoresistance in colorectal cancer through a moonlighting function

doi: 10.1038/s41467-024-55568-1

Figure Lengend Snippet: a Schematic illustration of the potential NLS sequence of the GCLM protein predicted by the cNLS Mapper tool. The strategy for mutating the NLS of the GCLM is also shown. b HCT116 cells overexpressed Flag-tagged GCLM WT or GCLM-NLS mutant (Mut) with or without importazole treatment (40 μM, 24 h). The localization of GCLM was detected by IF staining. Scale bar = 10 μm. c , d HCT116 cells overexpressed Flag-tagged GCLM WT or GCLM-NLS Mut with oxaliplatin or IPZ treatment (40 μM, 24 h). IB detected the nuclear and total GCLM expression ( c ) and Co-IP analysis showed the level of interaction between NKRF and GCLM ( d ). e – g GCLM-knockdown HCT116 or DLD1 cells overexpressed rGCLM WT or rGCLM-NLS Mut with or without oxaliplatin treatment (40 μM, 24 h). The transcriptional activity of NF-κB/p65 ( e ), cell viability ( f ) and apoptotic cells ( g ) were detected. h Co-localization of endogenous GCLM and importin α5 in HCT116 cells was detected by IF. Red arrowheads showed the co-localization. Scale bar = 10 μm. i HCT116 cells overexpressed Flag-tagged GCLM WT or GCLM-NLS Mut with or without oxaliplatin treatment (40 μM, 24 h). Co-IP analysis showing the GCLM-importin α5 interaction. IF staining of the localization of GCLM ( j ) and IB detection of nuclear and total GCLM expression ( k ) in HCT116 cells with control or importin α5 silencing in presence of oxaliplatin treatment (40 μM, 24 h). Scale bar = 10 μm. l , m GCLM-knockdown HCT116 cells overexpressed vector or rGCLM WT with control or importin α5 silencing under oxaliplatin treatment (40 μM, 24 h). The transcriptional activity of NF-κB/p65 ( l ), cell viability and apoptotic cells ( m ) were detected. IB experiments were repeated three times and n = 3 biologically independent experiments in ( e – g , l , m ). All the data are presented as the mean ± S.D. The P values were calculated by one-way ANOVA ( e , l , m ) and two-way ANOVA ( f , g ). IPZ importazole, Imp α5 importin α5.

Article Snippet: For Co-IP, the total cell lysate or cytoplasmic and nuclear fractions were incubated with anti-Flag, HA, GCLM, NKRF antibody or IgG and protein A/G beads (MedChemExpress, NJ, USA) overnight at 4 °C.

Techniques: Sequencing, Mutagenesis, Staining, Expressing, Co-Immunoprecipitation Assay, Knockdown, Activity Assay, Control, Plasmid Preparation

Journal: Nature Communications

Article Title: Nucleus-translocated GCLM promotes chemoresistance in colorectal cancer through a moonlighting function

doi: 10.1038/s41467-024-55568-1

Figure Lengend Snippet: a IP analysis showing the level of GCLM phosphorylated at threonine (pThr), serine (pSer) and tyrosine (pTyr) residues in 293 T cells treated with oxaliplatin or cisplatin (40 μM, 24 h). b Co-IP analysis showing the level of NKRF and importin α5 bound by GCLM in HCT116 cells treated with calf intestinal phosphatase (CIP) that resulted in GCLM dephosphorylation. c IP analysis showing the level of GCLM phosphorylated at Thr17, Thr106 and Thr164 in HCT116 cells treated with oxaliplatin or cisplatin (40 μM, 24 h). d IF staining showing the localization of Flag-GCLM in 293 T cells overexpressing Flag-tagged GCLM WT or T17A mutant with oxaliplatin treatment (40 μM, 24 h). Scale bar = 10 μm. e , f 293 T and HCT116 cells overexpressed the Flag-tagged GCLM WT or T17A, T17E mutant with or without oxaliplatin treatment (40 μM, 24 h). IB detection of nuclear and total GCLM expression ( e ) and Co-IP analysis showing the levels of importin α5 and NKRF interacting with GCLM ( f ). g 293 T cells were treated with the inhibitors of AKT (AKTi, MK-2206, 5 μM), ERK (ERKi, PD98059, 10 μM), JNK (JNKi, SP600125, 20 μM) or P38 MAPK (P38i, SB203580, 10 μM) for 24 h. Co-IP analysis showing the GCLM-importin α5 interaction and the level of GCLM phosphorylated at Thr17. h , i CRC cells were treated with control or P38 MAPK inhibitor (P38i, SB203580, 10 μM) in the presence or absence of oxaliplatin treatment (40 μM for HCT116, 80 μM for DLD1, 24 h). Co-IP analysis showing the level of importin α5 interacting with GCLM ( h ) and IB detection of nuclear and total GCLM expression ( i ). The transcriptional activity of NF-κB/p65 ( j ), cell viability ( k ) and apoptotic cells ( l ) in GCLM-knockdown HCT116 or DLD1 cells overexpressing rGCLM WT or T17A, T17E mutants with or without oxaliplatin treatment (40 μM, 24 h). IB experiments were repeated three times and n = 3 biologically independent experiments in ( j – l ). All the data are presented as the mean ± S.D. The P values were calculated by one-way ANOVA ( j ) and two-way ANOVA ( k , l ).

Article Snippet: For Co-IP, the total cell lysate or cytoplasmic and nuclear fractions were incubated with anti-Flag, HA, GCLM, NKRF antibody or IgG and protein A/G beads (MedChemExpress, NJ, USA) overnight at 4 °C.

Techniques: Co-Immunoprecipitation Assay, De-Phosphorylation Assay, Staining, Mutagenesis, Expressing, Control, Activity Assay, Knockdown

Journal: Nature Communications

Article Title: Nucleus-translocated GCLM promotes chemoresistance in colorectal cancer through a moonlighting function

doi: 10.1038/s41467-024-55568-1

Figure Lengend Snippet: a IHC staining and scores for nuclear GCLM expression (nGCLM, only focusing on GCLM staining in the nucleus, as shown by blue arrowheads) in paired primary CRC tumor tissues (T) and adjacent normal tissues (N) (n = 406, CRC tissue specimens). Scale bar = 50 μm. b IHC staining and scores for the level of GCLM phosphorylated at Thr17 (pT17-GCLM) in paired primary CRC tumor tissues (T) and adjacent normal tissues (N) (n = 200, CRC tissue specimens). Scale bar = 50 μm. c , d Overall survival (left) and disease-free survival (right) assays of patients with CRC based on nuclear GCLM ( a ) and pT17-GCLM ( b ) expression. e Overall survival assays of patients with GC (n = 235, GC tissue specimens) or ESCC (n = 276, ESCC tissue specimens) based on nuclear GCLM expression. f The correlation between nuclear GCLM and pT17-GCLM expression and the response of CRC patients to FOLFOX or XELOX chemotherapy (n = 58, CRC tissue specimens, PD progressive disease, SD stable disease, PR partial response, CR complete response). g IHC staining showing high and low expression of nuclear GCLM (nGCLM, blue arrowheads), pT17-GCLM, Ki67, Tunel (red arrowheads) and phospho-P38 MAPK (pP38) expression in CRC tumor tissues. Scale bar = 50 μm. h Correlations between nuclear GCLM expression and pT17-GCLM, Ki67, Tunel and pP38 MAPK expression. i Proposed working model based on this study. The model shows that P38 MAPK-mediated phosphorylated GCLM interacts with importin α5 and is translocated into the nucleus upon platinum drug treatment. Nuclear GCLM enhances NF-κB/p65 activity by interacting with NKRF to confer resistance to platinum-based chemotherapy in CRC cells. The data are presented as a box-and-whisker graph (minimum–maximum), and the horizontal line across the box indicates the median ( a , b right), and the percentage of total sample ( f , h ). We categorized proteins levels as low or high compared with the median value of IHC score ( c – e , f and h ). The P values were calculated by two-tailed paired Student’s t test ( a , b right), Kaplan–Meier analysis (log-rank test) ( c – e ), and two-sided chi-square test ( f , h ). GC, gastric carcinoma; ESCC, esophageal squamous cell carcinoma.

Article Snippet: For Co-IP, the total cell lysate or cytoplasmic and nuclear fractions were incubated with anti-Flag, HA, GCLM, NKRF antibody or IgG and protein A/G beads (MedChemExpress, NJ, USA) overnight at 4 °C.

Techniques: Immunohistochemistry, Expressing, Staining, TUNEL Assay, Activity Assay, Whisker Assay, Two Tailed Test

Journal: Neurobiology of Disease

Article Title: Neuroprotective activity of ursodeoxycholic acid in CHMP2B Intron5 models of frontotemporal dementia

doi: 10.1016/j.nbd.2020.105047

Figure Lengend Snippet: UDCA Identifies GCLC As a Novel Regulator of CHMP2B Intron5 Toxicity. A. The mRNA abundance of gclc from pan-neuronally expressing CHMP2B Intron5 third instar larval brains shows a significant upregulation when raised on UDCA (600 μM) supplemented food compared to vehicle (ethanol). Mean ± SEM, n = 5 per genotype, fold change values in log 2 scale. ⁎⁎⁎ q < 0.001. B. Representative images of the Drosophila eye phenotype caused by CHMP2B Intron5 expression with eye-specific GMR -Gal4 driver ( GMR -Gal4, UAS-CHMP2B Intron5 ) and amelioration by co-expression of UAS- GCLC . C. Quantification of the eye phenotype from (B) genotypes. n = 100. D, E. Co-expression of GCLC (UAS- GCLC 6 ) ameliorates unregulated synaptic growth characterised by increased bouton number (D) and NMJ length (E) at the third instar larval NMJ (Muscle 6/7, hemisegment A3) in CHMP2B Intron5 ( nSyb -Gal4) expressing larvae. ANOVA with post-hoc Tukey comparison between groups ⁎ p < .05, ⁎⁎ p < .01. F. Pan-neuronal ( nSyb -Gal4) expression of GCLC rescues aberrant crawling behaviour in 3rd instar wandering larvae pan-neuronally expressing CHMP2B Intron5 . ANOVA with post-hoc Dunnett's comparison to wild type controls ⁎⁎⁎ p < .001 and Tukey comparison between groups ### p < .001. G. Representative micrographs of mature neurons expressing FLAG-tagged CHMP2B Wildtype or CHMP2B Intron5 ± plasmids expressing the catalytic (GCLC) and modifying (GCLM) subunits of glutamate cysteine ligase (GCL). H, I Quantification of total arbour size (H) and cumulative number of intersections per unit distance (I) in CHMP2B Wildtype or CHMP2B Intron5 ± GCLC/M. Data represents mean ± SEM analysed using one-way ANOVA and Tukey's multiple comparisons post hoc test ( ## p < .01, ### p < .001). Scale bar = 50 μm. 20 neurons analysed, per condition, across 3 biological replicates.

Article Snippet: Neurons were transfected at 12 days in vitro (DIV) with Lipofectamine 2000 (11,668,019, Thermo Scientific) in transfection medium ( ) with either FLAG-tagged CHMP2B Wildtype or CHMP2B Intron5 for 5 h. The cDNAs for catalytic (GCLC, MC203908) and modifying (GCLM, MR225622) subunits of GCL were obtained from Origene.

Techniques: Expressing, Comparison

Journal: International journal of molecular sciences

Article Title: Arsenite-Mediated Transcriptional Regulation of Glutathione Synthesis in Mammalian Primary Cortical Astrocytes.

doi: 10.3390/ijms26115375

Figure Lengend Snippet: Figure 3. Effect of arsenite on astrocyte GCL mRNA and protein expression. Primary cortical astrocytes (n = 3 from 3 separate dissections) were treated with 10 µg/mL ActD (+ActD; gray bars) or its vehicle (−ActD; blue bars) for 30 min, after which NaAsO2 was spiked into experimental wells to a final concentration of 15µM. Four hours later, RNA was isolated, and (A) GCLC and (D) GCLM mRNA expression was determined by RT-qPCR. Data are expressed as mean + SEM fold change over untreated cells (−NaAsO2, −ActD; =1). Exact p-values are shown for each pairwise comparison determined by two-way ANOVA followed by Fisher’s uncorrected LSD test for multiple comparisons. Protein expression of (B) GCLC and (E) GCLM was assessed by Western blotting, as described in the Section 4. Exact p-values are shown for each pairwise comparison determined to be significant by a paired t-test. Representative blots for (C) GCLC and (F) GCLM are shown.

Article Snippet: After destaining, membranes were blocked in Odyssey blocking buffer (LI-COR Environmental, Lincoln, NE, USA) for 1 h at room temperature, followed by overnight incubation at 4 ◦C with primary antibodies against GCLc (0.93 ug/mL; rabbit polyclonal; ABclonal Technology, Woburn, MA, USA), GCLM (0.38 ug/mL; rabbit polyclonal; ABclonal), and GS (0.51 ug/mL; mouse monoclonal; Novus Biologicals, Centennial, CO, USA).

Techniques: Expressing, Concentration Assay, Isolation, Quantitative RT-PCR, Comparison, Western Blot

Journal: bioRxiv

Article Title: Endogenous formaldehyde scavenges cellular glutathione resulting in cytotoxic redox disruption

doi: 10.1101/2020.05.14.090738

Figure Lengend Snippet: a. Resazurin-based viability assay for HCT116 (left) and Nalm6 (right) Wild type (WT) and Δ ADH5 cells at 100 and 50 μM L-buthionine-sulfoximine (L-BSO), respectively (s.e.m., n=5). b. Western blot analysis of GCLM expression in clones edited by CRISPR/Cas9. c. ADH5 gene showing the exon targeted by CRISPR/Cas9 and the genetic modifications of the Δ ADH5 clone on which GCLM was inactivated. d. GCLM gene showing the exon targeted by CRISPR/Cas9 and the genetic modifications of the Δ GCLM clones used in this work.

Article Snippet: After 20 days, cells were expanded, and inactivation of the desire gene confirmed by western blot using GCLM (Atlas antibodies, #HPA023696) or ADH5 antibodies .

Techniques: Viability Assay, Western Blot, Expressing, Clone Assay, CRISPR

Journal: bioRxiv

Article Title: Endogenous formaldehyde scavenges cellular glutathione resulting in cytotoxic redox disruption

doi: 10.1101/2020.05.14.090738

Figure Lengend Snippet: a. Resazurin-based viability assay in Wild type (WT) and Δ ADH5 Nalm6 cells in presence of different concentrations of formaldehyde (FA) with or without 50 μM L-buthionine-sulfoximine (L-BSO) (mean ± s.e.m., n=6). b. HCT116 3D-spheroid formation in presence of 100 μM L-BSO and the indicated concentrations of FA. Pictures were taken 5 days after seeding cells on agarose-coated plates (scale bar 0.5 mm). c. Resazurin-based viability assay performed with Wild type, Δ ADH5 , Δ GCLM and Δ ADH5 Δ GCLM cells in response to increasing concentrations of FA (mean ± s.e.m., n=5, asterisks represent the statistical significance according to one-way ANOVA for multiple comparison using a Tukey-corrected test between Wild type and Δ GCLM ). d. Representative images of HCT116 3D-spheroid formation for the same cell lines described in (C). Pictures were taken at day 5 after seeding (scale bar 0.5 mm). e. Colony survival assay prepared seeding 600 of WT, Δ ADH5 , Δ GCLM and Δ GCLM Δ ADH5 cells in 6-well plates in presence of the indicated concentration of FA (mean ± s.e.m., n=5, one-way ANOVA using a Tukey’s multiple comparison test). f. Representative images of the colony survival assay quantified in e.

Article Snippet: After 20 days, cells were expanded, and inactivation of the desire gene confirmed by western blot using GCLM (Atlas antibodies, #HPA023696) or ADH5 antibodies .

Techniques: Viability Assay, Comparison, Clonogenic Cell Survival Assay, Concentration Assay

Journal: bioRxiv

Article Title: Endogenous formaldehyde scavenges cellular glutathione resulting in cytotoxic redox disruption

doi: 10.1101/2020.05.14.090738

Figure Lengend Snippet: a. Scheme showing the spontaneous reaction between formaldehyde (FA) and glutathione (GSH) yielding S-hydroxymethylglutathione (HSMGSH). b. Extracted ion chromatograms for [HSMGSH + H]+ ion at m/z 338.1022 ± 0.0500 generated from a non-spiked Wild type (WT) sample (green, left axis), a 20 μM spiked WT sample (black dotted line, right axis), and a 20 μM HSMGSH standard solution (blue, left axis). c. Product ion mass spectra of [HSMGSH + H] + precursor ion for a WT sample (green), and for a 20 μM HSMGSH standard solution (blue), using a collision cell voltage of 10 V. d. Box and whiskers plot for GSH content in WT and Δ ADH5 cells calculated as normalized chromatographic peak areas relative to the number of viable cells (n=9, Mann-Whitney test, *** P<0.0001). The box and whiskers plots are represented by a line in the box corresponding to the median; the edges are the 25th and 75th percentiles and the whiskers extend to the most extreme values in data. e. Left: Box and whiskers plot for HSMGSH content relative to GSH in WT and Δ ADH5 cells (n=9, Mann-Whitney test). Right: Net HSMGSH content in WT and Δ ADH5 cells calculated as normalized peak areas relative to the number of viable cells (n=9, Mann-Whitney test). f. Box and whiskers plot for total GSH (GSH disulfide (GSSG) plus GSH) content in WT and Δ ADH5 cells calculated as normalized peak areas relative to the number of viable cells (n=9, Mann-Whitney test). g. Bar plots for GSH content in WT (n=17), Δ ADH5 (n=15), Δ ADH5 /pADH5 (n=8), Δ GCLM (n=4) and Δ ADH5 Δ GCLM (n=4) cells, and in WT (n=7) and Δ ADH5 (n=7) cells exposed to 100 μM L-BSO for 48 h. Every dot is the percentage of fluorescence intensity in a single well relative to the average fluorescence of WT samples run the same day and corrected for viability determined using resazurin (mean ± s.e.m., unpaired t-test). h. Box and whiskers plot for GSSG content in WT and Δ ADH5 cells calculated relative to the number of viable cells (n=9, Mann-Whitney test). i. Box and whiskers plot for GSH:GSSG ratio in WT and Δ ADH5 cells (n=9, Mann-Whitney test). j. Flow cytometry representative plots from WT and Δ ADH5 cells harboring the cytosolic Grx1-roGFP2 reporter. Data was recorded 48 h post-FA exposure. k. Quantitation of oxidized Grx1-GFP2 (OxD (Grx1-roGFP2)) sensor from plots depicted in (J) (mean ± s.e.m., n=4, unpaired t-test).

Article Snippet: After 20 days, cells were expanded, and inactivation of the desire gene confirmed by western blot using GCLM (Atlas antibodies, #HPA023696) or ADH5 antibodies .

Techniques: Generated, MANN-WHITNEY, Fluorescence, Flow Cytometry, Quantitation Assay

Journal: Antioxidants (Basel, Switzerland)

Article Title: The Role of Fucoxanthin as a Potent Nrf2 Activator via Akt/GSK-3β/Fyn Axis against Amyloid-β Peptide-Induced Oxidative Damage.

doi: 10.3390/antiox12030629

Figure Lengend Snippet: Figure 3. Effects of fucoxanthin on expression of Nrf2 and Keap1 in Aβ25-35-induced PC12 cells. The cells were pretreated with the indicated concentrations of fucoxanthin for 1 h and stimulated with 10 µM Aβ25-35 for 4 h. (A) The nuclear and cytoplasm expression of Nrf2 as measured by western blot. (B) Relative ratio of nuclear and cytoplasmic Nrf2. (C) The total Nrf2 and Keap1 expression. Quantification of (D) total Nrf2 and (E) Keap1 protein expression levels. NE, nuclear extract; CE, cytoplasmic extract. β-Actin and PCNA were used as the loading control for whole cells and nuclear fractions, respectively, in western blot assay. (E) Keap1 protein expression levels. NE, nuclear extract; CE, cytoplasmic extract. mRNA (F,G) and protein (H,I) levels of phase-II enzymes including NQO1, GCLm, and TrxR1. The cells were pretreated with indicated concentrations of fucoxanthin for 1 h and stimulated with 10 µM Aβ25-35 for 24 h. The mRNA expression levels of phase-II enzymes were determined by RT-PCR analysis. For the normalization of mRNA expression GAPDH was used as the loading control. Results are indicated as mean ± S.D. and represent three independent experiments with 3 replications in each experiment. ## p < 0.01 and # p < 0.05 compared with the control groups; *** p < 0.001, ** p < 0.01 and * p < 0.05 compared with the Aβ25-35-treated alone.

Article Snippet: Blotted membranes were blocked in a 5% skim milk solution in Tris-buffered saline with Tween 20 (TBST) buffer at room temperature for 2 h, and probed overnight at 4 ◦C with the following primary antibodies: Bcl-2, Bax, NQO1, GSK-3β, p-Fyn, Fyn, β-actin (1:2000 dilution; all from Santa Cruz Biotechnology, Dallas, TX, USA), Keap1, p-Akt, Akt, p-GSK-3β (1:2000 dilutions; all from Cell Signaling Technology, Danvers, MA, USA), GCLm (1:2000 dilution; Cusabio Technology LCC, Wuhan, China), TrxR1, Nrf2, and PCNA (1:2000 dilution; all from GeneTex Inc., San Diego, CA, USA).

Techniques: Expressing, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Antioxidants (Basel, Switzerland)

Article Title: The Role of Fucoxanthin as a Potent Nrf2 Activator via Akt/GSK-3β/Fyn Axis against Amyloid-β Peptide-Induced Oxidative Damage.

doi: 10.3390/antiox12030629

Figure Lengend Snippet: Figure 4. Effects of fucoxanthin on Akt/GSK-3β/Fyn signaling in Aβ25-35-induced PC12 cells. The cells were pretreated with the indicated concentrations of fucoxanthin for 1 h and stimulated with 10 µM Aβ25-35 or 1 h. The expression of (A) p-Akt (Ser 473)/Akt, (B) p-GSK-3β (Ser 9)/GSK-3β, and (C) p-Fyn/Fyn. Western blotting results of (D) p-Akt (Ser 473)/Akt, (E) p-GSK-3β (Ser 9)/GSK-3β, (F) p-Fyn/Fyn, (G) Nrf2, and (H–K) phase-II enzymes (NQO1, GCLm, and TrxR1) expression co- treatment of the PI3K inhibitor LY294002 and fucoxanthin. After pretreatment with the PI3K inhibitor LY294002 for 30 min, the cells were treated with 5 µM fucoxanthin for 1 h, followed by Aβ25–35 treatment. Results are indicated as mean ± S.D. and represent three independent experiments with 3 replications in each experiment. ## p < 0.01 compared with the control groups; *** p < 0.001 and ** p < 0.01 compared with the Aβ25-35-treated alone; §§ p < 0.01 compared with the Aβ25–35 -treated alone; s p < 0.01 and R p < 0.05 compared with the fucoxanthin group without LY294002.

Article Snippet: Blotted membranes were blocked in a 5% skim milk solution in Tris-buffered saline with Tween 20 (TBST) buffer at room temperature for 2 h, and probed overnight at 4 ◦C with the following primary antibodies: Bcl-2, Bax, NQO1, GSK-3β, p-Fyn, Fyn, β-actin (1:2000 dilution; all from Santa Cruz Biotechnology, Dallas, TX, USA), Keap1, p-Akt, Akt, p-GSK-3β (1:2000 dilutions; all from Cell Signaling Technology, Danvers, MA, USA), GCLm (1:2000 dilution; Cusabio Technology LCC, Wuhan, China), TrxR1, Nrf2, and PCNA (1:2000 dilution; all from GeneTex Inc., San Diego, CA, USA).

Techniques: Expressing, Western Blot, Control