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: Frontiers in Pharmacology

Article Title: Formononetin Improves the Survival of Random Skin Flaps Through PI3K/Akt-Mediated Nrf2 Antioxidant Defense System

doi: 10.3389/fphar.2022.901498

Figure Lengend Snippet: (A) The protein expression of HO-1, NQO1, GCLc, GCLm, TrxR1 and Keap1 was detected by western blot (B) The expressing levels of proteins relate to antioxidant/phase II detoxification enzymes. All data represent mean ± SD. * p ≤ 0.05 and ** p ≤ 0.01 compared to the control group.

Article Snippet: Primary antibodies against NQO1, TrxR1, HO-1, Keap1 IL-1β, TNF-α and Lamin B were from Abcam (Cambridge, UK); antibodies against GCLc and GCLm were purchased from Boster Biological Technology (Wuhan, China); antibodies against Nrf2, GAPDH, PI3K, P-Pi3k, AKT, and P-AKT were purchased from Cell Signaling Technologies (Danvers, MA, United States); antibodies directed vascular endothelial growth factor (VEGF) and SOD2 were obtained from Affinity Biosciences (Cincinnati, OH, United States).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Formononetin Improves the Survival of Random Skin Flaps Through PI3K/Akt-Mediated Nrf2 Antioxidant Defense System

doi: 10.3389/fphar.2022.901498

Figure Lengend Snippet: (A,B) Western blot along with its quantification revealed that the level of P-PI3K, P-Akt and Nrf2 as treated above (C,D) Western blot along with its quantification revealed that the level of HO-1, NQO1, GCLc, GCLm and TrxR1 (E,F) The IHC result of SOD2 and HO-1 expression in skin flaps (scale bar: 50 μm) ** p ≤ 0.01 compared to the control group; ## p ≤ 0.01 compared to the FMNT-H group.

Article Snippet: Primary antibodies against NQO1, TrxR1, HO-1, Keap1 IL-1β, TNF-α and Lamin B were from Abcam (Cambridge, UK); antibodies against GCLc and GCLm were purchased from Boster Biological Technology (Wuhan, China); antibodies against Nrf2, GAPDH, PI3K, P-Pi3k, AKT, and P-AKT were purchased from Cell Signaling Technologies (Danvers, MA, United States); antibodies directed vascular endothelial growth factor (VEGF) and SOD2 were obtained from Affinity Biosciences (Cincinnati, OH, United States).

Techniques: Western Blot, Expressing, Control