Journal: Redox Biology

Article Title: SARS-CoV2 infection impairs the metabolism and redox function of cellular glutathione

doi: 10.1016/j.redox.2021.102041

Figure Lengend Snippet: Nrf2, Membrane transporters, and GCLC in SARS-CoV2 infected VERO-E6 cells treated with Nelfinavir (Nel) or Remdesivir (Rem). Immunoblot of Nrf2 protein expression ( A , left panels) was assessed 6 hpi and 24 hpi, and by semi-quantitative fluorescence analysis 48 hpi ( A , right panels). Fluorophores were FITC (green) for Nrf2 protein labelling, DAPI (blue) for nuclei and Phalloidin-Alexa Fluor595 (orange) for the cytosolic space. Immunoblot of xCT and MRP1 membrane transport proteins ( B ), and GCLC protein ( C ) were carried out as described in the section Methods 24 hpi. Infection conditions and treatments with antivirals were the same of . Control test with untreated cells (CTL -) vs infected cells or treatments: §p < 0.05, §§p < 0.01. Infected cells + DMSO vs antivirals *p < 0.05, **p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used were: Nrf2 Polyclonal Antibody, FITC Conjugated (bs-1074R-FITC, Bioss Antibodies, 1:50); NQO1 (A180, Mouse mAb #3187, CST, 1:50); GST-pi (610,718, mouse mAb, BD Biosciences, 1:500); PE anti-human IL-6 Antibody (BioLegend, 1:1000); PE anti-human IL-10 Antibody (BioLegend, 1:1000).

Techniques: Infection, Western Blot, Expressing, Fluorescence

Journal: Oncogenesis

Article Title: Increased expression of NAF1 contributes to malignant phenotypes of glioma cells through promoting protein synthesis and associates with poor patient survival

doi: 10.1038/s41389-019-0134-2

Figure Lengend Snippet: Upon knocking down of c-Myc ( a , b ) and NRF2 ( c , d ) in glioma cell lines SF295 and U87 using siRNAs, the protein and mRNA expression levels of NAF1, c-Myc, and NRF2 were measured by western blot and qRT-PCR assays. Shown is representative of three independently preformed western blot experiments. GAPDH and β-actin were used as the normalized controls for western blot and qRT-PCR assays, respectively. Data were shown as mean ± SD, * P < 0.05; ** P < 0.01 ( n = 3). Upon ectopic expression of c-Myc ( e , f ) and NRF2 ( g , h ) in glioma cell lines SF295 and U87, the protein and mRNA expression levels of NAF1, c-Myc, and NRF2 were determined by western blot and qRT-PCR assays. Shown is representative of three independently preformed western blot experiments. GAPDH and β-actin were used as the normalized controls for western blot and qRT-PCR assays, respectively. Data were shown as mean ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001 ( n = 3). The Dual-Luciferase Reporter assay system was used to evaluate the impact of ectopic expression of c-Myc ( i ) and NRF2 ( j ) on the promoter activity of NAF1 in SF295 and U87 cells with the empty vector or control lentivirus as the controls. All the ratios of the Luc/Renilla activity were expressed as means ± SD. *** P < 0.001 ( n = 3). k SF295 cells expressing c-Myc and NRF2 and control cells were subjected to ChIP-qPCR assays using corresponding primary antibodies. P1–P4 indicated four different regions of NAF1 promoter (P1: −100/−26; P2: −519/−410; P3: −981/−543; P4: −1648/−1551) (left panel). Fold enrichment was expressed as mean ± SD (middle and right panels). * P < 0.05; ** P < 0.01 ( n = 3)

Article Snippet: NC16 pCDNA3.1 FLAG NRF2 (Plasmid #36971), pCDNA-3xHA-hTERT (Plasmid #51637), and corresponding empty vector were obtained from Addgene (Cambridge, MA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation, Control, ChIP-qPCR

Journal: Oncogenesis

Article Title: Increased expression of NAF1 contributes to malignant phenotypes of glioma cells through promoting protein synthesis and associates with poor patient survival

doi: 10.1038/s41389-019-0134-2

Figure Lengend Snippet: a , b Protein expression levels of c-Myc, NRF2, and TERT upon NAF1 knockdown or ectopic expression in SF295 and U87 cells were determined by western blot analysis with GAPDH as a loading control. c , d The qRT-PCR assay was carried out to detect mRNA expression of c-Myc , NRF2 , and TERT upon depletion or overexpression of NAF1 in SF295 and U87 cells. β-Actin was used as an endogenous control. Data were shown as mean ± SD. ** P < 0.01; *** P < 0.001 ( n = 3). e , f Western blot analysis was used to evaluate protein expression of POLR1A and POLR2A upon knockdown or ectopic expression of NAF1 in SF295 and U87 cells. GAPDH was used as an endogenous control. The western blot is representative of three independently preformed experiments

Article Snippet: NC16 pCDNA3.1 FLAG NRF2 (Plasmid #36971), pCDNA-3xHA-hTERT (Plasmid #51637), and corresponding empty vector were obtained from Addgene (Cambridge, MA).

Techniques: Expressing, Knockdown, Western Blot, Control, Quantitative RT-PCR, Over Expression

Journal: Oncogenesis

Article Title: Increased expression of NAF1 contributes to malignant phenotypes of glioma cells through promoting protein synthesis and associates with poor patient survival

doi: 10.1038/s41389-019-0134-2

Figure Lengend Snippet: NAF1 plays a crucial role in maintaining the yield of mature H/ACA RNPs. During malignant transformation of glioma cells, increased expression of NAF1 promotes U17 snoRNA processing, 18S rRNA maturation, and the assembly of 40 S subunits, thereby enhancing protein synthesis, including some key molecules associated with malignant progression of gliomas, such as c-Myc, NRF2, TERT, POLR1A, and POLR2A. Meanwhile, c-Myc, NRF2, and TERT in turn transcriptionally upregulate NAF1 expression, while POLR1A and POLR2A also can active the transcription of 45 S rRNA , c-Myc , NRF2 , TERT , and H/ACA snoRNA. These observations indicate that there exist positive feedback loops between NAF1 and these key molecules. In addition, NAF1 can maintain telomere length by increasing the levels of TERT and TERC at transcriptional or post-transcriptional levels. Altogether, these molecular events will contribute to glioma tumorigenesis and progression

Article Snippet: NC16 pCDNA3.1 FLAG NRF2 (Plasmid #36971), pCDNA-3xHA-hTERT (Plasmid #51637), and corresponding empty vector were obtained from Addgene (Cambridge, MA).

Techniques: Transformation Assay, Expressing

Journal: Journal of Nanobiotechnology

Article Title: Self-immolative nanocapsules precisely regulate depressive neuronal microenvironment for synergistic antidepression therapy

doi: 10.1186/s12951-023-02008-9

Figure Lengend Snippet: In vivo antidepressive effects of nanocapsules. A In vivo Cy7.5 fluorescence of C57BL/6J mice and CUMS mice after intravenous injection with VCNCs-Cy7.5, CNCs-Cy7.5, and VNCs-Cy7.5 at 400 μg 5-HT/kg for 3 h and 7 h. Living Cy 5.5 fluorescence images of mice after treatment with VCNCs-Cy5.5, CNCs-Cy5.5, and VNCs -Cy5.5 (C 5-HT : 400 μg /kg) for 3 h. The representative western blot analysis of B Nrf 2 in the hippocampus of brain across all groups. The other two replicates were presented in Additional file : Fig. S38. Reverse transcription quantitative polymerase chain reaction analysis (RT-qPCR) of relative mRNA levels of C NLRP3 in the hippocampi of mice in all groups (n = 3). The levels of hippocampal D IL-6 in the mice hippocampi after treatment were detected using ELISA kits. The results were normalized by the protein concentration of each sample (n = 3). E ROS/DAPI staining and F GFAP / DAPI staining brains in groups subjected to different treatments. Analysis of hippocampal BDNF expression using G western blot, H RT-qPCR, I ELISA kits and J immunohistochemistry slides. The significant differences between groups were analyzed using the one-way ANOVA method, *P < 0.05, **P < 0.01, *** P < 0.001

Article Snippet: The sample solutions were then measured using IL-6 (Elabscience, #E-MSEL-M0001, China), IL-1β (Elabscience, #E-EL-M0037c, China), TNF-α (Elabscience, #E-EL-M3063, China), BDNF (Elabscience, #E-EL-M0203c, China), Nrf2 (Cusabio, #CSB-E16188m, China), and 5-HT ELISA kits (Elabscience, #E-EL-0033c China) according to the manufacturer’s instructions.

Techniques: In Vivo, Fluorescence, Injection, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Protein Concentration, Staining, Expressing, Immunohistochemistry

Journal: Journal of Nanobiotechnology

Article Title: Self-immolative nanocapsules precisely regulate depressive neuronal microenvironment for synergistic antidepression therapy

doi: 10.1186/s12951-023-02008-9

Figure Lengend Snippet: Effects of nanocapsules on in vivo 5-HT level and neuroprotection. A 5-HT IHC and B nissil and HE staining of brains after administering with fluoxetine, VCNCs, CNCs, and VNCs. C The levels of hippocampal 5-HT in the mice hippocampi after treatment were detected using ELISA kits. The results were normalized by the protein concentration of each sample (n = 3). The significant differences between groups were analyzed using the one-way ANOVA method, *P < 0.05. D The in vivo therapeutic outcomes of nanocapsules

Article Snippet: The sample solutions were then measured using IL-6 (Elabscience, #E-MSEL-M0001, China), IL-1β (Elabscience, #E-EL-M0037c, China), TNF-α (Elabscience, #E-EL-M3063, China), BDNF (Elabscience, #E-EL-M0203c, China), Nrf2 (Cusabio, #CSB-E16188m, China), and 5-HT ELISA kits (Elabscience, #E-EL-0033c China) according to the manufacturer’s instructions.

Techniques: In Vivo, Staining, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: Aging

Article Title: Pharmacological recapitulation of the lean phenotype induced by the lifespan-extending sulfur amino acid-restricted diet.

doi: 10.18632/aging.206237

Figure Lengend Snippet: Figure 3. The SAAR diet and BSO exert tissue-specific effects on Nrf2 and Phgdh. The SAAR diet increased Nrf2 (A) and Phgdh (B) protein expressions in the liver, which ultimately resulted in higher serine concentrations (C). Unlike the SAAR diet, BSO did not increase Nrf2 and Phgdh in the liver but increased both in the kidneys (D, E). Regardless of the changes in Nrf2 and Phgdh, BSO increased serine concentrations in livers and kidneys (C–F). NAC reversed SAAR-induced changes in Nrf2, Phgdh, and serine (A–F). Note: Sample size = 5-6. Statistical methods are similar to those in Figure 2.

Article Snippet: Host species Antibody dilutions Primary Antibodies Nrf2 Cell Signaling Technology 20733 Rabbit 1:1000 in 5% BSAa Phgdh Cell Signaling Technology 13428 Rabbit 1:1000 in 5% milka β-Actin Sigma A5441 Mouse 1:15000 in 5% milka Vinculin Proteintech 66305-1-Ig Mouse 1:10000 in 5% milka Secondary Antibodies Anti-Rabbit-HRP Cell Signaling Technology 7074 Goat 1:4000 in 5% milk, 1hb Anti-Mouse-HRP Bio-Rad 170-6516 Goat 1:20000 in 5% milk, 30 minb aAll incubations were performed overnight at 4° C. bAll incubations were performed at room temperature. www.aging-us.com 22 AGING Supplementary Table 3.

Techniques:

Journal: Bioengineered

Article Title: Long noncoding RNA BBOX1-AS1 promotes the progression of gastric cancer by regulating the miR-361-3p/Mucin 13 signaling axis.

doi: 10.1080/21655979.2022.2072629

Figure Lengend Snippet: Figure 5. MUC13 was targeted by miR-361-3p. (a) MUC13 and CLDN4 were predicted and screened as the target genes of miR-361- 3p using GEPIA and ENCORI. (b) RNA pull-down analysis on the interaction between MUC13 and CLDN4 of miR-361-3p. **P < 0.001 vs Bio-NC. (c) The binding sites of MUC13 on miR-361-3p were predicted by StarBase. (d) The luciferase reporter analysis on the relationship between miR-361-3p and MUC13. **P < 0.001 vs miR-NC. (e, f) The expression level of MUC13 in GC clinical samples (e) and GC cell lines (f) was detected by qRT-PCR. (g) Pearson analysis revealed the expression relationship between miR-361-3p and MUC13.

Article Snippet: These membranes were incubated with primary antibodies against GAPDH (1:1,000; #bs-10900 R; Bioss, Beijing, China) and MUC13 (1:1,000; #bs-1,074 R; Bioss, Beijing, China) at 4°C overnight and then incubated with the secondary antibody of horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; #ab6721; Abcam) for 1 h at 25°C.

Techniques: Binding Assay, Luciferase, Expressing, Quantitative RT-PCR

Journal: Bioengineered

Article Title: Long noncoding RNA BBOX1-AS1 promotes the progression of gastric cancer by regulating the miR-361-3p/Mucin 13 signaling axis.

doi: 10.1080/21655979.2022.2072629

Figure Lengend Snippet: Figure 6. MiR-361-3p knockdown promoted GC progression by targeting MUC13. (a) The expression level of miR-361-3p was evaluated in groups of si-NC, inhibitor-NC, inhibitor, si-MUC13, and si-MUC13+ inhibitor by qRT-PCR. (b-d) The cell proliferation (b), invasion (c), and apoptosis (d) were measured in groups of si-NC, inhibitor-NC, inhibitor, si-MUC13, and si-MUC13+ inhibitor using CCK-8, Transwell and flow cytometry assays, respectively. **P < 0.001 vs si-NC, &&P < 0.001 vs inhibitor-NC. ##P < 0.001 vs si-lnc +inhibitor.

Article Snippet: These membranes were incubated with primary antibodies against GAPDH (1:1,000; #bs-10900 R; Bioss, Beijing, China) and MUC13 (1:1,000; #bs-1,074 R; Bioss, Beijing, China) at 4°C overnight and then incubated with the secondary antibody of horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; #ab6721; Abcam) for 1 h at 25°C.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry