Journal: Chinese Medicine

Article Title: Honokiol acts as an AMPK complex agonist therapeutic in non-alcoholic fatty liver disease and metabolic syndrome

doi: 10.1186/s13020-023-00729-5

Figure Lengend Snippet: AMPK activation is independent of classical pathways. A Western blots of p-AMPKα, AMPKα, p-ACC, ACC, p-LKB1, LKB1, p-CAMKK2, CAMKK2, p-TAK1, TAK1, and PP2C of primary hepatocytes subjected to PA stimulation at the indicated conditions. n = 3 replicates. B Western blots of p-AMPKα, AMPKα, p-ACC, ACC, p-LKB1, LKB1, p-CAMKK2, CAMKK2, p-TAK1, TAK1, and PP2C in the livers of HFD-fed mice treated with vehicle or honokiol. n = 5 mice per group. C Western blots of p-AMPKα, AMPKα, p-ACC, ACC, p-LKB1, LKB1, p-CAMKK2, CAMKK2, p-TAK1, TAK1, and PP2C in the livers of CDAHFD-fed mice treated with vehicle or honokiol. n = 5 mice per group. D Western blots of p-AMPKα, AMPKα, p-ACC, ACC, p-LKB1, LKB1, p-CAMKK2, CAMKK2, p-TAK1, TAK1, and PP2C in the livers of MCD-fed mice treated with vehicle or honokiol. n = 5 mice per group. E Representative image of the oxygen consumption rate (OCR) of primary hepatocytes subjected to DMSO or honokiol at the indicated conditions. n = 3 replicates. Omy, oligomycin, F1F0 ATP synthase inhibitor. FCCP, mitochondrial uncoupler. Rot, retenone, complex I inhibitor. AA, antimycin A, complex II inhibitor. F ATP, ADP, and AMP in the livers of HFD-fed mice treated with vehicle or honokiol. n = 6 mice per group. Student’s t -test was applied for statistical analysis. G ATP, ADP, and AMP in the livers of CDAHFD-fed mice treated with vehicle or honokiol. n = 6 mice per group. Student’s t -test was applied for statistical analysis. H ATP, ADP, and AMP in the livers of MCD-fed mice treated with vehicle or honokiol. n = 6 mice per group. Student’s t -test was applied for statistical analysis

Article Snippet: Primary antibodies targeting PP2C (#ab211660) were procured from Abcam (Cambridge, UK).

Techniques: Activation Assay, Western Blot

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) GDF15 levels; B) ACE2 levels ; C) ratio GDf15/ACE2 ; The box plots represent the maximum and minimum levels (whiskers), the upper and lower quartiles, and the median. The length of each box represents the interquartile range. Dots represent outliers. Statistical significance between groups was determined using the ANOVA test. ** p <0.001, # p <0.001 UCI vs Control, n/s (non significant); GDF15 (Growth differentiation factor 15); ACE2 (angiotensin-converting enzyme 2).

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Control

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: GDF15 is positive associated with age (A), while ACE2 is not correlated with age (B). Each dot represents an individual value. The solid blue line represents the regression line. The grey shade represents the confidence interval.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: The box plots represent the maximum and minimum levels (whiskers), the upper and lower quartiles, and the median. The length of each box represents the interquartile range. Dots represent outliers. Statistical significance between groups was determined using the ANOVA test. ** p <0.001, # p <0.001 UCI vs Control, n/s (non significant); ACE2 (angiotensin-converting enzyme 2).

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Control

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) SARS-CoV-2 cell entry assay strategy: Lentiviral-based replication-defective pseudovirus were generated in HEK293T cells from lentiviral parental genes, SARS-CoV-2 Spike and encapsulating a mCherry reporter. Since the entry steps of the SARS-CoV-2 pseudovirions are governed by the coronavirus Spike protein at their surface, they enter cells in a similar fashion to native counterparts. A549 airway cells were transfected with exogenous GFP-hACE2 enabling SARS-CoV-2 pseudovirus to effectively infect the cells with mCherry reporter. Double-positive GFP/mCherry cells were quantified by flow cytometry to assess viral infection capacity. B,C) Following the strategy described in A, A549 cells expressing GFP-hACE2 were assayed for cell entry by SARS-CoV-2 pseudovirus expressing either empty vector (Δ Spike) or Spike protein corresponding to origin variant (Wuhan-1) or variants-of-concern Alpha, Beta, Delta or Zeta. A representative flow cytometry experiment is shown. Bars demonstrate mean and Standard Error of Mean while each data point represents a unique experiment; ****P < 0.0001; **P < 0.01; *P < 0.1 by t test.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Generated, Transfection, Flow Cytometry, Infection, Expressing, Plasmid Preparation, Variant Assay

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A549 cells were transfected with either GFP-ACE2 WT, GFP-ACE2 polymorphisms or GFP alone. Then, ACE2 protein expression was analyzed by A) Western Blot with MA5-32307 antibody B) Immunocytochemistry (red) with either MA5-32307 antibody (left panel) or MAB933 antibody (right panel). Nuclei was stained with DAPI (blue). Transfected cells contain GFP (green). CT: secondary antibody control to detect unspecific binding. Images were acquired with Cell Observer-Zeiss. Scale bar: 50 μm

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Transfection, Expressing, Western Blot, Immunocytochemistry, Staining, Control, Binding Assay

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) Studying the effect of ACE2 SNPs. Non-synonymous ACE2 single nucleotide polymorphism were selected among those fulfilling the triple criteria of high allelic frequency (Allele freq > 1.00e-4; Allele count > 20); involved in ACE2-claw S-protein RBD-binding interface and previously associated to clinical outcome. B, C) Following the strategy described in , A549 cells expressing either GFP-ACE2 either WT or polymorphisms were assayed for cell entry by SARS-CoV-2 pseudovirus expressing either empty vector (Δ Spike) or Spike protein corresponding to origin variant (Wuhan-1) or variants-of-concern Alpha, Beta, Delta or Zeta. A representative flow cytometry experiment is shown. Bars demonstrate mean and Standard Error of Mean while each data point represents a unique experiment; ***P < 0.001, **P < 0.01, *P < 0.1, ns P >0.1 to WT by t test.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Binding Assay, Expressing, Plasmid Preparation, Variant Assay, Flow Cytometry

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) Heatmap showing the distribution of ACE2 variants in the hospitalization severity groups. Coloured squares indicate de presence of the ACE2 variants. Red: Promoting; Green: Protective; Yellow: No-effect. B) Frequencies of ACE2 SNPs among hospitalization severity groups. Bars represent frequencies of the SNP in each group C) Frequencies of ACE2 SNPS among susceptibility groups.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: GDF15 and ACE2 levels across ACE2 variants.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: GDF15 and ACE2 levels among ICU and non-ICU COVID-19 patients and ACE2 genotypes.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: Genotype 0 corresponding to subjects that did not carry any variant; Genotype 1, subjects that carry at least one promoting variant; Genotype 2, subjects that carry at least one protective variant; Genotype 3, subjects that carry at least one promoting and one protective variant ( ; ). We only found differences in the levels of the ACE2 mRNA ( P <0.05).

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Variant Assay

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: Then, PCR1, PCR2 and PCR3 were performed in order to amplify the regions comprising the studied SNPs. PCR products were sequenced and aligned against reference ACE2 (NM_021804). PCR1: residues from Ser3 to Met249 (743 bp), PCR2: residues Phe308 to Arg621 (944 bp), PCR3: residues from Val670 to Val752 (250 bp) A) Agarose gel electrophoresis with the PCR products of several patients was performed to confirm specificity. B) Representative image of the alignment of the sequenced (forward and reverse) PCR products against reference ACE2 using SnapGene® Software. In particular, the image corresponds to the PCR2 of the non-ICU patient 10 that presents the L351V variant.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Agarose Gel Electrophoresis, Software, Variant Assay

Journal: International Journal of Biological Sciences

Article Title: CD44 3'-Untranslated Region Functions as a Competing Endogenous RNA to Enhance NK Sensitivity of Liver Cancer Stem Cell by Regulating ULBP2 Expression

doi: 10.7150/ijbs.35216

Figure Lengend Snippet: CSCs derived from liver cancer were susceptible to NK cell mediated cytotoxicity in correlation with CD44 expression. (A) CD44 high iCSC, CD44 int iCSC, HepG2, and Hep3B were used in the 4 hours NK cell cytotoxicity assay with NK-92 cells at different E:T ratios. Data are showed in means ± SD from three independent experiments which are performed in triplicate (ns: not significant, **: p < 0.01). (B) IFN-γ release in the supernatants of cytotoxic assay (Fig. A, E:T ratio = 15:1) was determined by ELISA. Data were presented as mean ± SD (n=3) (*: p<0.05, **: p<0.01). (C) CD44 transcript level of CD44 high iCSC/CD44 int iCSC stably expressing dCas9-KRAB and sgRNA against CD44 (CD44-knockdown, CD44-kd) was analyzed by qRT-PCR. Levels are represented relative to those found in control-infected cells as means ± SD (n=3) (**: p < 0.01). (D) Identical cells (Fig. C) were used in the 4 hours NK cell cytotoxicity assay with NK-92 cells at different E:T ratios. Data are showed in means ± SD from three independent experiments which are performed in triplicate (**: p < 0.01). (E) IFN-γ release in the supernatants of cytotoxic assay (Fig. D, E:T ratio = 15:1) was determined by ELISA. Data were presented as mean ± SD (n=3) (**: p<0.01).

Article Snippet: Concentrations of secreted IFN-γ were determined using Human Interferon gamma ELISA Kit (ab46048; Abcam, Cambridge, MA, USA).

Techniques: Derivative Assay, Expressing, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Knockdown, Quantitative RT-PCR, Control, Infection

Journal: International Journal of Biological Sciences

Article Title: CD44 3'-Untranslated Region Functions as a Competing Endogenous RNA to Enhance NK Sensitivity of Liver Cancer Stem Cell by Regulating ULBP2 Expression

doi: 10.7150/ijbs.35216

Figure Lengend Snippet: CD44 regulated ULBP2 expression, which then further influenced the susceptibility of CSCs to NK cell mediated cytotoxicity. (A ) MICA/B, ULBP1-3, CD48, CD112, CD155, and ICAM1 transcript levels of CD44 high iCSC/CD44 int iCSC stably expressing dCas9-KRAB and sgRNA against CD44 (CD44-knockdown, CD44-kd) were analyzed by qRT-PCR. Levels are represented relative to those found in control-infected cells as means ± SD (n=3) (**: p < 0.01). (B) CD44 and ULBP2 protein levels of identical cells (Fig. A) were analyzed by Western blotting. β-actin served as a loading control. (C) CD44 high iCSC and CD44 int iCSC were used in the 4 hours NK cell cytotoxicity assay with NK-92 cells at different E:T ratios. Assays were performed either in the presence of M311 mAb (anti-ULBP2) or control IgG. Data are showed in means ± SD from three independent experiments which are performed in triplicate (**: p < 0.01). (D) IFN-γ release in the supernatants of cytotoxic assay (Fig. C, E:T ratio = 15:1) was determined by ELISA. Data were presented as mean ± SD (n=3) (**: p<0.01).

Article Snippet: Concentrations of secreted IFN-γ were determined using Human Interferon gamma ELISA Kit (ab46048; Abcam, Cambridge, MA, USA).

Techniques: Expressing, Stable Transfection, Knockdown, Quantitative RT-PCR, Control, Infection, Western Blot, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cell Communication and Signaling

Article Title: METTL13 facilitates cell growth and metastasis in gastric cancer via an eEF1A/HN1L positive feedback circuit

doi: 10.1007/s12079-022-00687-x

Figure Lengend Snippet: METTL13 modulated HN1L expression in GC. a Genes that showed positively association with METTL13 were surveyed by COEXPEDIA database ( http://www.coexpedia.org/ ). qRT-PCR was performed to test the expression of METTL13 and HN1L in METTL13 knockdown cells ( b ) and METTL13 overexpression cells ( c ). d HN1L or METTL13 mRNA expression was upregulated in GC tissues than that in the normal tissues based on GEPIA online analysis for TCGA database. e Positive correlation between METTL13 and HN1L expression at mRNA levels were analyzed using GEPIA online tools. f Western blotting was applied to test HN1L protein level upon METTL13 knockdown or overexpression in GC cells. g CCK-8 assays showed that cell viability caused by overexpression of METTL13 was partially attenuated by introducing siRNA targeting HN1L into SCG7901 cells. Data are given as mean ± SD. * P < 0.05; ** P < 0.01

Article Snippet: The primary antibodies used in this study are as follows: METTL13 (#ab186002, Abcam), HN1L (#ab200571, Abcam), eEF1A (#2551, Cell Signaling Technology), β-actin (#81178, Santa Cruz Biotechnology).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, CCK-8 Assay

Journal: Journal of Cell Communication and Signaling

Article Title: METTL13 facilitates cell growth and metastasis in gastric cancer via an eEF1A/HN1L positive feedback circuit

doi: 10.1007/s12079-022-00687-x

Figure Lengend Snippet: eEF1A was involved in the regulation of HN1L by METTL13. a Knockdown efficacy for eEF1A1 or eEF1A2 was verified by qRT-PCR in AGS and MGC803 cells. b HN1L expression was analyzed by qRT-PCR in eEF1A1 or eEF1A2 knockdown cells. c Mutation site of eEF1A1/eEF1A2. d mRNA level of eEF1A1 or eEF1A2 was tested by qRT-PCR in SGC7901 cells transfected with pENTER-eEF1A1 or pENTER-eEF1A2 plasmid. And overexpression of eEF1A1 or eEF1A2 led to expression upregulation of HN1L by qRT-PCR analysis. No obvious change of HN1L expression was observed between wild type eEF1A and eEF1A K55A mutant cells. e Western blotting indicated that HN1L was decreased when eEF1A1 or eEF1A2 was silenced. f Western blot results were shown, indicating both wild-type and K55A mutant eEF1A upregulated HN1L expression. g Knockdown of eEF1A could partially offset the upregulation of HN1L in METTL13-overexpressed cells. Data are given as mean ± SD. * P < 0.05; ** P < 0.01

Article Snippet: The primary antibodies used in this study are as follows: METTL13 (#ab186002, Abcam), HN1L (#ab200571, Abcam), eEF1A (#2551, Cell Signaling Technology), β-actin (#81178, Santa Cruz Biotechnology).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Over Expression, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mesenchymal stem cells protect breast cancer cells through regulatory T cells: role of mesenchymal stem cell-derived TGF-beta.

doi: 10.4049/jimmunol.0903143

Figure Lengend Snippet: FIGURE 4. Granzyme B analysis and effector cell phenotypes. A, PBMCs were cocultured with gamma-irradiated T47D and/or gamma-irradiated MSCs. After 5 d, PBMCs were lysed, and Western blots for granzyme B were performed. Membranes were stripped and reprobed for b-actin. B, Granzyme B ELISA was performed on supernatants from 5-d conditioned media. Calculations were performed on a per-cell basis, using a standard curve of absorbance at 450 nm versus granzyme B concentration. pp # 0.05 versus T47D alone. C, Flow cytometry was performed for effector cell phenotypes by cell surface staining with CD8-FITC for CTLs (left panels) and CD56-APC for NK cells (right panels). Data are shown as overlays with comparison against isotype controls. Figures represent studies with n = 4. Each experiment was performed with cells from a different donor.

Article Snippet: Human Granzyme B ELISA Kit was purchased from Abcam (Cambridge, MA).

Techniques: Irradiation, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Flow Cytometry, Staining, Comparison