Journal: bioRxiv

Article Title: Hypoimmunogenic human motor neurons induced from iPSCs in vivo substantially ameliorate ALS disease in large animal models

doi: 10.1101/2025.09.03.673895

Figure Lengend Snippet: A) Schematic diagram of engineered HIP-NILB-iPSCs created by BioRender. ( B) Immunofluorescence staining of NANOG, OCT4, SOX2 and SSEA4 in WT iPSCs and HIP-NILB-iPSCs. Scale bar, 100 μm. (C) H&E staining of three germ layers in WT iPSCs and HIP-NILB-iPSCs-derived teratoma. Framed areas represent different germ layers regions that were enlarged in I-III. Scale bar, 500 μm or 50 μm. (D) Apoptosis assay of HIP-NILB-iPSCs and its quantification. WT iPSCs were used as a control. (E) Immunofluorescence staining of MAP2 and ChAT in HIP-NILB-iPSCs induced MNs (HIP-NILB-iPSCs-MNs). NILB-iPSCs induced MNs (NILB-iPSCs-MNs) were used as a control. Scale bar, 100 μm. (F) HLA class I and II expression in NILB-iPSCs-MNs and HIP-NILB-iPSCs-MNs with or without IFN-γ treatment. Isotype is a negative control with matched primary antibody. (G) PBMCs cytotoxicity against HIP-NILB-iPSCs-MNs by measuring LDH release. PBMCs cocultured with NILB-iPSCs-MNs were used as a control for G-I. (H) ELISA assay for IFN-γ secretion in PBMCs cocultured with HIP-NILB-iPSCs-MNs. (I) CFSE analysis of T cells (CD3 + ) proliferation in PBMCs when cocultured with HIP-NILB-iPSCs-MNs. PBMCs is negative control (Neg.), PBMCs activated by PHA is positive control (Pos.). (J) Primary NK cells cytotoxicity against HIP-NILB-iPSCs-MNs by measuring LDH release. NK cells cocultured with NILB-iPSCs-MNs were used as a control for J-L. (K) ELISA assay for IFN-γ secretion in NK cells cocultured with HIP-NILB-iPSCs-MNs. (L) NK degranulation assay by quantifying CD107a surface expression in CD56 + NK cells cocultured with HIP-NILB-iPSCs-MNs. NK cells, Neg., NK cells treated with PHA, Pos.. (M) The phagocytic activity of macrophages against NILB-iPSCs-MNs and HIP-NILB-iPSCs-MNs is characterized by Deep Red Cell Tracker-labeled THP-1 derived M1 macrophages. (N) Inflammatory-factor array indicates low inflammatory responses of HIP-NILB-iPSCs upon LPS stimulation. (O) BLI signals over time for NILB-iPSCs, HIP-NILB-iPSCs without or with Dox-treatment (HIP-NILB-iPSCs +Dox) engrafted in allogeneic humanized-PBMCs NCG mice (n=5). HIP-NILB-iPSCs +Dox group represents 12 hours pre-induction of Dox in vitro prior to transplantation, and continued 3 days Dox induction in vivo after transplantation. Experiments were independently repeated at least three times. Data are presented as mean ± SEM, p values were determined using a two-tailed, unpaired Student’s t-test (g, h, i, j, k, l, m) or Welch’s t-test (D), * p < 0.05, ** p < 0.01, *** p < 0.001, N.S, not significant.

Article Snippet: The supernatants were collected and a human IFN-γ ELISA Kit (MultiSciences, EK180) was used to measure the IFN-γ secretion.

Techniques: Immunofluorescence, Staining, Derivative Assay, Apoptosis Assay, Control, Expressing, Negative Control, Enzyme-linked Immunosorbent Assay, Positive Control, Degranulation Assay, Activity Assay, Labeling, In Vitro, Transplantation Assay, In Vivo, Two Tailed Test

Journal: bioRxiv

Article Title: Hypoimmunogenic human motor neurons induced from iPSCs in vivo substantially ameliorate ALS disease in large animal models

doi: 10.1101/2025.09.03.673895

Figure Lengend Snippet: (A) HLA class I and II expression in HIP-NILB-iPSCs with or without IFN-γ treatment. Isotype is a negative control with matched primary antibody. (B) PBMCs cytotoxicity against HIP-NILB-iPSCs by measuring LDH release. (C) ELISA assay for IFN-γ secretion in PBMCs cocultured with HIP-NILB-iPSCs. (D) CFSE analysis of T cells (CD3 + ) proliferation in PBMCs when cocultured with HIP-NILB-iPSCs. PBMCs is negative control (Neg.), PBMCs activated by PHA is positive control (Pos.). (E) Primary NK cells cytotoxicity against HIP-NILB-iPSCs by measuring LDH release. (F) ELISA assay for IFN-γ secretion in Primary NK cells cocultured with HIP-NILB-iPSCs. (G) NK degranulation assay by quantifying CD107a surface expression in CD56 + NK cells cocultured with HIP-NILB-iPSCs. NK cells alone as Neg., NK cells treated with PHA as Pos.. (H) Activation of HMC3 cells (CD68 + ) in IBA1 + when incubated with HIP-NILB-iPSCs. HMC3 alone as Neg., LPS-stimulated HMC3 as Pos.. (I) HMC3 cells cytotoxicity against HIP-NILB-iPSCs by measuring LDH release. (J) The phagocytic activity of THP-1 derived macrophages against HIP-NILB-iPSCs is characterized by the percentage of double positive of Deep Red and FITC. (K) Gene set enrichment analysis (GSEA) shows the enriched signaling pathway in HIP-NILB-iPSCs against NILB-iPSCs. NES represents the normalized enrichment score. All experiments were independently duplicated three times. Data are mean ± SEM, p values were determined using a two-tailed, unpaired Student’s t-test (b, c, e, f, g, h, i, j) or Welch’s t-test (D), * p < 0.05, ** p < 0.01, *** p < 0.001, N.S, not significant.

Article Snippet: The supernatants were collected and a human IFN-γ ELISA Kit (MultiSciences, EK180) was used to measure the IFN-γ secretion.

Techniques: Expressing, Negative Control, Enzyme-linked Immunosorbent Assay, Positive Control, Degranulation Assay, Activation Assay, Incubation, Activity Assay, Derivative Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Hypoimmunogenic human motor neurons induced from iPSCs in vivo substantially ameliorate ALS disease in large animal models

doi: 10.1101/2025.09.03.673895

Figure Lengend Snippet: (A) Schematic representation of the experiment design created by BioRender. (B) Representative images of merged GFP and hNuclei labelling HIP-NILB-iPSCs-derivatives in the lumbar, thoracic, cervical spinal cords and motor cortex of three HIP-NILB-iPSCs treated pigs. Scale bar, 50 μm. (C) Quantification of the ratio of GFP + hNuclei + cells to total cells (DAPI + ) in the lumbar, thoracic and cervical spinal cords of three HIP-NILB-iPSCs treated pigs (n=3). (D) Representative images show HIP-NILB-iPSCs-derivatives surround the central canal in the lumbar spinal cord of pig-2# (3 months after grafting), which were present in all three treated pigs. Scale bar, 100 μm. (E) Representative images show HIP-NILB-iPSCs-derivatives predominantly localized in the ventral horn (Ⅰ) rather than in the dorsal horn (Ⅱ), and white matter (Ⅲ) of the lumbar spinal cord of pig-2#, which were present in all three treated pigs. Scale bars, 500 μm or 50 μm. (F) Representative immunofluorescence images show expression of MAP2 and ChAT in HIP-NILB-iPSCs-derivatives located in ventral horn of the lumbar spinal cord of pig-2#, which were present in all three treated pigs. Scale bar, 50 μm. (G) Nissl staining of pig lumbar spinal cord in WT, ALS and HIP-NILB-iPSCs treated pigs and magnified images for the ventral horn area. Scale bars, 2 mm or 100 μm. (H) Quantification of neuron number in unilateral ventral horn of WT, ALS and HIP-NILB-iPSCs treated pigs (n=3). (I) Western blot analysis of NeuN in the lumbar spinal cord of WT, ALS and HIP-NILB-iPSCs treated ALS pigs (n=3). (J) Quantitation of STEM121 + ChAT + human MNs and STEM121 - ChAT + pig MNs cell numbers in the lumbar spinal cord of WT, ALS and HIP-NILB-iPSCs treated pigs (n=3). (K) Representative immunofluorescence images displaying nuclear localization of TDP-43 in TUJ1 + neurons in HIP-NILB-iPSCs treated pig-2#, which were present in all three treated pigs. WT and ALS pigs were used as controls. Scale bar, 50 μm. (L) ELISA of serum glutamate in WT, ALS and HIP-NILB-iPSCs-treated pigs (n=3). (M) Representative H&E and Masson staining show mild atrophy and fibrosis in gastrocnemius from hindlimbs of pig-2#, which were present in all three treated pigs. WT and ALS pigs were used as controls. Scale bar, 100 μm. (N) Creatine kinase activity in the serum of WT, ALS and HIP-NILB-iPSCs treated pigs (n=3). (O) Representative EMG signals showing spontaneous activities in gastrocnemius muscle of WT, ALS and HIP-NILB-iPSCs treated pigs (n=3). Statistical analysis was performed with one-way ANOVA followed by Tukey’s multiple comparisons. Data was mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, N.S, not significant.

Article Snippet: The supernatants were collected and a human IFN-γ ELISA Kit (MultiSciences, EK180) was used to measure the IFN-γ secretion.

Techniques: Immunofluorescence, Expressing, Staining, Western Blot, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: bioRxiv

Article Title: Hypoimmunogenic human motor neurons induced from iPSCs in vivo substantially ameliorate ALS disease in large animal models

doi: 10.1101/2025.09.03.673895

Figure Lengend Snippet: (A) Nissl staining of lumbar spinal cord in WT, ALS and HIP-NILB-iPSCs treated rabbits, and representative magnified images for ventral horn area (n=3). Scale bars, 1 mm or 100 μm. (B) Quantifications of gray to white matter ratio and average neuron number in the unilateral ventral horn (n=3). (C) Western blot analysis of NeuN expression in spinal cords of WT, ALS and HIP-NILB-iPSCs treated rabbits (n=3). (D) Quantitation of STEM121 + ChAT + human MNs and STEM121 - ChAT + rabbit MNs cell numbers in the lumbar spinal cord of three HIP-NILB-iPSCs treated rabbits. WT and ALS rabbits were used as controls. (E) Representative immunofluorescence images displaying nuclear localization of TDP-43 in TUJ1 + neurons in HIP-NILB-iPSCs treated. WT and ALS rabbits were used as controls. Scale bar, 50 μm. (F) Western blot analysis of the soluble and insoluble TDP-43 in supernatants and pellets of rabbit lumbar spinal cords. (G) ELISA assay for serum glutamate concentration in WT, ALS and HIP-NILB-iPSCs treated rabbits (n=3). (H) Representative H&E and Masson staining images showing the atrophy and fibrosis of gastrocnemius muscle in WT, ALS and HIP-NILB-iPSCs treated rabbits (n=3). Scale bar, 100 μm. (I) Creatine kinase activity in the serum of WT, ALS and HIP-NILB-iPSCs treated rabbits (n=3). (J) Representative EMG signals showing spontaneous activities in gastrocnemius muscle of WT, ALS and HIP-NILB-iPSCs treated rabbits. (K) Gait analysis and quantification of stride and sway length in hind limbs of WT (n=4), ALS (n=5) and HIP-NILB-iPSCs treated rabbits (n=5) at two months after grafting. (L) Analysis of tension index of hind limbs over time in WT (n=4), ALS (n=3) and HIP-NILB-iPSCs treated rabbits (n=3). The statistics analysis was performed with two-way ANOVA followed by Tukey’s multiple comparisons, and the significant differences were labeled between ALS and HIP-NILB-iPSCs group in different time points. (M) Representative immunofluorescence staining of CHRNA7, SYN, Vimentin and MBP in lumbar spinal cord of WT (n=3), ALS (n=4) and HIP-NILB-iPSCs treated rabbits (n=3), and their quantifications. Scale bar, 100 μm. (N) Representative immunofluorescence staining of NLRP3, IBA1 and GFAP in lumbar spinal cord of WT (n=3), ALS (n=4) and HIP-NILB-iPSCs treated rabbits (n=3), and their quantifications. Scale bars, 100 μm or 50 μm. (O-P) (O) Western blot analysis and (P) Real-time PCR analysis of CHRNA7, SYN, Vimentin, MBP, NLRP3, IBA1, GFAP in lumbar spinal cords of WT (n=3), ALS (n=3) and HIP-NILB-iPSCs treated rabbits (n=3). (Q-R) Real-time PCR analysis of (Q) neurotrophic factors ( NTF3 , NTF4 , NGF , BDNF ) and (R) inflammatory factors ( IFNG , IL17A , CD86 , CD204 and TGFB2 ) in lumbar spinal cords of WT (n=3), ALS (n=3) and HIP-NILB-iPSCs treated rabbits (n=3). The statistical data were analyzed by one-way ANOVA followed by Dunnett’s T3 multiple comparisons (N-NLRP3) or Tukey’s multiple comparisons (b, d, g, i, k, m, n-IBA1, n-GFAP, p, q, r) unless mentioned otherwise. Data was mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, N.S, not significant.

Article Snippet: The supernatants were collected and a human IFN-γ ELISA Kit (MultiSciences, EK180) was used to measure the IFN-γ secretion.

Techniques: Staining, Western Blot, Expressing, Quantitation Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Concentration Assay, Activity Assay, Labeling, Real-time Polymerase Chain Reaction

Journal: Asian Journal of Pharmaceutical Sciences

Article Title: Bone mesenchymal stem cell-derived exosomes involved co-delivery and synergism effect with icariin via mussel-inspired multifunctional hydrogel for cartilage protection

doi: 10.1016/j.ajps.2023.100799

Figure Lengend Snippet: Cytoprotective effects of ICA@Exos for cartilage repair. (A) The proliferation promoting effects of ICA with different concentrations. (B) The proliferation promoting effects of ICA, BMSC-Exos and ICA@Exos. (C) The migration promoting effects of ICA, BMSC-Exos and ICA@Exos. (D) The detection of MMP13 content in the inflammation-induced SW1353 cell supernatant treated with ICA, BMSC-Exos and ICA@Exos by ELISA kits. (E) The detection of MMP13 content in the inflammation-induced SW1353 cells treated with ICA, BMSC-Exos and ICA@Exos by WB. (F) The statistical graphs of WB results by Image J. Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001 and # P < 0.05.

Article Snippet: MMP13 in the supernatants was measured quantitatively by ELISA Kit (MultiSciences, China) according to the manufacturer's instructions.

Techniques: Migration, Enzyme-linked Immunosorbent Assay

Journal: Allergy, Asthma & Immunology Research

Article Title: The TL1A-DR3 Axis in Asthma: Membrane-Bound and Secreted TL1A Co-Determined the Development of Airway Remodeling

doi: 10.4168/aair.2022.14.2.233

Figure Lengend Snippet: (A and B) TL1A expression profile graph in GSE18965 and differential expression analysis between asthmatics patients and healthy controls. (C) Expression of TL1A by immunohistochemical collagen levels by Masson staining in controls and subjects with asthma. Original magnification ×400. (D) Quantification of TL1A staining and collagen volume fraction between 2 groups. (E and F ) Protein and mRNA expression of TL1A in lung tissue of mice 3 days after TL1A or PBS treatment. (G and H ) Immunofluorescence detection of TL1A protein. (I) Expression of soluble TL1A in induced sputum detected by ELISA. (H) Expression of soluble TL1A in BALF detected by ELISA in each group (n = 10). Data are expressed as mean ± standard deviation. TL1A, tumor necrosis factor ligand-related molecule 1A; PBS, phosphate buffered saline; BALF, bronchoalveolar lavage fluid. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the corresponding group.

Article Snippet: The ELISA kits used were as follows: Mouse IL-4 ELISA Kit (70-EK204/2-96; MultiSciences, Hangzhou, China), Mouse IL-5 ELISA Kit (70-EK105-96; MultiSciences), Mouse IL-13 ELISA Kit (70-EK204/2-96, 70-EK213/2-96; MultiSciences), Mouse IL-4 ELISA Kit, Mouse TNFSF15 ELISA Kit (ZC-55212; ZCI BIO, Shanghai, China), and Human TNFSF15 ELISA Kit (ZC-35762; ZCI BIO).

Techniques: Expressing, Quantitative Proteomics, Immunohistochemical staining, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Standard Deviation, Saline

Journal: Allergy, Asthma & Immunology Research

Article Title: The TL1A-DR3 Axis in Asthma: Membrane-Bound and Secreted TL1A Co-Determined the Development of Airway Remodeling

doi: 10.4168/aair.2022.14.2.233

Figure Lengend Snippet: (A-D) H&E and Masson staining were used to observe histopathological changes after TL1A treatment in the mouse model of asthma. Histopathological lesions were analyzed quantitatively. (E-G) Protein expression of fibrosis indicator (collagen I and α-SMA) after treatment and subsequent quantitative analysis. (H-I) Immunofluorescence detection of fibrosis “indicator” proteins and their quantitative analyses. (J) Expression of IL-4, IL-5, and IL-13 in the blood detected by ELISA. (K) AHR was measured by airway reactivity to increased doses of inhaled methacholine treatment. Administration of TL1A can increase methacholine-induced AHR. TL1A, tumor necrosis factor ligand-related molecule 1A; OVA, ovalbumin; H&E, hematoxylin and eosin; α-SMA, α-smooth muscle actin; IL, interleukin; AHR, airway hyper-responsiveness; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. * P < 0.05, ** P < 0.01.

Article Snippet: The ELISA kits used were as follows: Mouse IL-4 ELISA Kit (70-EK204/2-96; MultiSciences, Hangzhou, China), Mouse IL-5 ELISA Kit (70-EK105-96; MultiSciences), Mouse IL-13 ELISA Kit (70-EK204/2-96, 70-EK213/2-96; MultiSciences), Mouse IL-4 ELISA Kit, Mouse TNFSF15 ELISA Kit (ZC-55212; ZCI BIO, Shanghai, China), and Human TNFSF15 ELISA Kit (ZC-35762; ZCI BIO).

Techniques: Staining, Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: A Network Pharmacology-Based Investigation into the Mechanism of Quercetin Combined with Rosuvastatin in Delaying Diabetic Nephropathy via Inhibiting NRK-52E Cell Ferroptosis

doi: 10.2147/DMSO.S524983

Figure Lengend Snippet: Effect of QCT on NRK-52E cell injury in DN. ( A and B ) CCK-8 was used to detect the effect of different concentrations of QCT (0, 6.25, 12.5, 25, 50, 100 μg/mL) on the viability of NRK-52E cells under normal (5.5 mM glucose) ( A ) and high glucose (30 mM glucose) ( B ) conditions. ( C ) Cells were treated under normal conditions (5.5 mM glucose), high glucose (30 mM glucose), low-dose QCT (10 μg/mL) and high-dose QCT (50 μg/mL). And then, ELISA assay assessing the cellular expression levels of IL-6, TNF-α, and TGF-β in each group. ns represents no significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cytokine levels were quantified using: Secreted IL-6: Human IL-6 ELISA Kit (Elabscience, E-EL-R0015c, sensitivity 9.38 pg/mL, intra-assay CV <8%); Intracellular TGF-β: Cells lysed with RIPA buffer (Beyotime, P0013B); Rat TGF-β1 ELISA Kit (Cusabio, CSB-E04727r, detection limit 15.6 pg/mL); TNF-α: Rat TNF-α ELISA Kit (MultiSciences, 70-EK382RB, linear range 4.68–300 pg/mL).

Techniques: CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: A Network Pharmacology-Based Investigation into the Mechanism of Quercetin Combined with Rosuvastatin in Delaying Diabetic Nephropathy via Inhibiting NRK-52E Cell Ferroptosis

doi: 10.2147/DMSO.S524983

Figure Lengend Snippet: Effect of RSV on NRK-52E cell injury in DN. ( A and B ) CCK-8 was used to detect the effect of different concentrations of RSV (0, 6.25, 12.5, 25, 50, 100 μg/mL) on the viability of NRK-52E cells under normal (5.5 mM glucose) ( A ) and high glucose (30 mM glucose) ( B ) conditions. ( C ) Cells were treated under normal conditions (5.5 mM glucose), high glucose (30 mM glucose), low-dose RSV (5 μg/mL) and high-dose RSV (25 μg/mL). And then, ELISA assay assessing the cellular expression levels of IL-6, TNF-α, and TGF-β in each group. ns represents no significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cytokine levels were quantified using: Secreted IL-6: Human IL-6 ELISA Kit (Elabscience, E-EL-R0015c, sensitivity 9.38 pg/mL, intra-assay CV <8%); Intracellular TGF-β: Cells lysed with RIPA buffer (Beyotime, P0013B); Rat TGF-β1 ELISA Kit (Cusabio, CSB-E04727r, detection limit 15.6 pg/mL); TNF-α: Rat TNF-α ELISA Kit (MultiSciences, 70-EK382RB, linear range 4.68–300 pg/mL).

Techniques: CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: A Network Pharmacology-Based Investigation into the Mechanism of Quercetin Combined with Rosuvastatin in Delaying Diabetic Nephropathy via Inhibiting NRK-52E Cell Ferroptosis

doi: 10.2147/DMSO.S524983

Figure Lengend Snippet: Effect of QCT combined with RSV on NRK-52E cell ferroptosis in DN. ( A – E ) Cells were treated under high glucose (30 mM glucose), QCT (50 μg/mL) and RSV (25 μg/mL). And then, ELISA assay assessing the cellular expression levels of IL-6, TNF-α, and TGF-β in each group ( A ). Colorimetric assay was used to detect cellular SOD, MDA and iron ion levels ( B – D ); qRT-PCR was used to detect cellular GPX4 and SLC7A11 expression in each group of NRK-52E cell ( E ). ns represents no significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cytokine levels were quantified using: Secreted IL-6: Human IL-6 ELISA Kit (Elabscience, E-EL-R0015c, sensitivity 9.38 pg/mL, intra-assay CV <8%); Intracellular TGF-β: Cells lysed with RIPA buffer (Beyotime, P0013B); Rat TGF-β1 ELISA Kit (Cusabio, CSB-E04727r, detection limit 15.6 pg/mL); TNF-α: Rat TNF-α ELISA Kit (MultiSciences, 70-EK382RB, linear range 4.68–300 pg/mL).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Colorimetric Assay, Quantitative RT-PCR

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: METTL3/METTL14 Transactivation and m 6 A-Dependent TGF-β1 Translation in Activated Kupffer Cells

doi: 10.1016/j.jcmgh.2021.05.007

Figure Lengend Snippet: Global m 6 A modification level is increased in the HFD rat model. ( A ) Serum endotoxin concentration was measured by Endotoxin Test Kit; n = 6; mean ± SD; Student’s t test; ∗ P < .05. ( B , C ) (Liver inflammatory cytokines mRNA expression and concentrations were measured by qPCR and enzyme-linked immunosorbent assay; n = 6; mean ± SD; Student’s t test; ∗ P < .05. ( D ) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in plasma were measured by biochemical automatic analyzer; n = 6; mean ± SD; Student’s t test; ∗ P < .05. ( E ) Inflammatory NF-κB pathway–related protein content was detected by Western blot; n = 3; mean ± SD; Student’s t test; ∗ P < .05. ( F ) Sirius Red (red area) and Masson (blue area) staining in rat liver during HFD administration; n = 3. ( G ) Percentage in area of positive staining for Sirius Red. Positive area was quantified using ImageJ software; n = 3; mean ± SD; Student’s t test; ∗ P < .05. ( H ) Liver fibrosis–related proteins content were measured by Western blot; n = 3; mean ± SD; Student’s t test; ∗ P < .05. ( I ) Collagen Ⅰ expression was assessed by immunofluorescence. Nucleus was stained with DAPI; magnification = 200×; n = 3. ( J , K ) Global m 6 A level of rat liver mRNA was detected by dot blot and high-performance liquid chromatography with tandem mass spectrometry; n = 3; mean ± SD; Student’s t test; ∗ P < .05. ( L ) METTL3, METTL14, and fat mass and obesity–associated protein (FTO) protein content in rat liver during HFD treatment and statistics; n = 6; mean ± SD; Student’s t test; ∗ P < .05. α-SMA, α-smooth muscle actin; CON, control diet with 10% of energy from fat.

Article Snippet: IL-1β, IL-6, TNF-α, and TGF-β1 concentrations in the liver and in cell culture supernatant were quantified using enzyme-linked immunosorbent assay kits purchased from Multisciences (Hangzhou, China) according to the manufacturer’s instructions.

Techniques: Modification, Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Software, Immunofluorescence, Dot Blot, High Performance Liquid Chromatography, Mass Spectrometry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: METTL3/METTL14 Transactivation and m 6 A-Dependent TGF-β1 Translation in Activated Kupffer Cells

doi: 10.1016/j.jcmgh.2021.05.007

Figure Lengend Snippet: Global m 6 A modification level is increased in activated KCs. ( A ) IL-6 and TGF-β1 content in KC supernatant were measured by enzyme-linked immunosorbent assay; n = 6; mean ± SD; Student’s t test; ∗ P < .05. ( B ) TGF-β1 expression was assessed by immunofluorescence. Nucleus was stained with DAPI; magnification = 40×; n = 3. ( C ) RNA-sequencing Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of transcripts expression compared CON with LPS; n = 2. ( D–F ) RNA-sequencing (n = 2) and qPCR (n = 3) show that compared with TGF-β2 and TGF-β3, IL-1β/IL-6/TNF-α/TGF-β1 were significantly upregulated in LPS group; mean ± SD; Student’s t test; ∗ P < .05. ( G , H ) Global m 6 A level of activated KCs was detected by dot blot and high-performance liquid chromatography with tandem mass spectrometry; n = 3; mean ± SD; Student’s t test; ∗ P < .05. I, METTL3, METTL14, and fat mass and obesity–associated protein (FTO) protein content in activated KCs during LPS treatment and statistics; n = 3; mean ± SD; Student’s t test; ∗ P < .05.

Article Snippet: IL-1β, IL-6, TNF-α, and TGF-β1 concentrations in the liver and in cell culture supernatant were quantified using enzyme-linked immunosorbent assay kits purchased from Multisciences (Hangzhou, China) according to the manufacturer’s instructions.

Techniques: Modification, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Staining, RNA Sequencing Assay, Dot Blot, High Performance Liquid Chromatography, Mass Spectrometry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: METTL3/METTL14 Transactivation and m 6 A-Dependent TGF-β1 Translation in Activated Kupffer Cells

doi: 10.1016/j.jcmgh.2021.05.007

Figure Lengend Snippet: METTL3/METTL14 promote TGF-β1 translation in an m 6 A catalytic activity–dependent manner. ( A ) Protein expression of METTL3/METTL14 in METTL3/METTL14 knockdown KCs; n = 3; mean ± SD; 2-way analysis of variance followed by Tukey’s test for multiple comparisons; ∗ P < .05. ( B ) m 6 A modification was assessed by slot blot; n = 6. ( C ) Western blot was performed to detect TGF-β1 and phosphorylated p65 protein content in METTL3/METTL14 knockdown cells; n = 3; mean ± SD; 2-way analysis of variance followed by Tukey’s test for multiple comparisons; ∗ P < .05. ( D ) TGF-β1 content in KC supernatant was measured by enzyme-linked immunosorbent assay kit; n = 3; mean ± SD; 2-way analysis of variance followed by Tukey’s test for multiple comparisons; ∗ P < .05. ( E , F ) Protein content of CD68 and TGF-β1 were detected by immunofluorescence. DAPI was used for nuclear staining. Cells analyzed with a confocal laser scanning microscope (40×magnification) with Z-scan analysis. ( G ) Protein expression of TGF-β1 in METTL3 overexpression KCs; n = 3; mean ± SD; Student’s t test; ∗ P < .05. ( H ) Stellate cells (JS1) were cultured with conditioned medium from LPS-activated WT or METTL3/METTL14 knockdown KCs. n = 3; mean ± SD; 2-way analysis of variance followed by Tukey’s test for multiple comparisons; ∗ P < .05.

Article Snippet: IL-1β, IL-6, TNF-α, and TGF-β1 concentrations in the liver and in cell culture supernatant were quantified using enzyme-linked immunosorbent assay kits purchased from Multisciences (Hangzhou, China) according to the manufacturer’s instructions.

Techniques: Activity Assay, Expressing, Modification, Dot Blot, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Laser-Scanning Microscopy, Over Expression, Cell Culture

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: METTL3/METTL14 Transactivation and m 6 A-Dependent TGF-β1 Translation in Activated Kupffer Cells

doi: 10.1016/j.jcmgh.2021.05.007

Figure Lengend Snippet: METTL14 cKO blocks LPS-induced TGF-β1 upregulation. ( A ) Hematoxylin and eosin staining in METTL14 cKO mice liver when LPS administration; n = 3. ( B ) Primary KCs was isolated from treatment with or without LPS in METTL14 cKO and WT mice, METTL14 and TGF-β1 expression were measured by Western blot; n = 3; mean ± SD; 2-way analysis of variance followed by Tukey’s test for multiple comparisons; ∗ P < .05. ( C ) TGF-β1 concentrations in the liver was measured by enzyme-linked immunosorbent assay kit; n = 4; mean ± SD; 2-way analysis of variance followed by Tukey’s test for multiple comparisons; ∗ P < .05. ( D , E ) Validation of m 6 A modification in TGF-β1 using SELECT when treatment with LPS in METTL14 cKO mice; n = 3; mean ± SD; Student’s t test; ∗ P < .05. ( F ) Sirius Red staining in METTL14 cKO mice liver when CCl4 administration; n = 3. ( G ) Primary KC METTL14 and TGF-β1 expression were detected by Western blot; n = 3; mean ± SD; 2-way analysis of variance followed by Tukey’s test for multiple comparisons; ∗ P < .05. ( H ) TGF-β1 concentrations in the liver was measured by enzyme-linked immunosorbent assay kit; n = 4; mean ± SD; 2-way analysis of variance followed by Tukey’s test for multiple comparisons; ∗ P < .05. ( I , J ) m 6 A modification in TGF-β1 was detected by SELECT in CCl4-treated METTL14 cKO mice; n = 3; mean ± SD; 2-way analysis of variance followed by Tukey’s test for multiple comparisons; ∗ P < .05.

Article Snippet: IL-1β, IL-6, TNF-α, and TGF-β1 concentrations in the liver and in cell culture supernatant were quantified using enzyme-linked immunosorbent assay kits purchased from Multisciences (Hangzhou, China) according to the manufacturer’s instructions.

Techniques: Staining, Isolation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Modification