Journal: Acta Biochimica et Biophysica Sinica

Article Title: Epigallocatechin-3-gallate inhibits osteogenic differentiation of vascular smooth muscle cells through the transcription factor JunB

doi: 10.3724/abbs.2024060

Figure Lengend Snippet: EGCG inhibits VSMC osteogenic differentiation in CKD mouse aortas (A) Immunofluorescence staining was used to determine the expressions of α-SMA, SM-MHC, MMP2, MMP9, ALP, and Runx2 in CKD mouse aortas. Scale bar: 20 μm. (B) MMP2, MMP9, α-SMA, and ALP expressions in mouse aortas were analyzed by western blot analysis. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ns: not significantly different between the indicated groups (n = 3 or 6).

Article Snippet: RIPA lysis buffer, a BCA protein assay kit, an EdU staining kit, an ALP activity assay kit, and a Lipofectamine 8000 kit were obtained from Beyotime (Shanghai, China).

Techniques: Immunofluorescence, Staining, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Epigallocatechin-3-gallate inhibits osteogenic differentiation of vascular smooth muscle cells through the transcription factor JunB

doi: 10.3724/abbs.2024060

Figure Lengend Snippet: EGCG inhibits mineral deposition and the osteogenic differentiation of HASMCs in vitro (A) Mineral deposition in HASMCs was assessed by Alizarin Red S staining. (B,C) Calcium content and ALP activity were measured in HASMCs. (D) Western blot analysis of MAC-related proteins in HASMCs. (E) Immunofluorescence staining was used to determine the protein expressions of α-SMA, SM-MHC, MMP2, MMP9, ALP, and Runx2. Scale bar: 20 μm. (F‒H) After treatment with EGCG (20 μM) for 2, 7, and 14 days, the mRNA expressions of α-SMA, ALPL, and Runx2 in HASMCs were determined by qRT-PCR. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ns: not significantly different between the indicated groups (n = 5).

Article Snippet: RIPA lysis buffer, a BCA protein assay kit, an EdU staining kit, an ALP activity assay kit, and a Lipofectamine 8000 kit were obtained from Beyotime (Shanghai, China).

Techniques: In Vitro, Staining, Activity Assay, Western Blot, Immunofluorescence, Quantitative RT-PCR

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Epigallocatechin-3-gallate inhibits osteogenic differentiation of vascular smooth muscle cells through the transcription factor JunB

doi: 10.3724/abbs.2024060

Figure Lengend Snippet: EGCG improves MAC by downregulating JunB (A,B) Schematic diagrams of the experimental design for HASMCs with JunB overexpression or knockdown that was co-treated with EGCG. (C,D) Calcium content and ALP activity were measured in HASMCs. (E) Western blot analysis of MAC-related proteins in HASMCs. (F) The results of the EdU staining assay and quantification. Representative EdU-positive cells (red) and Hoechst (blue) are shown. Scale bar: 200 μm. Data are shown as the mean ± SEM. **P < 0.01, ns: not significantly different between the indicated groups (n = 3 or 5).

Article Snippet: RIPA lysis buffer, a BCA protein assay kit, an EdU staining kit, an ALP activity assay kit, and a Lipofectamine 8000 kit were obtained from Beyotime (Shanghai, China).

Techniques: Over Expression, Knockdown, Activity Assay, Western Blot, Staining

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Epigallocatechin-3-gallate inhibits osteogenic differentiation of vascular smooth muscle cells through the transcription factor JunB

doi: 10.3724/abbs.2024060

Figure Lengend Snippet: The Ras/Raf/MEK/ERK signaling pathway is involved in the EGCG-mediated inactivation of JunB (A) Western blot analysis of JunB after treatment with EGCG and Ras/Raf/MEK/ERK signaling pathway inhibitors (PD98059 and sorafenib). (B) A schematic diagram of the experimental design of HASMCs cotreated with inhibitors. (C‒E) Western blot analysis of JunB and p-JunB in whole-cell lysates, cytoplasm, and nuclei. (F) Immunofluorescence staining was used to examine the distribution and expressions of JunB and p-JunB in HASMCs. Scale bar: 20 μm. (G) A schematic diagram of the experimental design of JunB-overexpressing HASMCs co-treated with inhibitors. (H) Calcium deposition in HASMCs was detected by Alizarin Red S staining. (I,J) Calcium content and ALP activity were measured in HASMCs. Data are shown as the mean ± SEM. **P < 0.01 (n = 5).

Article Snippet: RIPA lysis buffer, a BCA protein assay kit, an EdU staining kit, an ALP activity assay kit, and a Lipofectamine 8000 kit were obtained from Beyotime (Shanghai, China).

Techniques: Western Blot, Immunofluorescence, Staining, Activity Assay

Journal: Cell Death & Disease

Article Title: Role of microRNA-335 carried by bone marrow mesenchymal stem cells-derived extracellular vesicles in bone fracture recovery

doi: 10.1038/s41419-021-03430-3

Figure Lengend Snippet: Antibodies for western blot assay.

Article Snippet: α-SMA , ab5694, Abcam , 1:1000.

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Role of microRNA-335 carried by bone marrow mesenchymal stem cells-derived extracellular vesicles in bone fracture recovery

doi: 10.1038/s41419-021-03430-3

Figure Lengend Snippet: A Representative radiologic images of WT and CD9 −/− fracture mice after EV treatment at 0, 1, 2, and 4 weeks after EV treatment. The femurs of WT mice and CD9 −/− mice were harvested 2 weeks after fracture. B HE staining, toluidine blue staining, immunochemistry of BMP2 were performed to access bone fracture healing. C RT-qPCR, and D western blot analysis were performed to determine mRNA and protein levels of α-SMA, OCN, GDF-10, and FGF-2. Data are expressed as mean ± standard deviation. Two-way ANOVA and Tukey’s multiple comparisons test were used to determine statistical significance. * p < 0.05. n = 8 in each group.

Article Snippet: α-SMA , ab5694, Abcam , 1:1000.

Techniques: Staining, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Cell Death & Disease

Article Title: Role of microRNA-335 carried by bone marrow mesenchymal stem cells-derived extracellular vesicles in bone fracture recovery

doi: 10.1038/s41419-021-03430-3

Figure Lengend Snippet: The EVs were extracted from BMSCs transfected with miR-335 inhibitor or its negative control Mock for 48 h, and treated on WT and CD9 −/− fracture mice on the 1st and 8th days after the fracture. A Representative radiologic images of WT and CD9 −/− fracture mice at 0, 1, 2, and 4 weeks after EV treatment. B HE staining, Toluidine blue staining, immunochemistry of BMP2 were performed to observe the femoral fracture healing of WT and CD9 −/− fracture mice treated with EVs-inhibitor. C RT-qPCR and D western blot analysis were performed to determine mRNA and protein levels of α-SMA, OCN, GDF-10, and FGF-2 in femurs of mice 2 weeks after fracture. Data are expressed as mean ± standard deviation. Two-way ANOVA and Tukey’s multiple comparisons test were used to determine statistical significance. * p < 0.05. n = 8 in each group.

Article Snippet: α-SMA , ab5694, Abcam , 1:1000.

Techniques: Transfection, Negative Control, Staining, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Cell Death & Disease

Article Title: Role of microRNA-335 carried by bone marrow mesenchymal stem cells-derived extracellular vesicles in bone fracture recovery

doi: 10.1038/s41419-021-03430-3

Figure Lengend Snippet: The EVs were extracted from BMSCs and treated on MC3T3 and MG63 cells for 24 h, respectively. A MC3T3 and MG63 cell viability was determined by MTT assay. B TUNEL staining was performed to determine apoptosis of MC3T3 and MG63 cells. C Immunofluorescence staining measured the expression of F-actin in cells. D ALP activity in MC3T3 and MG63 cells was determined by ALP assay. E Alizarin red staining was performed to validate MC3T3 and MG63 bone differentiation ability. F Immunofluorescence staining measured the expression of Col I in cells. G , H RT-qPCR and western blot analysis were performed to determine mRNA and protein levels of α-SMA, OCN, GDF-10, and FGF-2. Data are expressed as mean ± standard deviation. Two-way ANOVA and Tukey’s multiple comparisons test were used to determine statistical significance. * p < 0.05. Four independent experiments were performed.

Article Snippet: α-SMA , ab5694, Abcam , 1:1000.

Techniques: MTT Assay, TUNEL Assay, Staining, Immunofluorescence, Expressing, Activity Assay, ALP Assay, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Cell Death & Disease

Article Title: Role of microRNA-335 carried by bone marrow mesenchymal stem cells-derived extracellular vesicles in bone fracture recovery

doi: 10.1038/s41419-021-03430-3

Figure Lengend Snippet: The EVs were extracted from BMSCs transfected with miR-335 Inhibitor or Mock for 48 h, and treated on MC3T3 and MG63 cells for 24 h. A – B MC3T3 and MG63 cell viability was determined by MTT assay. C TUNEL staining was performed to determine apoptosis of MC3T3 and MG63 cells. D ALP activity in MC3T3 and MG63 cells was determined by ALP assay. E Alizarin red staining was performed to validate MC3T3 and MG63 bone differentiation ability. F Immunofluorescence staining measured the expression of Col I in cells; G , H RT-qPCR and western blot assays were performed to determine mRNA and protein levels of α-SMA, OCN, GDF-10, and FGF-2. Data are expressed as mean ± standard deviation. Two-Way ANOVA and Tukey’s multiple comparisons test were used to determine statistical significance. * p < 0.05. Four independent experiments were performed.

Article Snippet: α-SMA , ab5694, Abcam , 1:1000.

Techniques: Transfection, MTT Assay, TUNEL Assay, Staining, Activity Assay, ALP Assay, Immunofluorescence, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Scientific Reports

Article Title: Impaired MC3T3-E1 osteoblast differentiation triggered by oncogenic HRAS is rescued by the farnesyltransferase inhibitor Tipifarnib

doi: 10.1038/s41598-025-91592-x

Figure Lengend Snippet: Overexpression of recombinant HRAS G12S and G12V mutants impaired osteoblast differentiation in MC3T3-E1 preosteoblast cell line. ( a ) Architecture of the lentivirus integration cassette harboring either HRAS wild type (WT) or G12S or G12V mutants (created with SnapGene Viewer). ( b ) The effect of recombinant HRAS WT, G12S and G12V on downstream signaling was analyzed 24 h after doxycycline (DOX) induction. Activation of MAPK and Akt pathways was analyzed by immunoblotting with specific antibodies for activating phosphorylations of Erk1/2 and Akt (Ser473 and Thr308). Total Akt and Erk1/2 blots were used as control for their phosphorylated-forms, whereas vinculin (Vcl) and GFP served as loading and recombinant protein expression controls, respectively. Immunoblot signals were quantified using BioRad ImageLab software ( n = 3 independent experiments). Original blots are presented in Supplementary Fig. 3. ( c – f ) MC3T3-E1 and -overexpressing recombinant HRAS were differentiated into osteoblasts using osteogenic induction media. ( c ) Qualitative and quantitative assessment of osteoblast differentiation 7 days after osteogenic induction. Alp activity was measured using Alp assay kit and normalized to cell viability measured by Presto Blue assay kit ( n = 3). Qualitative assessment was performed by imaging 24-well plates with Canon Sigma DG macro camera. ( d ) Qualitative assessment of mineralization by Alizarin Red S staining was performed 28 days after the induction of osteoblast differentiation ( n = 3) using Leica DMI6000 microscope at 10x magnification. ( e ) mRNA expression of osteoblast differentiation marker genes Runx2 , Sp7 , Alp , and Col1a1 7 days after osteogenic induction ( n = 3, each in triplicates). ( f ) Analyses of MAPK and Akt activation, and the expression of Runx2 osteoblast differentiation marker by immunoblotting 7 days after osteogenic induction with specific antibodies for activating phosphorylations of Erk1/2, Akt (Ser473 and Thr308) and osteoblast marker Runx2. Akt and Erk1/2 immunoblots were used as control for their phospho-forms, whereas vinculin (Vcl) and GFP served as loading and recombinant protein expression controls, respectively. Original blots are presented in Supplementary Fig. 4. Immunoblot signals from n = 3 independent experiments were quantified using BioRad Image Lab software. For quantified data, plots and statistical analyses were performed using GraphPad Prism 10. Data are represented as single data points of n = 3 independent experiments. Asterisks indicate a statistically significant difference from controls ( P < 0.05, two-way ANOVA).

Article Snippet: Membranes were blocked with 5% BSA in TBST for 1 h at room temperature followed by incubation with primary antibodies (anti-ERK1/2 [#9102, 1:1,000; Cell Signaling], anti-pERK1/2 Thr202/Tyr204 [#4370, 1:1,000; Cell Signaling], anti-AKT [#9272, 1:1,000, Cell Signaling], anti-pAKT Ser473 [#4060, 1:1,000, Cell Signaling], anti-pAKT Thr308 [#2965, 1:1,000, Cell Signaling], anti-OPN [#AF808, 1:1,000, R&D], anti-RUNX2 [#12556,1:1,000, Cell Signaling], anti-GFP [#2956, 1:1,000; Cell Signaling], anti-vinculin [#sc-73614, 1:2,000; Santa Cruz]) overnight at 4 °C and 1 h at room temperature with secondary antibodies (horseradish peroxidase-conjugated goat anti-mouse IgG [#P0447; Dako] and anti-rabbit IgG [#A6154; Invitrogen]).

Techniques: Over Expression, Recombinant, Activation Assay, Western Blot, Control, Expressing, Software, Activity Assay, ALP Assay, Imaging, Staining, Microscopy, Marker

Journal: Scientific Reports

Article Title: Impaired MC3T3-E1 osteoblast differentiation triggered by oncogenic HRAS is rescued by the farnesyltransferase inhibitor Tipifarnib

doi: 10.1038/s41598-025-91592-x

Figure Lengend Snippet: Tipifarnib rescued osteoblast differentiation in cell lines expressing recombinant HRAS mutants. ( a ) Schematic representation of MAPK pathway and inhibitors used in this study (created using Adobe Illustrator). ( b – c ) MC3T3-E1 cells kept under osteogenic induction for 7 days, in the presence or absence of DOX and Tipifarnib (0.5 µM). Immunoblotting analyses of Erk1/2 and Akt activation were performed with specific antibodies for activating phosphorylations of Erk1/2, Akt (Ser473 and Thr308, respectively). Akt and Erk1/2 immunoblots were used as control for their phospho-forms, whereas Vcl serves as loading control and GFP as control for expression of recombinant protein. Original blots are presented in Supplementary Fig. 6. Signal intensity of 3 independent experiments was quantified using BioRad Image Lab software, normalized to loading control and total protein abundance, respectively. ( b ) Expression of osteoblast specific markers Runx2 , Sp7 , Alp and Col1a1 relative to housekeeping genes was measured by qRT-PCR ( n = 3). ( c ) Immunoblotting analyses of Runx2 expression, whereas vinculin (Vcl) and GFP served as loading and recombinant protein expression controls, respectively. Immunoblot signals from n = 3 independent experiments were quantified using BioRad Image Lab software. Original blots are presented in Supplementary Fig. 7. ( a – c ) Statistical analyses were performed using GraphPad Prism 10. Data are represented as single data points of n = 3 independent experiments. Asterisks indicate a statistically significant difference from controls ( P < 0.05, two-way ANOVA). ( d ) Assessment of mineralization by Alizarin Red S staining was performed 27 days after osteogenic and DOX induction, and in the presence or absence of Tipifarnib (0.5 µM). The percentage of the well area covered with mineralized extracellular matrix was calculated using ImageJ ( n = 9 independent experiments).

Article Snippet: Membranes were blocked with 5% BSA in TBST for 1 h at room temperature followed by incubation with primary antibodies (anti-ERK1/2 [#9102, 1:1,000; Cell Signaling], anti-pERK1/2 Thr202/Tyr204 [#4370, 1:1,000; Cell Signaling], anti-AKT [#9272, 1:1,000, Cell Signaling], anti-pAKT Ser473 [#4060, 1:1,000, Cell Signaling], anti-pAKT Thr308 [#2965, 1:1,000, Cell Signaling], anti-OPN [#AF808, 1:1,000, R&D], anti-RUNX2 [#12556,1:1,000, Cell Signaling], anti-GFP [#2956, 1:1,000; Cell Signaling], anti-vinculin [#sc-73614, 1:2,000; Santa Cruz]) overnight at 4 °C and 1 h at room temperature with secondary antibodies (horseradish peroxidase-conjugated goat anti-mouse IgG [#P0447; Dako] and anti-rabbit IgG [#A6154; Invitrogen]).

Techniques: Expressing, Recombinant, Western Blot, Activation Assay, Control, Software, Quantitative Proteomics, Quantitative RT-PCR, Staining

Journal: Aging

Article Title: CRYAB suppresses ferroptosis and promotes osteogenic differentiation of human bone marrow stem cells via binding and stabilizing FTH1.

doi: 10.18632/aging.205851

Figure Lengend Snippet: Figure 1. CRYAB positively regulates the osteogenic differentiation of BMSCs. (A) mRNA expression level of CRYAB in osteoporosis samples was detected using qRT-PCR. (B, C) mRNA expression levels of CRYAB, OCN, OPN, RUNX2, and COLI when CRYAB was overexpressed or silenced were detected using qRT-PCR (n=3). (D, E) Protein expression levels of CRYAB, OCN, OPN, RUNX2, and COLI when CRYAB was overexpressed or silenced were detected using the Western blotting method (n=3). (F) The activity of ALP was detected using an Alkaline Phosphatase Assay Kit (n=3). *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: The information of antibodies was as follows: anti-OCN antibody (Proteintech, 23418-1-AP), OPN antibody (Proteintech, 22952-1-AP), RUNX2 (Abcam, UK, ab192256), COLI antibody (Proteintech, 14695-1-AP), CRYAB antibody (Proteintech, 15808-1-AP), FTH1 antibody (Cell Signaling Technology, 4393) and antiGAPDH antibody (Abcam, ab8245).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, ALP Assay

Journal: Aging

Article Title: CRYAB suppresses ferroptosis and promotes osteogenic differentiation of human bone marrow stem cells via binding and stabilizing FTH1.

doi: 10.18632/aging.205851

Figure Lengend Snippet: Figure 5. Knockdown of FTH1 induces ferroptosis and suppresses osteogenic differentiation of BMSCs. (A) The efficiency of FTH1 knockdown was validated using Western blotting method (n=3). (B, C) Cellular Fe and ROS levels were detected after FTH1 knockdown in BMSCs (n=3). (D, E) The cell viabilities were detected using CCK-8 assay (n=3). (F) mRNA expression levels of OCN, OPN, RUNX2, and COLI when FTH1 was silenced were detected using qRT-PCR (n=3). (G) The activity of ALP was detected using an Alkaline Phosphatase Assay Kit (n=3). ns: no significance; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: The information of antibodies was as follows: anti-OCN antibody (Proteintech, 23418-1-AP), OPN antibody (Proteintech, 22952-1-AP), RUNX2 (Abcam, UK, ab192256), COLI antibody (Proteintech, 14695-1-AP), CRYAB antibody (Proteintech, 15808-1-AP), FTH1 antibody (Cell Signaling Technology, 4393) and antiGAPDH antibody (Abcam, ab8245).

Techniques: Knockdown, Western Blot, CCK-8 Assay, Expressing, Quantitative RT-PCR, Activity Assay, ALP Assay

Journal: Aging

Article Title: CRYAB suppresses ferroptosis and promotes osteogenic differentiation of human bone marrow stem cells via binding and stabilizing FTH1.

doi: 10.18632/aging.205851

Figure Lengend Snippet: Figure 6. CRYAB regulates ferroptosis and osteogenic differentiation of BMSCs in an FTH1-dependent manner. (A) The protein levels of CRYAB and FTH1 were detected using a Western blotting assay (n=3). (B–D) Cellular Fe and ROS levels and cell viability were detected (n=3). (E) mRNA expression levels of OCN, OPN, RUNX2, and COLI were detected using qRT-PCR (n=3). (F) The activity of ALP was detected using an Alkaline Phosphatase Assay Kit (n=3). *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: The information of antibodies was as follows: anti-OCN antibody (Proteintech, 23418-1-AP), OPN antibody (Proteintech, 22952-1-AP), RUNX2 (Abcam, UK, ab192256), COLI antibody (Proteintech, 14695-1-AP), CRYAB antibody (Proteintech, 15808-1-AP), FTH1 antibody (Cell Signaling Technology, 4393) and antiGAPDH antibody (Abcam, ab8245).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Activity Assay, ALP Assay

Journal: Cells

Article Title: Expression of Toll-like Receptors in Stem Cells of the Apical Papilla and Its Implication for Regenerative Endodontics

doi: 10.3390/cells12202502

Figure Lengend Snippet: Expression of TLR3 and its effect on SCAP proliferation and osteogenic differentiation. ( A ) TLR3 immunoreactivity is visualized in green and found highly expressed in cultured SCAP, the cytoskeleton is visualized in red by the staining of phalloidin (Phalloid). The staining for TLR3 was absent in samples lacking the primary antibody (No 1°). ( B ) Poly (I:C) evoked a concentration and time-dependent increase in SCAP proliferation ( n = 6–9/group/time). ( C ) Activation of TLR3 by poly (I:C) reduced alkaline phosphatase activity in a concentration-dependent manner ( n = 6). ( D ) Activation of TLR3 by poly (I:C) resulted in reduced mineralization, which was reversed and increased by a TLR3 inhibitor detected by a quantitative Alizarin red assay. ns = not statistically significant; * = p < 0.05; ** = p < 0.01 and **** = p < 0.0001 by two-way ANOVA (panel B ) and one-way ANOVA (panels C , D ) with Bonferroni’s post-hoc test. Scale bar = 200 μm.

Article Snippet: Real-time RT-PCR reactions for the following targets: dentin sialophosphoprotein ( DSSP , assay Hs00171962_m1), alkaline phosphatase ( ALP , assay Hs03046558_s1), TLR3 (assay Hs01551079_g1) and 18S (assay #Hs99999901_s1) were performed using the TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific), and amplification was performed on an ABI7500 Fast Real-Time PCR System (Thermo Fisher Scientific).

Techniques: Expressing, Cell Culture, Staining, Concentration Assay, Activation Assay, Activity Assay

Journal: Cells

Article Title: Expression of Toll-like Receptors in Stem Cells of the Apical Papilla and Its Implication for Regenerative Endodontics

doi: 10.3390/cells12202502

Figure Lengend Snippet: Knockdown of TLR3 with SiRNA for 3 days reverses the inhibitory effects of poly (I:C) on differentiation into a mineralizing phenotype of SCAP culture in presence of differentiation media for an additional 7 days. ( A ) A designed siRNA against TLR3 resulted in approximately 80% reduction in mRNA expression while a control siRNA (SC, scrambled sequence) had no effect ( n = 3–4/group). ( B ) Knockdown of TLR3 reversed the inhibition of ALP mRNA expression evoked by poly (I:C) ( n = 4/group). ( C ) Knockdown of TLR3 expression by siRNA reversed poly (I:C) inhibition and increased the expression of the odontoblast marker DSPP ( n = 6/group). ns = not statistically significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001 and **** = p < 0.0001. one-way ANOVA with Bonferroni’s post-hoc test.

Article Snippet: Real-time RT-PCR reactions for the following targets: dentin sialophosphoprotein ( DSSP , assay Hs00171962_m1), alkaline phosphatase ( ALP , assay Hs03046558_s1), TLR3 (assay Hs01551079_g1) and 18S (assay #Hs99999901_s1) were performed using the TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific), and amplification was performed on an ABI7500 Fast Real-Time PCR System (Thermo Fisher Scientific).

Techniques: Knockdown, Expressing, Control, Sequencing, Inhibition, Marker

Journal: Frontiers in Cell and Developmental Biology

Article Title: H19 Promotes Osteoblastic Transition by Acting as ceRNA of miR-140-5p in Vascular Smooth Muscle Cells

doi: 10.3389/fcell.2022.774363

Figure Lengend Snippet: | H19 promotes osteogenic differentiation and mineralization of VSMCs treated with osteogenic medium. (A,B) Expression of H19, calcification marker genes Runx2 and ALP, and Satb2 during the osteogenic differentiation of VSMCs. VSMCs were treated with osteogenic medium (OM) that contains 10 mM ß -glycerophosphate ( β -GP) for 7 days to induce osteogenic differentiation. (A) Expression of H19, Runx2, ALP, and Satb2 were determined by qRT-PCR. (B) Expression of Runx2, ALP, a -SMA and Satb2 were determined by Western blotting. The data were presented as densitometric ratios normalized to ß -actin [ (B) , right panel]. (C) Alizarin Red S staining were measured in VSMCs incubated with ß -GP for 21 days. Representative microscopic views are shown. Data were presented as ratio of positive staining area (lower panel). (D–F) VSMCs were treated with OM along with either lentivirus containing full length of H19 cDNA (LvH19) or control cDNA (LvCtrl). The protein expression of Runx2, ALP, and Satb2 were determined by Western blotting in VSMCs after treatment for 2 days (D) . Data were presented as densitometric ratios normalized to GAPDH [ (D) , right panel]. The ALP activity was determined by an Elisa kit in VSMCs after treatment 2 days (E) . Alizarin Red S staining were measured in VSMCs after treatment for 21 days. Representative microscopic views are shown (F) . Scale bar represents 200 µm. Data were presented as ratio of positive staining area [ (F) , right panel]. (G–I) VSMCs were incubated with OM medium along with either H19 siRNA (siH19) or control siRNA (siCtrl). The protein expression of Runx2, ALP, and Satb2 were determined by Western blotting after treatment for 2 days (G) . Data were presented as densitometric ratios normalized to GAPDH [ (G) , right panel]. The ALP activity was determined by an Elisa kit in VSMCs after treatment for 2 days (H) . Alizarin Red S staining were measured in after treatment for 21 days. Representative microscopic views are shown (I) . Scale bar represents 200 µm. Data were presented as ratio of positive staining area [ (I) , right panel]. Each experiment was repeated for three times. The data represent the mean ± SD of triplicates. * p < 0.05.

Article Snippet: Runx2 (ab23981, 1:1,000) and ALP (ab229126, 1:1,000) antibodies were purchased from Abcam (Cambridge, United Kingdom).

Techniques: Expressing, Marker, Quantitative RT-PCR, Western Blot, Staining, Incubation, Control, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: H19 Promotes Osteoblastic Transition by Acting as ceRNA of miR-140-5p in Vascular Smooth Muscle Cells

doi: 10.3389/fcell.2022.774363

Figure Lengend Snippet: miR-140-5p antagonises osteogenic differentiation of VSMCs. (A) qRT-PCR was performed to evaluate the expression of miR-140-5p in VSMCs cultured in OM medium. (B) qRT-PCR was performed to evaluate the expression of miR-140-5p in VSMCs transfected with specific miR-140-5p mimics or inhibitor. (C) Western blotting was performed to determine the protein expression of Runx2 and ALP in VSMCs transfected with specific miR-140-5p mimics or inhibitor. The data were presented as densitometric ratios normalized to GAPDH [ (C) , lower panel]. (D) qRT-PCR was performed to determine the protein expression of Runx2 and ALP in VSMCs transfected with specific miR-140-5p mimics or inhibitor. (E) The ALP activity was evaluated by specific Elisa kits in VSMCs transfected with specific miR-140-5p mimics or inhibitor. (F) Alizarin Red S staining were measured in ß -GP-treated VSMCs transfected with specific miR-140-5p mimics or inhibitor for 21 days. Representative microscopic views are shown. Scale bar represents 200 µm. Data were presented as ratio of positive staining area [ (F) , right panel]. Each experiment was repeated for three times. The data represent the mean ± SD of triplicates. * p < 0.05.

Article Snippet: Runx2 (ab23981, 1:1,000) and ALP (ab229126, 1:1,000) antibodies were purchased from Abcam (Cambridge, United Kingdom).

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Transfection, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: H19 Promotes Osteoblastic Transition by Acting as ceRNA of miR-140-5p in Vascular Smooth Muscle Cells

doi: 10.3389/fcell.2022.774363

Figure Lengend Snippet: miR-140-5p antagonises osteogenic differentiation of VSMCs by targeting Satb2. (A) Schematic view of miR-140-5p putative binding sites in WT and MUT Satb2 3′-UTR. (B) Western blotting was performed to determine the protein expression of Satb2 in VSMCs transfected with specific miR-140-5p mimics or inhibitor. The data were presented as densitometric ratios normalized to GAPDH [ (B) , lower panel]. (C) VSMCs were transfected with luciferase report vector containing WT or MUT Satb2 3′-UTR following by transfection of miR-140-5p mimics or control mimics. Luciferase activity was determined 48 h post transfection and luciferase activity was normalised to Renilla luciferase activity. (D) Western blotting was performed to determine the protein expression of Satb2 and Runx2 in VSMCs transfected with specific miR-140-5p inhibitor with or without Satb2 siRNA. The data were presented as densitometric ratios normalized to GAPDH [ (D) , lower panel]. (E) The ALP activity was determined by an Elisa kit in VSMCs infected with inhibitor of miR-140-5p in the presence of Satb2 siRNA or not. (F) Alizarin Red S staining were measured in ß -GP-treated VSMCs infected with inhibitor of miR-140-5p in the presence of Satb2 siRNA or not for 21 days. Representative microscopic views are shown. Scale bar represents 200 µm. Data were presented as ratio of positive staining area [ (F) , right panel]. Each experiment was repeated for three times. The data represent the mean ± SD of triplicates. * p < 0.05.

Article Snippet: Runx2 (ab23981, 1:1,000) and ALP (ab229126, 1:1,000) antibodies were purchased from Abcam (Cambridge, United Kingdom).

Techniques: Binding Assay, Western Blot, Expressing, Transfection, Luciferase, Plasmid Preparation, Control, Activity Assay, Enzyme-linked Immunosorbent Assay, Infection, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: H19 Promotes Osteoblastic Transition by Acting as ceRNA of miR-140-5p in Vascular Smooth Muscle Cells

doi: 10.3389/fcell.2022.774363

Figure Lengend Snippet: Knockdown of Satb2 inhibits osteogenic differentiation of VSMCs. OM-treated VSMCs were transfected with control siRNA (siCtrl), Satb2 siRNA (siSatb2), or vehicle control for 48 h. (A) Western blotting was performed to evaluate Satb2 and Runx2 expression. The data were presented as densitometric ratios normalized to GAPDH [ (A) , right panel]. (B) The ALP activity was determined by an Elisa kit. (C) Alizarin Red S staining were measured in ß -GP-treated VSMCs infected with siCtrl or siSatb2 for 21 days. Representative microscopic views are shown. Scale bar represents 200 µm. Data were presented as ratio of positive staining area [ (C) , right panel]. CM-treated VSMCs were infected with control lentivirus (LvCtrl), Satb2-expressing lentivirus (LvSatb2), or vehicle control for 48 h. (D) Western blotting was performed to evaluate Satb2, Runx2, pERK1/2, ERK1/2, p38MAPK, and p38MAPK expression. The data were presented as densitometric ratios normalized to ß -actin or phosphorylation/total protein [ (D) , right panel]. (E) The ALP activity was determined by an Elisa kit. Each experiment was repeated for three times. The data represent the mean ± SD of triplicates. * p < 0.05.

Article Snippet: Runx2 (ab23981, 1:1,000) and ALP (ab229126, 1:1,000) antibodies were purchased from Abcam (Cambridge, United Kingdom).

Techniques: Knockdown, Transfection, Control, Western Blot, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Staining, Infection, Phospho-proteomics

Journal: Frontiers in Cell and Developmental Biology

Article Title: H19 Promotes Osteoblastic Transition by Acting as ceRNA of miR-140-5p in Vascular Smooth Muscle Cells

doi: 10.3389/fcell.2022.774363

Figure Lengend Snippet: H19 promotes osteogenic differentiation of VSMCs by sponging miR-140-5p. VSMCs were transfected with LvH19, miR-140-5p, or corresponding negative control. (A) Western blotting was performed to evaluate Satb2 and Runx2 expression. The data were presented as densitometric ratios normalized to GAPDH [ (A) , lower panel]. (B) The ALP activity was determined by an Elisa kit. (C) Alizarin Red S staining were measured in ß -GP-treated VSMCs infected with LvCtrl or LvH19 in the presence of miR-140-5p mimics or not for 21 days. Representative microscopic views are shown. Scale bar represents 200 µm. Data were presented as ratio of positive staining area [ (C) , lower panel]. Each experiment was repeated for three times. The data represent the mean ± SD of triplicates. * p < 0.05.

Article Snippet: Runx2 (ab23981, 1:1,000) and ALP (ab229126, 1:1,000) antibodies were purchased from Abcam (Cambridge, United Kingdom).

Techniques: Transfection, Negative Control, Western Blot, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Staining, Infection

Journal: Aging

Article Title: CRYAB suppresses ferroptosis and promotes osteogenic differentiation of human bone marrow stem cells via binding and stabilizing FTH1.

doi: 10.18632/aging.205851

Figure Lengend Snippet: Figure 1. CRYAB positively regulates the osteogenic differentiation of BMSCs. (A) mRNA expression level of CRYAB in osteoporosis samples was detected using qRT-PCR. (B, C) mRNA expression levels of CRYAB, OCN, OPN, RUNX2, and COLI when CRYAB was overexpressed or silenced were detected using qRT-PCR (n=3). (D, E) Protein expression levels of CRYAB, OCN, OPN, RUNX2, and COLI when CRYAB was overexpressed or silenced were detected using the Western blotting method (n=3). (F) The activity of ALP was detected using an Alkaline Phosphatase Assay Kit (n=3). *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: The information of antibodies was as follows: anti-OCN antibody (Proteintech, 23418-1-AP), OPN antibody (Proteintech, 22952-1-AP), RUNX2 (Abcam, UK, ab192256), COLI antibody (Proteintech, 14695-1-AP), CRYAB antibody (Proteintech, 15808-1-AP), FTH1 antibody (Cell Signaling Technology, 4393) and antiGAPDH antibody (Abcam, ab8245).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, ALP Assay

Journal: Aging

Article Title: CRYAB suppresses ferroptosis and promotes osteogenic differentiation of human bone marrow stem cells via binding and stabilizing FTH1.

doi: 10.18632/aging.205851

Figure Lengend Snippet: Figure 5. Knockdown of FTH1 induces ferroptosis and suppresses osteogenic differentiation of BMSCs. (A) The efficiency of FTH1 knockdown was validated using Western blotting method (n=3). (B, C) Cellular Fe and ROS levels were detected after FTH1 knockdown in BMSCs (n=3). (D, E) The cell viabilities were detected using CCK-8 assay (n=3). (F) mRNA expression levels of OCN, OPN, RUNX2, and COLI when FTH1 was silenced were detected using qRT-PCR (n=3). (G) The activity of ALP was detected using an Alkaline Phosphatase Assay Kit (n=3). ns: no significance; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: The information of antibodies was as follows: anti-OCN antibody (Proteintech, 23418-1-AP), OPN antibody (Proteintech, 22952-1-AP), RUNX2 (Abcam, UK, ab192256), COLI antibody (Proteintech, 14695-1-AP), CRYAB antibody (Proteintech, 15808-1-AP), FTH1 antibody (Cell Signaling Technology, 4393) and antiGAPDH antibody (Abcam, ab8245).

Techniques: Knockdown, Western Blot, CCK-8 Assay, Expressing, Quantitative RT-PCR, Activity Assay, ALP Assay

Journal: Aging

Article Title: CRYAB suppresses ferroptosis and promotes osteogenic differentiation of human bone marrow stem cells via binding and stabilizing FTH1.

doi: 10.18632/aging.205851

Figure Lengend Snippet: Figure 6. CRYAB regulates ferroptosis and osteogenic differentiation of BMSCs in an FTH1-dependent manner. (A) The protein levels of CRYAB and FTH1 were detected using a Western blotting assay (n=3). (B–D) Cellular Fe and ROS levels and cell viability were detected (n=3). (E) mRNA expression levels of OCN, OPN, RUNX2, and COLI were detected using qRT-PCR (n=3). (F) The activity of ALP was detected using an Alkaline Phosphatase Assay Kit (n=3). *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: The information of antibodies was as follows: anti-OCN antibody (Proteintech, 23418-1-AP), OPN antibody (Proteintech, 22952-1-AP), RUNX2 (Abcam, UK, ab192256), COLI antibody (Proteintech, 14695-1-AP), CRYAB antibody (Proteintech, 15808-1-AP), FTH1 antibody (Cell Signaling Technology, 4393) and antiGAPDH antibody (Abcam, ab8245).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Activity Assay, ALP Assay

Journal: Frontiers in Endocrinology

Article Title: Super-Enhancer-Associated Long Non-Coding RNA LINC01485 Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Regulating MiR-619-5p/RUNX2 Axis

doi: 10.3389/fendo.2022.846154

Figure Lengend Snippet: LNC01485 expression was up-regulated during osteogenic differentiation of hBMSCs. (A, C) ALP staining (A) and ALP activity assay (C) of hBMSCs before osteogenic induction and 7 days after induction. (B, D) hBMSCs were stained with Alizarin Red S (B) before osteogenic induction and at 14 days after induction, and the staining results were analyzed semi-quantitatively (D) . (E-I) The protein expression levels of RUNX2 (E, F) , COL1A1 (E, G) , OSX (E, H) , and OPN (E, I) level on Days 0, 7, 14, and 21 of osteogenic induction were detected by Western blot and quantified analysis by normalized to GAPDH. (J-O) The mRNA expression levels of RUNX2 (J) , OPN (K) , OCN (L) , COL1A1 (M) , OSX (N) , and LINC01485 (O) before and after osteogenic differentiation were determined by qRT-PCR. (P-R) Expression correlation analysis between LINC01485 and osteogenic genes RUNX2 (P) , OPN (Q) , and OCN (R) during osteogenic differentiation. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies were as follows: RUNX2 (1:1000, Cell Signaling Technology, Cat# 12556s), Osterix (1:1000, BOSTER, Cat# A02077-1), COL1A1 (1:1000, BOSTER, Cat# BA0325), OPN (1:1000, Abcam, Cat# ab8448), and GAPDH (1:20000, Proteintech, Cat# 60004–1-Ig).

Techniques: Expressing, Staining, ALP Activity Assay, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Endocrinology

Article Title: Super-Enhancer-Associated Long Non-Coding RNA LINC01485 Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Regulating MiR-619-5p/RUNX2 Axis

doi: 10.3389/fendo.2022.846154

Figure Lengend Snippet: LINC01485 regulates osteogenic differentiation of hBMSCs. (A, B) The overexpression (A) and interference (B) efficiency of LINC01485 was determined by qRT-PCR in hBMSCs after transduction with LV-LINC01485 and sh-LINC01485. (C–F) The mRNA levels of RUNX2 (C) , COL1A1 (D) , OSX (E) , and OCN (F) after 14 days of osteogenic induction in hBMSC infected with lentivirus by qRT-PCR. (G, H) Western blot analysis of the RUNX2 (G, I) , COL1A1 (G, I) , OSX (G, J) , and OPN (G, K) protein expression in hBMSCs infected with lentivirus after osteogenic induction 14 days later and the corresponding gray value quantitative analysis. (L, M) ALP staining (L) and ALP activity (M) of hBMSC cells infected with lentivirus after 7 days of osteogenic induction. (N, O) Alizarin Red S staining (N) and semi-quantitative analysis (O) of infected hBMSCs with lentivirus after 14 days osteogenic induction. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies were as follows: RUNX2 (1:1000, Cell Signaling Technology, Cat# 12556s), Osterix (1:1000, BOSTER, Cat# A02077-1), COL1A1 (1:1000, BOSTER, Cat# BA0325), OPN (1:1000, Abcam, Cat# ab8448), and GAPDH (1:20000, Proteintech, Cat# 60004–1-Ig).

Techniques: Over Expression, Quantitative RT-PCR, Transduction, Infection, Western Blot, Expressing, Staining, Activity Assay

Journal: Frontiers in Endocrinology

Article Title: Super-Enhancer-Associated Long Non-Coding RNA LINC01485 Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Regulating MiR-619-5p/RUNX2 Axis

doi: 10.3389/fendo.2022.846154

Figure Lengend Snippet: MiR-619-5p is down-regulated and inhibits osteogenesis during osteogenic induction of hBMSCs. (A) The relative expression levels of miR-619-5p before and after osteogenic differentiation were determined by qRT-PCR. (B) Correlation analysis of LINC01485 and miR-619-5p expression levels during osteogenic differentiation. (C, D) The mRNA level of miR-619-5p in hBMSCs transfected with miR-199a-5p mimic (C) and miR-199a-5p inhibitor (D) by qRT-PCR. (E–I) Western blot analysis of the RUNX2 (E, F) , COL1A1 (E, G) , OSX (E, H) , and OPN (E, I) protein expression in hBMSCs transfected with miR-619-5p mimic, miRNA mimic NC, miR-619-5p inhibitor, and miRNA inhibitor NC after osteogenic induction and the corresponding gray value quantitative analysis. (J) ALP staining analysis of hBMSCs transfected with miR-619-5p mimic, miRNA mimic NC, miR-619-5p inhibitor, and miRNA inhibitor NC after osteogenic induction. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies were as follows: RUNX2 (1:1000, Cell Signaling Technology, Cat# 12556s), Osterix (1:1000, BOSTER, Cat# A02077-1), COL1A1 (1:1000, BOSTER, Cat# BA0325), OPN (1:1000, Abcam, Cat# ab8448), and GAPDH (1:20000, Proteintech, Cat# 60004–1-Ig).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Staining

Journal: Frontiers in Endocrinology

Article Title: Super-Enhancer-Associated Long Non-Coding RNA LINC01485 Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Regulating MiR-619-5p/RUNX2 Axis

doi: 10.3389/fendo.2022.846154

Figure Lengend Snippet: LINC01485 acts as a ceRNA of miR-619-5p to regulate RUNX2 and osteogenic differentiation. (A–E) Western blot analysis of the RUNX2 (A, B) , COL1A1 (A, C) , OSX (A, D) , and OPN (A, E) protein expression in hBMSCs infected with sh-NC or sh-LINC01485 lentivirus along with miRNA inhibitor NC or miR-619-5p inhibitor after osteogenic induction and the corresponding gray value quantitative analysis. (F) ALP staining analysis of hBMSCs infected with sh-NC or sh-LINC01485 lentivirus along with miRNA inhibitor NC or miR-619-5p inhibitor after osteogenic induction. (G) ALP staining analysis of hBMSCs infected with LV-NC or LV-LINC01485 lentivirus along with miRNA mimic NC or miR-619-5p mimic after osteogenic induction. (H) Luciferase activity of RUNX2-WT upon transfection of pcDNA3.1, pcDNA3.1-LINC01485, or miR-619-5p mimic into HEK293T cells. ns, none significance. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies were as follows: RUNX2 (1:1000, Cell Signaling Technology, Cat# 12556s), Osterix (1:1000, BOSTER, Cat# A02077-1), COL1A1 (1:1000, BOSTER, Cat# BA0325), OPN (1:1000, Abcam, Cat# ab8448), and GAPDH (1:20000, Proteintech, Cat# 60004–1-Ig).

Techniques: Western Blot, Expressing, Infection, Staining, Luciferase, Activity Assay, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Macrophage Control of Incipient Bone Formation in Diabetic Mice

doi: 10.3389/fcell.2020.596622

Figure Lengend Snippet: Diabetic macrophages affect the quality of bone regeneration. (A) Representative 3D μCT images and light microscopic images of the demineralized and paraffin-embedded tissue sections of the mouse calvaria at 4 weeks post wounding stained with hematoxylin and eosin (H&E) and 3,3′-diaminobenzidine (DAB) for CD31. WT, wild-type mice; DB, db/db mice; WT+dbMO, WT mice treated with DB macrophages; DB+wtMO, DB mice treated with WT macrophages. The black arrows represent newly formed bone in the H&E images and CD31-positive cells in DAB staining images. Scale bar represents 100 μm in all images. (B) Volumetric quantitation of the 3D μCT data ( n = 6). The graph represents mean percentage bone volume regenerated to total volume of the defect ± SD. (C) The fold change represents alkaline phosphatase (ALP) activity in hMSCs treated with osteogenic medium (OS), osteogenic medium with WT macrophage conditioned medium (OS+wtCM), and osteogenic medium with conditioned medium from DB macrophages (OS+dbCM) with respect to hMSCs in growth medium at day 5 ( n = 3). *Statistical significance ( P < 0.05) as measured by Tukey's ad hoc test post ANOVA.

Article Snippet: ALP activity was quantified using Alkaline Phosphatase Assay Kit (Abcam) by measuring p -nitrophenyl ( p NP) based on the spectrophotometric absorbance at 405 nm.

Techniques: Staining, Quantitation Assay, Activity Assay

Journal: EMBO Reports

Article Title: NAT10-mediated acetylation of NIK mRNA in B cells promotes IgA production

doi: 10.1038/s44319-025-00509-2

Figure Lengend Snippet: ( A ) Sex and age of patients with IBD and healthy volunteers ( n = 30). ( B ) ELISA was used to quantify sIgA levels in serum from healthy donors and newly diagnosed patients with CD or UC ( n = 30). ( C ) NAT10 expression was analyzed by qPCR in IgA + CD19 + B cells isolated from the colonic tissues of healthy donors and newly diagnosed CD or UC patients ( n = 7). Results are shown as fold changes relative to Actb mRNA levels, normalized with Bio-Rad CFX Manager 3.1. All data are representative of biological replicates at least three independent experiments. Data are represented as the means ± SDs. The significance of differences ( B , C ) was determined using one-way ANOVA with Newman–Keuls post-hoc test. ** P < 0.01; *** P < 0.005. .

Article Snippet: NAT10 antibody (13365-1-AP) came from Proteintech, while antibodies against RelB (C-19), IKBα (C-21), p65 (C-20), ERK (K-23), phospho-ERK (E-4), JNK (C-17), p38 (H-147), p105/p50 (C-19), AKT1 (B-1), Lamin B (C-20), NIK (H248), and TRAF3 (C-20) were sourced from Santa Cruz Biotechnology.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Isolation

Journal: EMBO Reports

Article Title: NAT10-mediated acetylation of NIK mRNA in B cells promotes IgA production

doi: 10.1038/s44319-025-00509-2

Figure Lengend Snippet: ( A ) ELISA was used to quantify IgA levels in fecal samples from healthy donors and newly diagnosed patients with Crohn’s disease (CD) or ulcerative colitis (UC) ( n = 30). ( B ) The proportion of IgA + B cells within the total CD19 + B cell population from colonic tissues of healthy donors and newly diagnosed CD or UC patients ( n = 10) was assessed using flow cytometry. The percentages of cells in each gated region are indicated. ( C ) LC–MS/MS was employed to determine the relative ac 4 C/C content in total RNA extracted from IgA + and IgA - B cells from colonic tissues of healthy volunteers ( n = 10). ( D ) Immunoblotting (IB) was used to measure NAT10 protein levels in IgA + and IgA - B cells from the colonic tissues of healthy volunteers ( n = 3). ( E ) CD19 + B cells isolated from healthy volunteers were stimulated with BAFF (200 ng/ml) for the indicated times, and Nat10 mRNA levels were analyzed by RT-qPCR ( n = 5). The data are presented as fold changes relative to ACTB mRNA levels, normalized using Bio-Rad CFX Manager 3.1. ( F ) Following BAFF stimulation for the indicated times, total cellular extracts from CD19 + B cells isolated from healthy volunteers were subjected to immunoblotting to detect NAT10 protein. ( G ) NAT10 expression was analyzed by qPCR in total CD19 + B cells isolated from the colonic tissues of healthy donors and newly diagnosed CD or UC patients ( n = 10). Results are shown as fold changes relative to Actb mRNA levels, normalized with Bio-Rad CFX Manager 3.1. ( H ) NAT10 protein levels were assessed by immunoblotting in total CD19 + B cells from colonic tissues of healthy donors and newly diagnosed CD or UC patients ( n = 3). ( I ) A scatterplot showing the linear regression correlation between NAT10 mRNA levels in B cells from the colonic tissues of newly diagnosed CD patients ( n = 10) and their disease activity index (CDAI). All data are representative of biological replicates in three independent experiments. Data are represented as the means ± SDs. The significance of differences ( C ) was determined by t test, and those ( A , B , E , G ) were determined using one-way ANOVA with Newman–Keuls post-hoc test. ** P < 0.01; *** P < 0.005. .

Article Snippet: NAT10 antibody (13365-1-AP) came from Proteintech, while antibodies against RelB (C-19), IKBα (C-21), p65 (C-20), ERK (K-23), phospho-ERK (E-4), JNK (C-17), p38 (H-147), p105/p50 (C-19), AKT1 (B-1), Lamin B (C-20), NIK (H248), and TRAF3 (C-20) were sourced from Santa Cruz Biotechnology.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Isolation, Quantitative RT-PCR, Expressing, Activity Assay

Journal: EMBO Reports

Article Title: NAT10-mediated acetylation of NIK mRNA in B cells promotes IgA production

doi: 10.1038/s44319-025-00509-2

Figure Lengend Snippet: ( A ) NAT10 cKO genotyping PCR. Nat10-floxed mice were crossed with CD19-Cre mice to generate Nat10 fl/fl Cd19 Cre/+ (NAT10 cKO ), and Nat10 +/+ Cd19 Cre/+ (WT). ( B ) Immunoblotting (IB) assays showing specific ablation of NAT10 in the B cells of NAT10 cKO mice. ( C ) Tissue sections of heart, kidney, liver, and lung from 6-week-old WT and NAT10 cKO mice were performed with hematoxylin and eosin (H&E) staining ( n = 3). All data are representative of biological replicates at least three independent experiments. .

Article Snippet: NAT10 antibody (13365-1-AP) came from Proteintech, while antibodies against RelB (C-19), IKBα (C-21), p65 (C-20), ERK (K-23), phospho-ERK (E-4), JNK (C-17), p38 (H-147), p105/p50 (C-19), AKT1 (B-1), Lamin B (C-20), NIK (H248), and TRAF3 (C-20) were sourced from Santa Cruz Biotechnology.

Techniques: Western Blot, Staining

Journal: EMBO Reports

Article Title: NAT10-mediated acetylation of NIK mRNA in B cells promotes IgA production

doi: 10.1038/s44319-025-00509-2

Figure Lengend Snippet: ( A – C ) Flow cytometry was used to assess the percentages (numbers within quadrants) of different B cell subsets within the CD19 + splenic cells in the spleens of 6-week-old WT and NAT10 cKO mice ( n = 6). FM, follicular mature (IgM int IgD high ); FO, follicular (CD21 int CD23 high ); T1, transitional 1 (IgM high IgD low , CD21 low CD23 low ); T2, transitional 2 (IgM high IgD high ); MZ, marginal zone (IgM high IgD low , CD21 high CD23 low ). ( D ) Flow cytometry was also employed to analyze the proportions of recirculating (RC), immature (IM), and early developmental (ProPre) B cell stages ( n = 6). ( E ) Tissue sections of spleens from 6-week-old WT and NAT10 cKO mice were performed with hematoxylin and eosin (H&E) staining ( n = 3). Scale bar = 10 μm. All data are representative of biological replicates at least three independent experiments. Data are represented as the means ± SDs. The significance of differences ( A – D ) was determined by t test. ns no significance. .

Article Snippet: NAT10 antibody (13365-1-AP) came from Proteintech, while antibodies against RelB (C-19), IKBα (C-21), p65 (C-20), ERK (K-23), phospho-ERK (E-4), JNK (C-17), p38 (H-147), p105/p50 (C-19), AKT1 (B-1), Lamin B (C-20), NIK (H248), and TRAF3 (C-20) were sourced from Santa Cruz Biotechnology.

Techniques: Flow Cytometry, Staining

Journal: EMBO Reports

Article Title: NAT10-mediated acetylation of NIK mRNA in B cells promotes IgA production

doi: 10.1038/s44319-025-00509-2

Figure Lengend Snippet: ( A ) Diagram illustrating the experimental design. ( B ) Quantification of NP-specific antibody subclasses in the serum of 6-week-old WT and NAT10 cKO mice, following intraperitoneal immunization with NP-KLH or NP-Ficoll, measured by enzyme-linked immunosorbent assay (ELISA) ( n = 5). ( C ) Total antibody subclass levels in the serum of 6-week-old WT and NAT10 cKO mice immunized with sheep red blood cells (SRBCs), determined by ELISA ( n = 6). ( D ) Analysis of the proportion of IgA + B cells within the spleens of 12-month-old WT and NAT10 cKO mice immunized with SRBCs, evaluated 7 days post-immunization by FACS and subsequent quantification ( n = 6). ( E ) Immunofluorescence microscopy of IgA deposition in colon tissue sections from 12-month-old WT and NAT10cKO mice, with a subsequent quantification of immunoglobulin deposits ( n = 5). Scale bar = 10 μm. All data are representative of biological replicates at least three independent experiments. Data are represented as the means ± SDs. The significance of differences ( B – E ) was determined by t test. ** P < 0.01; *** P < 0.005. .

Article Snippet: NAT10 antibody (13365-1-AP) came from Proteintech, while antibodies against RelB (C-19), IKBα (C-21), p65 (C-20), ERK (K-23), phospho-ERK (E-4), JNK (C-17), p38 (H-147), p105/p50 (C-19), AKT1 (B-1), Lamin B (C-20), NIK (H248), and TRAF3 (C-20) were sourced from Santa Cruz Biotechnology.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Microscopy

Journal: EMBO Reports

Article Title: NAT10-mediated acetylation of NIK mRNA in B cells promotes IgA production

doi: 10.1038/s44319-025-00509-2

Figure Lengend Snippet: ( A ) Measurement of total antibody subclasses in the serum of 12-month-old WT and NAT10 cKO mice using ELISA ( n = 6). ( B ) ELISA analysis of baseline levels of autoantibodies against antinuclear antigen (ANA Ab) and double-stranded DNA (dsDNA Ab) in the serum of 12-month-old unimmunized WT and NAT10 cKO mice ( n = 5). ( C ) Body weight was tracked over a 7-day period in WT and NAT10 cKO mice that were intranasally administered either influenza virus H7N9 or PBS ( n = 7 per group). ( D ) Survival rates of H7N9-infected WT and NAT10 cKO mice were monitored for 7 days ( n = 11 per group). ( E ) Viral titers in the lungs were measured at 5 days post-infection (dpi) using a TCID50 assay ( n = 7 per group). ( F ) ELISA was performed to assess IgA concentrations in the bronchoalveolar lavage fluid (BAL) at 5 dpi ( n = 5). ( G ) Lung tissue sections from 6-week-old WT and NAT10 cKO mice infected with H7N9 were stained with H&E for histological analysis ( n = 5). All data are representative of biological replicates at least three independent experiments. Data are represented as the means ± SDs. The significance of differences ( A , B ) and ( E , F ) was determined by t test, and those ( C , D ) were determined using one-way ANOVA with Newman–Keuls post-hoc test. ** P < 0.01; *** P < 0.005. .

Article Snippet: NAT10 antibody (13365-1-AP) came from Proteintech, while antibodies against RelB (C-19), IKBα (C-21), p65 (C-20), ERK (K-23), phospho-ERK (E-4), JNK (C-17), p38 (H-147), p105/p50 (C-19), AKT1 (B-1), Lamin B (C-20), NIK (H248), and TRAF3 (C-20) were sourced from Santa Cruz Biotechnology.

Techniques: Enzyme-linked Immunosorbent Assay, Virus, Infection, TCID50 Assay, Staining

Journal: EMBO Reports

Article Title: NAT10-mediated acetylation of NIK mRNA in B cells promotes IgA production

doi: 10.1038/s44319-025-00509-2

Figure Lengend Snippet: ( A – C ) Weight loss ( A ) ( n = 6), disease activity score ( B ) ( n = 6), and survival rate ( C ) ( n = 10) in DSS-treated WT and NAT10 cKO mice. ( D , E ) Measurements of colon length ( D ) ( n = 5), and histopathological analysis using hematoxylin and eosin (H&E) staining ( E ) ( n = 5) on day 12 following DSS treatment in WT and NAT10 cKO mice. Scale bar = 200 μm. ( F ) Flow cytometry (FACS) analysis showing macrophage (CD11b + F4/80 + ) populations in representative plots from multiple mice ( n = 5). ( G ) Bacterial colony-forming units (CFU) counted in the feces of WT and NAT10 cKO mice on day 12 of colitis ( n = 5). ( H ) Optical density (O.D.) values reflecting the amount of bacteria bound by serum antibodies in WT and NAT10 cKO mice on day 12 post-DSS treatment ( n = 5). ( I ) Serum and mucus-rich fluid supernatants were collected, the latter transferred into 10 mL of phosphate-buffered saline (PBS), and secretory IgA (sIgA) levels in the colon were measured via ELISA ( n = 6). ( J ) The percentage of IgA + B cells among the B cell population in the mediastinal lymph nodes (mLN) and Peyer’s patches of WT and NAT10 cKO mice on day 12 post-DSS treatment were measured by FACS, along with the quantification of these results ( n = 5). All data are representative of biological replicates at least three independent experiments. Data are represented as the means ± SDs. The significance of differences ( B – J ) was determined by t test, and those ( A ) were determined using two-way ANOVA. ** P < 0.01; *** P < 0.005. .

Article Snippet: NAT10 antibody (13365-1-AP) came from Proteintech, while antibodies against RelB (C-19), IKBα (C-21), p65 (C-20), ERK (K-23), phospho-ERK (E-4), JNK (C-17), p38 (H-147), p105/p50 (C-19), AKT1 (B-1), Lamin B (C-20), NIK (H248), and TRAF3 (C-20) were sourced from Santa Cruz Biotechnology.

Techniques: Activity Assay, Staining, Flow Cytometry, Bacteria, Saline, Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: NAT10-mediated acetylation of NIK mRNA in B cells promotes IgA production

doi: 10.1038/s44319-025-00509-2

Figure Lengend Snippet: ( A – C ) Spleen B cells derived from 6-weeks-old WT and NAT10 cKO mice were stimulated as indicated. IB assays detecting the indicated NF-κB, MAPKs and AKT pathways and the loading control Lamin B in the nuclear extracts prepared from the αIgM stimulated WT and NAT10-deficient B cells. ( D ) IB analysis of TBK1 using total cell extracts from WT and NAT10-deficient B cells, stimulated as indicated. ( E ) Immunoassay of NAT10 −/− B cells stimulated with LPS (5 μg/ml) for 8 h, followed by infection with a retroviral vector expressing GFP and either WT NAT10 (NAT10-WT) or the NAT10-G641E mutant. Infected cells (GFP + ) were analyzed by FACS ( n = 4). All data are representative of biological replicates at least three independent experiments. Data are represented as the means ± SDs. The significance of differences ( E ) was determined using one-way ANOVA with Newman–Keuls post-hoc test. .

Article Snippet: NAT10 antibody (13365-1-AP) came from Proteintech, while antibodies against RelB (C-19), IKBα (C-21), p65 (C-20), ERK (K-23), phospho-ERK (E-4), JNK (C-17), p38 (H-147), p105/p50 (C-19), AKT1 (B-1), Lamin B (C-20), NIK (H248), and TRAF3 (C-20) were sourced from Santa Cruz Biotechnology.

Techniques: Derivative Assay, Control, Infection, Retroviral, Plasmid Preparation, Expressing, Mutagenesis

Journal: EMBO Reports

Article Title: NAT10-mediated acetylation of NIK mRNA in B cells promotes IgA production

doi: 10.1038/s44319-025-00509-2

Figure Lengend Snippet: ( A , B ) IB analysis of noncanonical NF-κB components in cytoplasmic (CE) and nuclear (NE) extracts from NAT10-deficient and WT B cells, stimulated with anti-CD40 (α-CD40; 1 μg/ml) or BAFF (200 ng/ml). ( C , D ) IB analysis using total cell extracts from WT and NAT10-deficient B cells, stimulated as indicated. ( E ) Immunoassay of NAT10 −/− B cells stimulated with LPS (5 μg/ml) for 8 h, followed by infection with a retroviral vector expressing GFP and either WT NAT10 (NAT10-WT) or the NAT10-G641E mutant. Infected cells (GFP + ) were sorted via flow cytometry and stimulated with BAFF, after which in vitro proliferation assays were performed ( n = 5). ( F ) Proportion of IgA + cells among purified NAT10 −/− B cells overexpressing the indicated genes, cultured with various combinations of IgA inducers, and subjected to in vitro IgA class switching. Analysis was performed using flow cytometry, based on intracellular IgA and GFP staining ( n = 4). ( G ) IB analysis of whole-cell lysates from NAT10 −/− B cells overexpressing the indicated genes as described in ( E ). All data are representative of biological replicates at least three independent experiments. Data are represented as the means ± SDs. The significance of differences ( E , F ) was determined using one-way ANOVA with Newman–Keuls post-hoc test. ** P < 0.01; *** P < 0.005. .

Article Snippet: NAT10 antibody (13365-1-AP) came from Proteintech, while antibodies against RelB (C-19), IKBα (C-21), p65 (C-20), ERK (K-23), phospho-ERK (E-4), JNK (C-17), p38 (H-147), p105/p50 (C-19), AKT1 (B-1), Lamin B (C-20), NIK (H248), and TRAF3 (C-20) were sourced from Santa Cruz Biotechnology.

Techniques: Infection, Retroviral, Plasmid Preparation, Expressing, Mutagenesis, Flow Cytometry, In Vitro, Purification, Cell Culture, Staining

Journal: EMBO Reports

Article Title: NAT10-mediated acetylation of NIK mRNA in B cells promotes IgA production

doi: 10.1038/s44319-025-00509-2

Figure Lengend Snippet: ( A ) qPCR analysis performed on RNA extracted from spleen B cells derived from WT and NAT10 cKO mice, stimulated with either anti-CD40 (α-CD40; 1 μg/ml) or BAFF (200 ng/ml) ( n = 4). ( B ) qPCR analysis of Map3k14 and Gfp mRNA in HEK293T cells, transfected with various combinations of expression vectors for hemagglutinin-tagged human NIK (HA-hNIK)-GFP and Flag-tagged NAT10 WT or the NAT10-G641E mutant ( n = 4). ( C ) IB analysis of specific proteins in HEK293T cells transfected with different plasmid constructs. ( D ) mRNA stability assays in WT and NAT10-deficient B cells pretreated with BAFF (200 ng/ml) for 24 h, followed by the addition of 3 μg/ml CHX to inhibit transcription. Cells were harvested at the indicated times post-CHX treatment, and the levels of NIK mRNA were quantified by qPCR ( n = 5). Results are expressed as fold changes relative to Actb mRNA, normalized using Bio-Rad CFX Manager 3.1. ( E ) RNA immunoprecipitation (RIP) was conducted using an anti-NAT10 antibody, followed by qPCR to detect RNA enrichment in NAT10 −/− B cells overexpressing either NAT10 WT or NAT10-G641E, and stimulated with BAFF (200 ng/ml) for 24 h ( n = 4). ( F ) Sequence alignment of ac4C modification sites in NIK mRNA targeted by NAT10. Canonical bases are highlighted in red, and mutated ones in blue. ( G , H ) qPCR ( G ) and IB ( H ) analyses of Map3k14 and Gfp mRNA levels in HEK293T cells transfected with various plasmid combinations ( n = 4). ( I ) mRNA stability assays in NIK −/− B cells infected with different plasmids expressing the specified proteins. These B cells were pretreated with BAFF (200 ng/ml) for 24 h before the addition of 3 μg/ml CHX to stop transcription. Cells were harvested at the indicated times post-CHX treatment, and NIK mRNA levels were assessed by qPCR ( n = 4). Data are shown as fold changes relative to Actb mRNA, normalized using Bio-Rad CFX Manager 3.1. ( J ) Proportion of IgA + cells among purified NIK −/− B cells overexpressing the indicated genes, cultured with different combinations of IgA-inducing factors, and analyzed for IgA class switching in vitro by flow cytometry using intracellular IgA and GFP staining ( n = 4). All data are representative of biological replicates at least three independent experiments. Data are represented as the means ± SDs. The significance of differences ( E ) was determined by t test, and those ( B , D , G , I , J ) were determined using one-way ANOVA with Newman–Keuls post-hoc test. ** P < 0.01; *** P < 0.005. .

Article Snippet: NAT10 antibody (13365-1-AP) came from Proteintech, while antibodies against RelB (C-19), IKBα (C-21), p65 (C-20), ERK (K-23), phospho-ERK (E-4), JNK (C-17), p38 (H-147), p105/p50 (C-19), AKT1 (B-1), Lamin B (C-20), NIK (H248), and TRAF3 (C-20) were sourced from Santa Cruz Biotechnology.

Techniques: Derivative Assay, Transfection, Expressing, Mutagenesis, Plasmid Preparation, Construct, RNA Immunoprecipitation, Sequencing, Modification, Infection, Purification, Cell Culture, In Vitro, Flow Cytometry, Staining