Journal: Cell communication and signaling : CCS

Article Title: MSC-derived mitochondria promote axonal regeneration via Atf3 gene up-regulation by ROS induced DNA double strand breaks at transcription initiation region.

doi: 10.1186/s12964-024-01617-7

Figure Lengend Snippet: Fig. 3 Atf3 is a key upstream target of mitochondrial transfer in regulating the expression of RAGs. (A) Schematic of the experiment. The expression of Atf3 in DRG cells was knocked down by injection of Atf3 siRNA in to L3/4/5 DRGs and electrotransfection. The non-targeting siRNA were employed as a control to Atf3 siRNA. The SNI models were constructed 2 days later. To construct the SNI model, the right thigh of each mouse was surgically exposed at the mid- thigh level to access the sciatic nerve. The sciatic nerve was then crushed with moderate force using a forceps for a duration of 10 s. For the non-targeting siRNA with mitochrondria injection therapy (non-targeting siRNA + Mito) and Atf3 siRNA with mitochrondria injection therapy (Atf3 siRNA + Mito) groups, 2 µL of mitochondria derived from 106 MSCs were injected into the crush sites of the injured nerves. The injured sciatic nerves were removed 4 days later. After fixation and clearing, the nerves were whole-mount stained utilizing primary anti-Tuj1 antibodies and fluorescent secondary antibodies, imaging by a fluorescence microscope; (B) Representative immunofluorescence images of whole-mount sciatic nerves in non-targeting siRNA, non-targeting siRNA + Mito, Atf3 siRNA and Atf3 siRNA + Mito groups (left). Quantification of the average length of the top 5 axons with the longest regeneration distance in each groups (right). (C) qPCR of the selected RAGs in DRG tissue from the above 4 groups. The mRNA expression was normalized against GAPDH. Data represent means ± SD. Statistically significant differences are indicated; n = 6; *P < 0.05, vs. Control

Article Snippet: The siRNAs used for Atf3 knockdown and non-targeting control were sourced from Santa Cruz Biotechnology, Inc., Santa Cruz, CA (#SC-29,758 and #SC-37,007).

Techniques: Expressing, Injection, Control, Construct, Derivative Assay, Staining, Imaging, Fluorescence, Microscopy, Immunofluorescence

Journal: Cell communication and signaling : CCS

Article Title: MSC-derived mitochondria promote axonal regeneration via Atf3 gene up-regulation by ROS induced DNA double strand breaks at transcription initiation region.

doi: 10.1186/s12964-024-01617-7

Figure Lengend Snippet: Fig. 4 Mitochondria promote the expression of ATF3 through ROS induced DSBs. Four days after constructing SNI models and administering mitochon drial injection therapy, the DRGs were surgically extracted and sectioned. The levels of reactive oxygen species (ROS) in the DRGs were evaluated using Hydrocyanine-Cy3 staining and relative fluorescence quantification among the Sham, SNI, SNI with mitochondrial injection therapy (SNI + Mito), and SNI with mitochondrial injection and oxidant scavenger N-acetylcysteine (NAC) treatment (SNI + Mito + NAC) groups. (A) Representative Hydrocyanine-Cy3 staining images of DRG tissue from the Sham, SNI, SNI + Mito and SNI + Mito + NAC groups (left). Relative quantification of Hydrocyanine-Cy3 fluorescence intensities of the above 4 groups. The levels of DNA double strand breaks and ATF3 protein expression in DRGs from the above 4 groups were detect by Western blot. (B) Western blot of protein expression levels of DSB markers of γ-H2AX and 53BP1, and ATF3 in DRGs from Sham, SNI, SNI + Mito and SNI + Mito + NAC groups (left). Densitometric analysis of the protein expression of γ-H2AX, 53BP1, and ATF3 among the above 4 groups (right). Protein expression was normalized against β-actin. Data represent means ± SD. Statistically significant differences are indicated; n = 3; *P < 0.05, vs. Control

Article Snippet: The siRNAs used for Atf3 knockdown and non-targeting control were sourced from Santa Cruz Biotechnology, Inc., Santa Cruz, CA (#SC-29,758 and #SC-37,007).

Techniques: Expressing, Injection, Staining, Fluorescence, Quantitative Proteomics, Western Blot, Control

Journal: Cell communication and signaling : CCS

Article Title: MSC-derived mitochondria promote axonal regeneration via Atf3 gene up-regulation by ROS induced DNA double strand breaks at transcription initiation region.

doi: 10.1186/s12964-024-01617-7

Figure Lengend Snippet: Fig. 5 MSC-derived mitochondrial transfer mediates DSBs at the Atf3 gene transcription initiation region. To detect double-strand breaks (DSBs) in vari ous regions of the Atf3 gene, the ChIP-PCR assay was employed, utilizing γ-H2AX and 53BP1 antibodies along with primers targeting the Atf3 promoter, exon 2, and 3’UTR. (A-C) ChIP-PCR analysis of γ-H2AX binding to the Atf3 promoter (A), exon 2 (B) and 3’UTR (C) regions in the sham, SNI and SNI with mitochrondrial injection therapy (SNI + Mito) groups. (D-F) ChIP-PCR analysis of 53BP1 binding to the Atf3 promoter (D), exon 2 (E) and 3’UTR (F) regions in the sham, SNI and SNI + Mito groups

Article Snippet: The siRNAs used for Atf3 knockdown and non-targeting control were sourced from Santa Cruz Biotechnology, Inc., Santa Cruz, CA (#SC-29,758 and #SC-37,007).

Techniques: Derivative Assay, Binding Assay, Injection

Journal: Cell communication and signaling : CCS

Article Title: MSC-derived mitochondria promote axonal regeneration via Atf3 gene up-regulation by ROS induced DNA double strand breaks at transcription initiation region.

doi: 10.1186/s12964-024-01617-7

Figure Lengend Snippet: Fig. 6 MSC-derived mitochondria-mediated DSBs are close to the CTCF binding site. To investigate the positional correlation between mitochondria- mediated double-strand breaks (DSBs) and CTCF binding sites, dorsal root ganglions (DRGs) were isolated from sham and SNI with mitochrondrial injec tion therapy (SNI + Mito) groups, and γH2AX ChIP-seq as well as immunoprecipitation were employed. (A) UCSC genome browser views denoting the disposition of γH2AX signals at Atf3 under the sham and SNI with mitochrondrial injection therapy (SNI + Mito). (B) The plot denotes the disposition of input-normalized γ-H2AX signals relative to CTCF sites that displayed γ-H2AX peaks in their vicinity. The dashed line denotes the profile of γ-H2AX in the sham group, whereas the solid line indicates γ-H2AX profiles in the SNI with mitochrondrial injection therapy (SNI + Mito) group. (C) DRGs from the sham and SNI + Mito groups were lysed and γ-H2AX was immunoprecipitated. The precipitates were analyzed by western blotting with the γ-H2AX and CTCF antibodies

Article Snippet: The siRNAs used for Atf3 knockdown and non-targeting control were sourced from Santa Cruz Biotechnology, Inc., Santa Cruz, CA (#SC-29,758 and #SC-37,007).

Techniques: Derivative Assay, Binding Assay, Isolation, ChIP-sequencing, Immunoprecipitation, Injection, Western Blot

Journal: Cell communication and signaling : CCS

Article Title: MSC-derived mitochondria promote axonal regeneration via Atf3 gene up-regulation by ROS induced DNA double strand breaks at transcription initiation region.

doi: 10.1186/s12964-024-01617-7

Figure Lengend Snippet: Fig. 7 Diagrammatic depiction of the therapeutic effect of MSC-derived mitochondria injection therapy on sciatic nerve injury. MSC-derived mitochon dria are retrogradely transferred to DRG neurons via axoplasmic transport, and DSBs at the transcription initiation region of the Atf3 gene are mediated by ROS accumulation, thereby rapidly releasing topological constraints on chromatin interactions, facilitating spatial interactions between enhancers and Atf3 promoters, and promoting Atf3 expression, thus promoting the expression of genes related to regeneration, and finally, promoting axon regeneration

Article Snippet: The siRNAs used for Atf3 knockdown and non-targeting control were sourced from Santa Cruz Biotechnology, Inc., Santa Cruz, CA (#SC-29,758 and #SC-37,007).

Techniques: Derivative Assay, Injection, Expressing

Journal: Journal of Personalized Medicine

Article Title: Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells

doi: 10.3390/jpm13040700

Figure Lengend Snippet: ATF3 expression is upregulated in ΔNp63 -overexpressing CECs.

Article Snippet: ATF3 siRNA (si- ATF3 ) was purchased from Santa Cruz Biotechnology (sc-29757; Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Journal of Personalized Medicine

Article Title: Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells

doi: 10.3390/jpm13040700

Figure Lengend Snippet: ΔNp63 upregulates ATF3 expression in CECs. ( A ) Human CECs were infected with Ad- ΔNp63 or Ad- GFP (control) for 6 h. Cells were harvested 2 days after infection, and total RNA was extracted to analyze the mRNA expression level of the genes; ( B ) Human CECs were treated with si- ΔNp63 , Ad- ΔNp63 , or control plasmids (si-control + Ad- GFP ). The Cells were harvested 2 days after transfection, and the cell lysates were used to assess the protein levels of ΔNp63 and ATF3 via western blot analysis. ** p < 0.01 versus control.

Article Snippet: ATF3 siRNA (si- ATF3 ) was purchased from Santa Cruz Biotechnology (sc-29757; Santa Cruz, CA, USA).

Techniques: Expressing, Infection, Control, Transfection, Western Blot

Journal: Journal of Personalized Medicine

Article Title: Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells

doi: 10.3390/jpm13040700

Figure Lengend Snippet: ΔNp63 increases ATF3 promoter activity in CECs. ( A ) The p63-binding sites in the human ATF3 promoter region (−1249 to +12) were deduced using p63 motif analysis software (p63scan algorithm). Position weight matrices (matrix sites) were matched to identify p63 motifs (−1044 to −1025); ( B ) Human (HuLmP1) or rabbit (RbLmP1) CECs were co-transfected with β-gal plasmids (a transfection control) and either pGL3-Basic, wt-Hu ATF3 , del-Hu ATF3 , wt-Hu ATF3 +Ad- ΔNp63 or del-Hu ATF3 +Ad- ΔNp63 for 16 h. After 48 h of transfection, the cells were harvested and their luciferase activity and β-gal activity were measured. ** p < 0.01.

Article Snippet: ATF3 siRNA (si- ATF3 ) was purchased from Santa Cruz Biotechnology (sc-29757; Santa Cruz, CA, USA).

Techniques: Activity Assay, Binding Assay, Software, Transfection, Control, Luciferase

Journal: Journal of Personalized Medicine

Article Title: Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells

doi: 10.3390/jpm13040700

Figure Lengend Snippet: ΔNp63 induces cell proliferation through an ATF3-dependent pathway. ( A ) Rabbit CECs were transfected with pCMV- ATF3 or control plasmids for 16 h. After transfection, cells were harvested and counted on days 1, 2, 3, and 4; ( B ) Cells were harvested on day 4 and analyzed for proliferation via BrdU assay and Ki-67 staining. Scale bar: 100 μm; ( C ) Ad- ΔNp63 -infected rabbit CECs were transfected with si- ATF3 or si-control for 16 h. Cells were harvested and counted on days 1, 2, 3, and 4 after transfection. ** p < 0.01.

Article Snippet: ATF3 siRNA (si- ATF3 ) was purchased from Santa Cruz Biotechnology (sc-29757; Santa Cruz, CA, USA).

Techniques: Transfection, Control, BrdU Staining, Staining, Infection

Journal: Journal of Personalized Medicine

Article Title: Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells

doi: 10.3390/jpm13040700

Figure Lengend Snippet: ATF3 regulates the expression of cell-cycle–related genes in CECs. ( A ) Human CECs were transfected with pCMV- ATF3 or control plasmids for 48 h. After transfection, the cells were harvested, and the expression of cell-cycle–related genes was analyzed by qRT-PCR; ( B ) Human CECs were transfected with pCMV- ATF3 or control plasmids for 16 h. Cells were harvested on day 4 after transfection and assessed for cyclin D1 and p27 KipP1 protein expression via western blot analysis. ** p < 0.01.

Article Snippet: ATF3 siRNA (si- ATF3 ) was purchased from Santa Cruz Biotechnology (sc-29757; Santa Cruz, CA, USA).

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

Journal: Journal of Personalized Medicine

Article Title: Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells

doi: 10.3390/jpm13040700

Figure Lengend Snippet: ATF3 did not alter the expression of keratinocyte differentiation-related proteins in human CECs. Human CECs were transfected with pCMV- ATF3 , si- ATF3 , or control plasmids for 16 h. Cells were harvested on day 4 after transfection and assessed for the expression of keratinocyte-differentiation–related proteins via western blot analysis.

Article Snippet: ATF3 siRNA (si- ATF3 ) was purchased from Santa Cruz Biotechnology (sc-29757; Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Control, Western Blot

Journal: Pharmacological research

Article Title: A positive feedback loop between AMPK and GDF15 promotes metformin antidiabetic effects.

doi: 10.1016/j.phrs.2022.106578

Figure Lengend Snippet: Fig. 4. Metformin increases GDF15 levels via AMPK activation. (a) Gdf15 mRNA levels, and (b) GDF15 secreted into the culture medium in C2C12 myotubes exposed to different concentrations of metformin (Met) for 24 h. (c) Gdf15 mRNA levels in C2C12 myotubes exposed to 0.5 mM metformin (Met) for 24, 48 or 72 h or (d) transfected with control (Ct) (scrambled) siRNA or AMPKα1/α2 siRNA and treated with 2 mM metformin for 24 h. (e) Gdf15 mRNA levels in C2C12 myotubes transfected with control (Ct) (scrambled) siRNA or ATF3 siRNA and treated with 2 mM metformin for 24 h. (f) Atf3 mRNA levels in C2C12 myotubes exposed to different concentrations of metformin (Met) for 24 h or (g) transfected with control (Ct) (scrambled) siRNA or AMPKα1/α2 siRNA and treated with 2 mM metformin for 24 h. (h) GDF15 mRNA levels in Huh-7 human hepatoma cells exposed to different concentrations of metformin (Met) for 24 h. (i) ATF3 mRNA levels in Huh-7 cells exposed to different concentrations of metformin (Met) for 24 h. (j) Gdf15 mRNA levels in mouse primary hepatocytes exposed to different concentrations of metformin (Met) for 24 h or (k) transfected with control (Ct) (scrambled) siRNA or AMPKα1/α2 siRNA and treated with 2 mM metformin for 24 h. (l) Gdf15 mRNA levels in mouse primary culture of hepatocytes exposed to 2 mM metformin (Met) for 24 h in the presence or absence of 30 µM compound C (Comp C). (m) Immunoblot analysis of GDF15, and total and phospho-AMPK in C2C12 in mouse primary culture of hepatocytes exposed to 2 mM metformin (Met) for 24 h in the presence or absence of 30 µM compound C (Comp C). (a,b,d-h) n = 5 per group. (c,i-l) n = 4 per group. Data are presented as the mean ± SEM. * *p < 0.01 and * **p < 0.001 vs. control. ###p < 0.001 vs. Ct siRNA + Met or Met.

Article Snippet: Differentiated myotubes were transiently transfected with 70 nM of siRNA against AMPK1/2 (Santa Cruz Biotechnology Inc., catalog sc45313), GDF15 (Santa Cruz Biotechnology Inc., catalog sc-39799), p53 (Santa Cruz Biotechnology Inc., catalog sc-29436), ATF3 (Santa Cruz Biotechnology Inc., catalog sc-29758), or control siRNA (Santa Cruz Biotechnology Inc., catalog sc-37007) in Opti-MEM medium (Thermo Fisher, MA), using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) (7 μl per 1.5-mL well) according to the manufacturer’s instructions.

Techniques: Activation Assay, Transfection, Control, Western Blot