Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Interleukin 13 Promotes Maturation and Proliferation in Metaplastic Gastroids

doi: 10.1016/j.jcmgh.2024.101366

Figure Lengend Snippet: Antibodies Used

Article Snippet: ATF3 , Rabbit , Novus, NBP1-85816 , 1:500.

Techniques:

Journal: PLoS ONE

Article Title: Bortezomib-Induced Painful Peripheral Neuropathy: An Electrophysiological, Behavioral, Morphological and Mechanistic Study in the Mouse

doi: 10.1371/journal.pone.0072995

Figure Lengend Snippet: Summary of the experimental plan.

Article Snippet: Following several rinses in PBS, the sections were incubated overnight at 4°C with rabbit anti-mouse ATF3 antibody (Santa Cruz Biotechnology, Dallas, TX), diluted 1:50 in PBS containing 1% NGS+0.2% Triton X-100.

Techniques: Immunohistochemistry, Flow Cytometry

Journal: bioRxiv

Article Title: ATF3 Preserves Skeletal Muscle Stem Cell Quiescence by Preventing Precocious Activation

doi: 10.1101/2022.08.12.503549

Figure Lengend Snippet: A Schematic for isolation of quiescent satellite cells (QSC) after in situ fixation, freshly isolated SCs (FISC) without prior fixation from muscles of Tg: Pax7-nGFP mice. FISCs were subsequently cultured and activated for 24 (ASC-24h), 48 (ASC-48h), or 72 h (DSC-72h). RNAs were extracted for RNA-Seq analysis. B Heat maps indicating gene expression levels (Log2[FPKM]) of AP-1 family TFs detected by the RNA-Seq. C-D Expression levels (FPKM) and genomic snapshots of Atf3 , Atf4 , Fos , FosB , JunB mRNAs from the above RNA-Seq. E RT-qPCR detection of Atf3 in the above cells. F Immunofluorescence (IF) staining of ATF3 and Pax7 protein on the above cells. Scale bar: 50 μm. G IF staining of ATF3 and Pax7 protein on single myofibers from EDL muscles immediately after isolation or cultured for 24h. Scale bar: 25 μm. All the bar graphs are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., no significance.

Article Snippet: 10 μg of antibodies against ATF3 (Santa Cruz Biotechnology), or normal mouse IgG (Santa Cruz Biotechnology) was used for immunoprecipitation.

Techniques: Isolation, In Situ, Muscles, Cell Culture, RNA Sequencing, Gene Expression, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: ATF3 Preserves Skeletal Muscle Stem Cell Quiescence by Preventing Precocious Activation

doi: 10.1101/2022.08.12.503549

Figure Lengend Snippet: A Breeding scheme for generating inducible Atf3 inducible conditional knock out ( Atf3 iKO), Pax7 CreER/+ ; ROSA EYFP/+ ; Atf3 fl/fl , and control (Ctrl), Pax7 CreER/+ ; ROSA EYFP/+ ; Atf3+/+ , littermates. B Schematic outline of the tamoxifen (TMX) administration used in the study and experimental design for testing the effect of short-term Atf3 deletion on barium chloride (BaCl 2 ) induced muscle regeneration process. C Loss of ATF3 protein in iKO FISC was confirmed by Western blot. α -Tubulin was used as a loading control. D No overt morphological difference or body weight change was observed in representative Ctrl vs iKO mice. E Left: H&E staining of the TA muscles collected at 3, 5 and 7 days post the 1 st round injury (dpi). Scale bar: 50 μm. Right: Cross-sectional areas (CSAs) of newly formed fibers were quantified from the TA muscles at 7dpi and the distribution is shown. n = 3 mice per group. F Left: IF staining of eMyHC (red) and Laminin (green) was performed on the above TA muscles. Scale bar: 50 μm. Right: the numbers of eMyHC+ fibers per view at 3dpi were quantified. n = 3 mice per group. G-H Left: H&E and eMyHC staining of the TA muscles collected at 3, 5 and 7 days post the 2 nd round injury. Scale bar: 50 μm. n = 3 mice per group. I-J H&E and eMyHC staining of the TA muscles collected at 3, 5 and 7 days post the 3 rd round injury. Scale bar: 50 μm. n = 3 mice per group. K Left: IF staining of Pax7 (red) and Laminin (green) on Ctrl and iKO TA muscles on 30 days post the 3 rd round of injury. Scale bar: 50 μm. Right: the numbers of Pax7+ SCs per view were quantified. n = 3 mice per group. L Schematic for engraftment. FISCs from donor mice (Ctrl/ iKO) were injected into pre-injured (1 day before) receptor nude mice. TA muscles were collected 21 days after engraftment for analysis. M Left: IF staining of YFP (green) and Laminin (red) on the TA muscles 21 days after engraftment. Scale bar: 50 μm. Right: the numbers of YFP+ fibers per view at 21 days after engraftment were quantified. n= 3 mice per group. N IF staining of Myod (red) and Laminin (green) on Ctrl and iKO TA muscles at 3 and 5 days post the 1 st , 2 nd and 3 rd rounds of injury. Scale bar: 50 μm; n = 3 mice per group. O Quantification of the numbers of Myod+ SCs per view on the above TA muscles (N). n = 3 mice per group. All the bar graphs are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., no significance.

Article Snippet: 10 μg of antibodies against ATF3 (Santa Cruz Biotechnology), or normal mouse IgG (Santa Cruz Biotechnology) was used for immunoprecipitation.

Techniques: Knock-Out, Control, Western Blot, Staining, Muscles, Injection

Journal: bioRxiv

Article Title: ATF3 Preserves Skeletal Muscle Stem Cell Quiescence by Preventing Precocious Activation

doi: 10.1101/2022.08.12.503549

Figure Lengend Snippet: A Schematic outline of the experimental design for testing the effect of long-term Atf3 deletion on muscle regeneration process. A 30 or 120 days chasing after TMX injection was given before BaCl 2 injection. B Left: H&E staining of the TA muscles collected at 3, 5 and 7 dpi after the 30 days chasing period. Scale bar: 50 μm. n = 3 mice per group. Right: CSAs of newly formed fibers were quantified from the above-stained TA muscle at 7dpi and the distribution is shown. n = 3 mice per group. C Left: IF staining of eMyHC (red) and Laminin (green) was performed on the above TA muscles. Scale bar: 50 μm. Right: Quantification of the numbers of eMyHC+ fibers per view at 5 and 7dpi. n = 3 mice per group. D Left: IF staining of Pax7 (red) and Laminin (green) was performed on the above TA muscles. Scale bar: 50 μm. Right: Quantification of the numbers of Pax7+ SCs per view. n = 3 mice per group. E-G The above assays/quantifications were performed on the Ctrl or iKO muscles collected at 3, 5 and 7 dpi after the 120 days chasing period. Scale bar: 50 μm. n = 3 mice per group. All the bar graphs are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., no significance.

Article Snippet: 10 μg of antibodies against ATF3 (Santa Cruz Biotechnology), or normal mouse IgG (Santa Cruz Biotechnology) was used for immunoprecipitation.

Techniques: Injection, Staining, Muscles

Journal: bioRxiv

Article Title: ATF3 Preserves Skeletal Muscle Stem Cell Quiescence by Preventing Precocious Activation

doi: 10.1101/2022.08.12.503549

Figure Lengend Snippet: A Schematic outline of the experimental design. RNA-Seq and H2B CUT&RUN were performed with FISCs isolated from Ctrl or iKO mice; ATF3 ChIP-Seq was performed with C2C12 overexpressing ATF3. The acquired datasets were then integrated for analyses. B Left: Differentially expressed genes (DEGs) were identified from the above Ctrl vs. iKO RNA-Seq. Right: The percentages of up and down-regulated genes. C&D Gene ontology (GO) analyses of the above up and down-regulated genes. The top 5 enriched GO terms ranked by gene ratio (proportion of genes annotated for each GO term) are shown. Dots are colored by adjusted P value of fold change (FC) and their size corresponds to the gene counts annotated to each GO term. E FPKM and Log2FC of H2b genes in iKO vs. Ctrl from the above RNA-Seq. F ChIP-Seq was performed using chromatins from C2C12 cells overexpressing ATF3, and the genomic distribution of 2871 ATF3 binding peaks is shown. G De novo motif prediction by DREME revealed the enrichment of canonical ATF3 motifs in the above binding regions. H The GO analysis for all the above genes with ATF3 binding. The top ten enriched GO terms ranked by gene ratio are shown. Dots are colored by adjusted P value (degree of enrichment) and their size corresponds to the gene counts annotated to each GO term. I Left: Venn diagrams showing the overlapping (72 genes) between the above identified ChIP-Seq target (2871) and the down-regulated genes (469) from the RNA-Seq. Right: GO analysis of the above 72 genes revealed an extreme enrichment of histone-related terms. J Genomic snapshots of 3 of the above identified H2b genes ( Hist1h2bb , Hist1h2bm and Hist2h2bb ) with ATF3 binding in their TSSs (ChIP-Seq tracks) and down-regulated by ATF3 deletion (RNA-Seq tracks). K ATF3, H2A, H2B, H3 and H4 proteins were measured by western blot in FISC from Ctrl and iKO mice. α-Tubulin was used as a loading control. L IF staining of H2A (green) and H2B (red) on the above FISCs. Scale bar: 100 μm; n = 3 mice per group. M H2B CUT&RUN was performed with FISCs isolated from Ctrl or iKO mice. The enrichment level in iKO vs. Ctrl was compared by analyzing gains and losses of H2B reads (fold change 1.5) in 10 kb bins. Pie charts showing the number of the bins with H2B changed (6589, 229 up and 6369 down) and unchanged (247114) (left and middle), and the genomic distribution of the down-regulated H2B signals (right). N Genomic snapshots of a 1098kb region Integrative Genomics Viewer (IGV) track of H2B signal in-Ctrl vs. iKO in Chr4. O Schematic of the RNA Polymerase II elongation process during DNA transcription P Venn diagrams showing the overlapping (252 genes) between the above regions with down-regulated H2B CUT&RUN signals (4333) and the up-regulated genes (1866) from the above (A) RNA-Seq. Q Metaplots showing average H2B CUT&RUN signals in Ctrl and iKO cell 3 kb upstream to 3 kb downstream of TSS. R GO analysis of the above 252 genes from (P). The top ten enriched GO terms ranked by gene ratio are shown. Dots are colored by adjusted P value (degree of enrichment) and their size corresponds to the gene counts annotated to each GO term. S Genomic snapshots of Eln gene which shows down-regulated H2B CUT&RUN signal and up-regulated RNA-Seq signal in iKO vs. Ctrl. Regions with H2B signal reduction in iKO vs. Ctrl are highlighted in blue frame.

Article Snippet: 10 μg of antibodies against ATF3 (Santa Cruz Biotechnology), or normal mouse IgG (Santa Cruz Biotechnology) was used for immunoprecipitation.

Techniques: RNA Sequencing, Isolation, ChIP-sequencing, Binding Assay, Western Blot, Control, Staining

Journal: bioRxiv

Article Title: ATF3 Preserves Skeletal Muscle Stem Cell Quiescence by Preventing Precocious Activation

doi: 10.1101/2022.08.12.503549

Figure Lengend Snippet: A Upper: Comet assay was performed on Ctrl or iKO SCs after 0, 1000 or 2000J UV treatment. Scale bar: 25 μm. Lower: Quantification of the length of comet tails; n = 3 mice per group. B IF staining of γH2AX (red) and Myod (green) was performed on SCs from Ctrl or iKO mice after cultured for 0, 24, 48 or 72hours. Scale bar: 50 μm. C Quantification of the percentages of Myod+ / γH2AX+ cells; n = 3 mice per group. D Left: β-Gal (blue) staining was performed on SCs from Ctrl or iKO mice after cultured for 9 days. Scale bar: 50 μm. Right: Quantification of the percentages of β-Gal+ cells; n = 3 mice per group. E Expression of the selected senescence marker genes including p16 , p21 and p53 in SCs from Ctrl and iKO was quantified by RT– qPCR . F p21 and p53 protein levels were detected by Western blotting in SCs from Ctrl and iKO mice. GAPDH was used as a loading control. G&H SCs from Ctrl and iKO mice were transfected with an H2B-overexpression or vector control plasmid. 96hours after transfection, expression of the indicated genes was detected by Western blotting and RT–qPCR. GAPDH was used as a loading control. I Left: The above transfected cells were cultured for 24 h before treated with EdU for 6 h and stained for EdU (red) and Pax7 (green). Scale bar: 50 μm. Right: Quantification of the percentage of EdU+/Pax7+ SCs; n = 3 mice per group. J Schematic model depicting the functional role of ATF3 in preserving SC quiescence and preventing SC precocious activation. In homeostatic muscle, the rapid induction of ATF3 upon minor stress promotes H2b expression to maintain proper nucleosome positioning and suppress the expression of activation genes, thus preventing the precocious activation of SCs. With ATF3 loss, H2B deficiency occurs which leads to altered nucleosomes positioning and the up-regulation of activation genes, causing SCs to break quiescence (QSC) and enter early activating stage (eASC). All the bar graphs are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., no significance.

Article Snippet: 10 μg of antibodies against ATF3 (Santa Cruz Biotechnology), or normal mouse IgG (Santa Cruz Biotechnology) was used for immunoprecipitation.

Techniques: Single Cell Gel Electrophoresis, Staining, Cell Culture, Expressing, Marker, Quantitative RT-PCR, Western Blot, Control, Transfection, Over Expression, Plasmid Preparation, Functional Assay, Preserving, Activation Assay