Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: Rac is dominant over Rho in the initial transcriptome-wide response to ECM stiffness and preferentially represses ATF3. (A,B) Venn diagrams of differentially expressed genes in MEFs cultured with 10% FBS for 1 h on stiff versus soft hydrogels, stiff hydrogels with or without EHT1864, or stiff hydrogels with or without CT04. (C,D) The genes regulated by ECM and Rac in A,B were compared to GO gene lists for transcription factors (TFs), transcription co-regulators (co-reg) and histone modifiers. (E,F) Log 2 (fold change) values and adjusted P -values of the genes regulated by ECM stiffness and Rac and contained within the indicated GO terms above. (G) Serum-starved MEFs were plated on soft or stiff FN-coated hydrogels with 10% FBS for 1 h with DMSO (Ctrl), EHT1864 or CT04. Atf3 mRNA levels were quantified by RT-qPCR. The graph shows mean+s.e.m. ( n =4) with results normalized to the expression level on soft hydrogels. * P <0.05; *** P <0.001 (two-tailed unpaired t -tests).

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: Dose-dependent effects of ECM stiffness on Rac–GTP, Atf3 mRNA, cyclin D1 mRNA and S phase entry. Serum-starved MEFs were incubated in DMEM containing 10% FBS on FN-coated hydrogels of increasing stiffness (∼2, 8, 15 and 25 kPa). (A) Rac–GTP levels determined at 1 h and graphed relative to Rac activity on the softest hydrogel. Results show mean±s.e.m. ( n =4). (B,C) Atf3 and cyclin D1 mRNA levels determined after 9 h and graphed relative to the mRNA levels on the softest hydrogel. Results show mean±s.e.m. ( n =3). (D) The percentage of EdU-positive nuclei determined at 24 h and graphed relative to EdU incorporation on the softest hydrogel. Results show mean±s.d. ( n =3). Statistical significance for each panel was determined by one-way ANOVA; asterisks show the results of Dunnett's post-tests relative to the softest hydrogel. * P <0.05; *** P <0.001; **** P <0.0001.

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Incubation, Activity Assay

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: ATF3 repression linked to stiffness-dependent cyclin D1 expression. (A) Serum-starved MEFs on soft or stiff FN-coated hydrogels in DMEM containing 10% FBS were treated with vehicle (DMSO) or EHT1864 for 9 h. Atf3 and cyclin D1 mRNA levels were determined from the same lysates and normalized to mRNA expression levels in cells on soft hydrogels. Results show mean±s.e.m. ( n =3). (B) MEFs were infected with adenoviruses encoding GFP (control) or Rac V12 , serum-starved, and cultured and analyzed as in panel A. Results show mean±s.e.m. ( n =3). (C) MEFs infected with adenoviruses (Ad) encoding GFP (control) or ATF3 were serum-starved, incubated on FN-coated hydrogels with 10% FBS for 15 h, and analyzed by immunoblotting for cyclin D1 and ATF3 with GAPDH as the loading control. (D) Quantification of the immunoblot results in C. The graph shows mean+s.d. with results normalized to GAPDH abundance and plotted relative to the normalized cyclin D1 signal on the soft hydrogels ( n =3). (E,F) Serum-starved ROSA and ATF3 KO MEFs were incubated on stiff FN-coated hydrogels with DMSO (Ctrl) or EHT1864 for 9 h. Lysates were analyzed for the levels of ATF3 and cyclin D1 by immunoblotting. GAPDH was used as the loading control. Panel E shows results from ROSA clone R12 and ATF3 KO clone 1-20, and panel F shows quantification of the combined results from ROSA clones R11, R12 and R15 and ATF3 KO clones 1-20 and 1-29. Data were accrued from four independent experiments, and the graph shows mean+s.d. with results normalized to GAPDH abundance and plotted relative to the normalized cyclin D1 signal in the ROSA control. (G) S phase entry was analyzed by EdU incorporation in ROSA clones (R3, R11, R12 and R15) and ATF3 KO clones (1-20, 1-29, 1-44, 1-48 and 1-49) after serum starvation and incubation on stiff FN-coated hydrogels in DMEM containing 10% FBS for 24 h with DMSO (Ctrl) or EHT1864. Results show mean+s.d. n =7 for the ROSA clones and n =8 for the ATF3 KO clones. (H) Model showing that cyclin D1 is regulated by ECM stiffness and Rac through ATF3. * P <0.05; ** P <0.01; **** P <0.0001 (D, two-tailed unpaired t -tests; F and G, one-tailed unpaired t -tests).

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Expressing, Infection, Control, Cell Culture, Incubation, Western Blot, Clone Assay, Two Tailed Test, One-tailed Test

Journal: Cell reports

Article Title: Optic nerve regeneration screen identifies multiple genes restricting adult neural repair

doi: 10.1016/j.celrep.2021.108777

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were incubated with tissue sections overnight at 4°C: anti-Iba1 (1:200, Abcam), anti-CD68 (1:250, Bio-Rad), anti-GFAP (1:500, Abcam), anti-DLK (1:100, Genetex), anti-Atf3 (1:200, Novus Biologicals), anti-GFP (1:500, Abcam), anti-Il22Ra1 (1:200, Bioss), anti-Rbpms (1:100; PhosphoSolutions), anti-βIII-tubulin antibody (1:500, Promega).

Techniques: Virus, Recombinant, SYBR Green Assay, DNA Extraction, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Gene Expression, shRNA, Expressing, Plasmid Preparation, Software