Journal: Stem Cell Reports

Article Title: Enhancing Hematopoiesis from Murine Embryonic Stem Cells through MLL1-Induced Activation of a Rac/Rho/Integrin Signaling Axis

doi: 10.1016/j.stemcr.2019.12.009

Figure Lengend Snippet: hMLL1 Induction Specifically Promotes Integrin-Mediated Cell Adhesion and Further Expands CFU (A) Flow cytometry to detect Cd49d expression in c-Kit + /Cd41 + gated EB cells. Quantification of one representative experiment from three independent differentiation experiments is shown on the right. Data show the average ± SEM, n = triplicate cultures. (B) Experimental procedure to test integrin function. Day 6 EB Cd41 + enriched WT or hMLL1-inducible cells were cultured on integrin ligand (fibronectin or Vcam1)-coated plates for the indicated time and tested for adhesion or CFU content. (C) Cell adhesion of Cd41 + enriched progenitors to fibronectin and Vcam1. Data are representative of four independent experiments and presented as the average of triplicate cultures ± SEM. (D) CFU assay using sorted Cd41 + EB progenitors following 24 h adhesion. Adherent cells were harvested with dissociation buffer and pooled with remaining suspension cells, then counted for CFU assay. Left: both hMLL1-inducible and WT cells were adhered to fibronectin fragment and then followed by CFU assay. One representative experiment of three is shown as the average ± SEM, n = 3 triplicate cultures. Right: quantification of CFU fold changes with Dox, fibronectin fragment, or Rac1 inhibitor (NSC23766, 10 μM). The graph show data pooled from 3 to 4 independent experiments (n = 3 for WT; n = 4 for hMLL1i, with each data point representing the average of triplicate cultures) representing the overall average ± SEM.

Article Snippet: We confirmed and extended these results using independently sorted samples, including integrins ( Itgb2 , Itgal , Itga4 ), Rac/Rho small GTPases ( Rac1 , Rac2 , Rhoa ), kinases ( Akt1 , Pi3kcd ), regulatory subunits or cytoskeleton proteins ( Myl12a/b , Actb , Arp3 ) ( C and D).

Techniques: Flow Cytometry, Expressing, Cell Culture, Colony-forming Unit Assay, Suspension

Journal: Cancer research

Article Title: HACE1 prevents lung carcinogenesis via inhibition of RAC-family GTPases

doi: 10.1158/0008-5472.CAN-19-2270

Figure Lengend Snippet: (A) Amino acid (aa) sequence alignments of murine RAC1, RAC2, and RAC3. Amino acids highlighted in red indicate differences among the family members. The amino acid positions are indicted. (B) Relative mRNA expression of Rac1, Rac2 and Rac3 normalized to β-Actin expression in primary lung tumor cells, isolated from KRasG12D;Hace1+/+Rac1+/+Rac2+/+ mice (n=5) at week 7 post lung cancer induction, followed by RT-qPCR analysis. (C) In vitro ubiquitylation assay. Recombinant GST-HACE1 was incubated with GTP- or GDP-preloaded His-tagged RAC1 or RAC2 in the presence of E1, E2 (Ubch7), ubiquitin and ATP. As a control, catalytic dead HACE1C876S was used. Blots show RAC1 and RAC2 (detected via the His-tag), HACE1 and ubiquitin after 3 h incubation. Ubiquitylated RAC1 and RAC2 are indicated (white arrows). (D) Kaplan-Meier survival curves of KRasG12D;Hace1+/+Rac1+/+Rac2+/+ (n=23), KRasG12D;Hace1–/– (n=15), KRasG12D;Rac1fl/fl (n=25), KRasG12D;Hace1–/–Rac1fl/fl (n=19), KRasG12D;Rac2–/– (n=23) and KRasG12D;Hace1–/–Rac2–/– (n=7), KRasG12D;Rac1fl/flRac2–/– (n=19) and KRasG12D;Hace1–/–Rac1fl/flRac2–/– (n=20) mice. Mice were intratracheally instilled with Adeno-Cre virus on the indicated day 0. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 (log-rank test). (E) Representative H&E stained-lung sections and (F) tumor-to-lung ratios at week 18 post lung cancer induction for KRasG12D;Hace1+/+Rac1+/+Rac2+/+, KRasG12D;Rac1fl/fl, KRasG12D;Hace1–/–Rac1fl/fl, KRasG12D;Rac1fl/flRac2–/– and KRasG12D;Hace1–/–Rac1fl/flRac2–/– mice. Scale bars, 1 mm for 10x images and 50μm for 40x images of lung sections. * P<0.05, ** P<0.01 (One-way ANOVA, Tukey’s post-hoc test, n≥5 mice per cohort). Data in (B) and (F) are presented as mean values ± SEM.

Article Snippet: His-tagged recombinant human RAC1 (Cytoskeleton, RC01) and RAC2 (Cytoskeleton, RC02) were preloaded with GTP or GDP as described ( 41 ).

Techniques: Sequencing, Expressing, Isolation, Quantitative RT-PCR, In Vitro, Ubiquitin Assay, Recombinant, Incubation, Staining

Journal: Cancer research

Article Title: HACE1 prevents lung carcinogenesis via inhibition of RAC-family GTPases

doi: 10.1158/0008-5472.CAN-19-2270

Figure Lengend Snippet: (A) Kaplan-Meier curves of overall survival and (B) disease-free survival for lung adenocarcinoma patients, based on HACE1 and RAC1 mRNA expression. (C) Kaplan-Meier curves of overall survival and (D) disease-free survival for lung adenocarcinoma patients, based on HACE1, RAC1 and RAC2 mRNA expression. (E) Schematic representation of genetic alterations in HACE1, RAC1, RAC2 and RAC3 in lung adenocarcinoma patients from the TCGA (PanCancer Atlas) data set for 507 cases. Color coding indicates mutation types: red, amplification; blue, homozygous deletion; yellow, missense mutation; black, truncating mutation. Percentages (%) of cases with alteration in HACE1, RAC1, RAC2 and RAC3 are indicated. Only altered cases are shown. (F) Heatmap of gene expression profiles of HACE1, RAC1, RAC2 and RAC3 in lung adenocarcinoma patients. Each row represents the expression of either HACE1, RAC1, RAC2 or RAC3. Each line corresponds to one lung cancer patient. Z-score (RNA Seq V2 RSEM) is shown from 10 (red, highest expression) to -2 (blue, lowest expression). The mRNA expression level in a single sample is depicted according to the color scale. (G) Correlation matrix showing Spearman’s rank correlation of HACE1, RAC1, RAC2 and RAC3 mRNA expression profiles. Correlation coefficients are shown in white and the associated p-values in black (statistically significant values with P<0.05 in bold). Orange and blue colors indicate positive and negative correlations, respectively, beige indicates no correlation.

Article Snippet: His-tagged recombinant human RAC1 (Cytoskeleton, RC01) and RAC2 (Cytoskeleton, RC02) were preloaded with GTP or GDP as described ( 41 ).

Techniques: Expressing, Mutagenesis, Amplification, RNA Sequencing Assay

Journal: Cancer research

Article Title: HACE1 prevents lung carcinogenesis via inhibition of RAC-family GTPases

doi: 10.1158/0008-5472.CAN-19-2270

Figure Lengend Snippet: HACE1 ubiquitylates GTP-RAC1 when bound to the NADPH oxidase complex, leading to RAC1 degradation and thereby controlling ROS production (top, left). HACE1 deficiency results in an accumulation of GTP-bound RAC1, increased NADPH oxidase activity and enhanced levels of genotoxic cellular ROS, promoting cancer progression (top, right). Additionally, deregulated RAC1 could promote tumor development by ROS-independent mechanisms. In the absence of the more abundant RAC1, the activity of GTP-RAC2 when bound to the NADPH oxidase complex is controlled by HACE1, leading to decreased cellular ROS levels (bottom, left). When HACE1 and RAC1 are both ablated, active GTP-RAC2 can compensate and promote cancer progression (bottom, right).

Article Snippet: His-tagged recombinant human RAC1 (Cytoskeleton, RC01) and RAC2 (Cytoskeleton, RC02) were preloaded with GTP or GDP as described ( 41 ).

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Characterization of the erythrocyte GTPase Rac1 in relation to Plasmodium falciparum invasion

doi: 10.1038/s41598-020-79052-0

Figure Lengend Snippet: Rac expression in human erythrocytes. ( A ) Proteins extracted from of 5 × 10 7 erythrocyte membranes were hybridized with a monoclonal antibody specific for Rac1 (R1-ab#2). The signal was detected at about 21 kDa, as expected. ( B ) In a different gel, same amounts of erythrocyte membranes were probed with the monoclonal anti-Rac2 specific antibody; no signal was detected. The same filter was then probed with an anti-Rac antibody (recognizing Rac1-3 proteins) (R1-ab#1), used as a loading control (bottom). The full lanes are shown in Fig. .

Article Snippet: Purified His-tagged Rac2 protein (Sino Biological) was used as a positive control.

Techniques: Expressing