Journal: eLife

Article Title: Doublecortin engages the microtubule lattice through a cooperative binding mode involving its C-terminal domain

doi: 10.7554/eLife.66975

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Rhodamine-labeled α/β-tubulin , Cytoskeleton , T240 , .

Techniques: Recombinant, Plasmid Preparation, Purification, Software, Mass Spectrometry

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: In vitro ubiquitylation assay 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, Ubiquitin Proteomics, Control, Virus, 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: In vitro ubiquitylation assay His-tagged recombinant human RAC1 (Cytoskeleton, RC01) and RAC2 (Cytoskeleton, RC02) were preloaded with GTP or GDP as described ( 41 ).

Techniques: Expressing, Mutagenesis, Amplification, Gene Expression, RNA Sequencing

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

Techniques: Activity Assay

Journal: Redox Biology

Article Title: A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy

doi: 10.1016/j.redox.2021.102186

Figure Lengend Snippet: PGAM5 interferes with PINK1 processing independently of its phosphatase activity. A. PGAM5 mutant and isoform lacking phosphatase activity induce Parkin translocation. PC6 cells were transfected with Parkin-EYFP and PGAM5 wt, PGAM5 F244D or short PGAM5 isoform (isoform 2, UniProt #Q96HS1-2). ****P < 0.0001, n = 6 dishes, 20 fields per dish, Welch's ANOVA followed by Dunnett's T3 multiple comparisons test. B. PGAM5 mutant lacking PARL/OMA1 cleaving site does not induce Parkin translocation. PC6 cells were transfected with Parkin-EYFP and PGAM5 or PGAM5 S24F. ****P < 0.0001, n = 6 dishes, 20 fields per dish, Welch's ANOVA followed by Dunnett's T3 multiple comparisons test. C. TOMM7 overexpression increases the fraction of full-length PGAM5 in HEK293 cells expressing PGAM5-flag and TOMM7-myc. D. TOMM7 overexpression protects partially against PGAM5 induced Parkin translocation. PC6 cells were transfected with Parkin-EYFP, PGAM5 wt or/and TOMM7. ****P < 0.0001, n = 9 dishes, 20 fields per dish, Welch's ANOVA followed by Dunnett's T3 multiple comparisons test. E. OMA1 and PARL protect partially against PGAM5 induced Parkin translocation. PC6 cells were transfected with Parkin-EYFP, PGAM5 wt and PARL or OMA1. ****P < 0.0001, n = 6 dishes, 20 fields per dish, One-way ANOVA followed by Sidak's multiple comparisons test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: PARL-FLAG , Addgene , Cat# 13639.

Techniques: Activity Assay, Mutagenesis, Translocation Assay, Transfection, Over Expression, Expressing

Journal: Redox Biology

Article Title: A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy

doi: 10.1016/j.redox.2021.102186

Figure Lengend Snippet:

Article Snippet: PARL-FLAG , Addgene , Cat# 13639.

Techniques: Recombinant, Lysis, Extraction, Blocking Assay, Isolation, DC Protein Assay, Caspase-Glo Assay, shRNA, Generated, Plasmid Preparation, Synthesized, Variant 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: (A) Illustration of the KRasG12D-driven lung adenocarcinoma mouse model. Mice carrying the conditional Lox-Stop-Lox (LSL)-KRasG12D allele develop lung adenocarcinomas upon intratracheal instillation of Adeno-Cre which excises the Stop cassette allowing the constitutive expression of oncogenic KRasG12D. (B) Kaplan-Meier survival curve of KRasG12D;Hace1+/+ (n=10) and littermate KRasG12D;Hace1–/– (n=14) mice injected with Adeno-Cre on day 0. **** P<0.0001 (log-rank test). (C) Representative pictures of haematoxylin and eosin (H&E) stained-lung sections and (D) tumor-to-lung ratios at week 8 and 16 post lung cancer induction for KRasG12D;Hace1+/+Rac1+/+, KRasG12D;Hace1–/–, KRasG12D;Rac1fl/fl, and KRasG12D;Hace1–/–Rac1fl/fl mice. Scale bars, 1 mm for 10x images and 50μm for 40x images of lung sections. * P<0.05, ** P<0.01, **** P<0.0001 (One-way ANOVA, Tukey’s post-hoc test, n 5 mice per cohort). (E) Numbers of benign (hyperplasias and adenomas), pre-invasive (atypical adenomatous hyperplasias) and malignant (minimally invasive adenocarcinomas (MIA / “pre-adenocarcinoma”), adenocarcinoma) tumor foci at week 8 post lung cancer induction for KRasG12D;Hace1+/+Rac1+/+, KRasG12D;Hace1–/–, KRasG12D;Rac1fl/fl and KRasG12D;Hace1–/–Rac1fl/fl mice. * P<0.05, ** P<0.01, **** P<0.0001 (Two-way ANOVA, Tukey’s post-hoc test, n≥4 mice per cohort). Data in (D) and (E) are presented as mean values ± SEM.

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

Techniques: Expressing, Injection, 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) Representative pictures of Ki67 and γH2AX immunostaining of lungs at week 16 post lung cancer induction for KRasG12D;Hace1+/+Rac1+/+, KRasG12D;Hace1–/–, KRasG12D;Rac1fl/fl and KRasG12D;Hace1–/–Rac1fl/fl mice. Scale bars, 50μm. (B) Quantification of Ki67+ and (C) γH2AX+ tumor cells at week 8 (left panels) and 16 (right) post lung cancer induction for KRasG12D;Hace1+/+Rac1+/+, KRasG12D;Hace1–/–, KRasG12D;Rac1fl/fl and KRasG12D;Hace1–/–Rac1fl/fl mice. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 (One-way ANOVA, Tukey’s post-hoc test, n 3 mice per cohort). Data in (B) and (C) are presented as mean values SEM.

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

Techniques: Immunostaining

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) Representative images of DHE-stained lung sections and (B) quantification of the DHE fluorescence intensity at week 16 post lung cancer induction for KRasG12D;Hace1+/+Rac1+/+, KRasG12D;Hace1–/–, KRasG12D;Rac1fl/fl and KRasG12D;Hace1–/–Rac1fl/fl mice (Student’s two-tailed t-test, n≥5 mice per cohort). Red staining indicates the presence of ROS. Scale bars, 100μm. Data in (B) are presented as mean values SEM. (C) Detection of active RAC1 in Hace1 mutant lung tumor cells. Tumor cells, isolated from KRasG12D;Hace1+/+ (n=2) and KRasG12D;Hace1–/– (n=3) mice at week 12 post lung cancer induction, were treated with EGF (50ng/ml) for 5 minutes, followed by GST-PAK pull-down of active GTP-RAC1 and immunoblotting for total RAC1 and HACE1. β-Actin is shown as loading control.

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

Techniques: Staining, Fluorescence, Two Tailed Test, Mutagenesis, Isolation, Western Blot, Control

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: In vitro ubiquitylation assay 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, Ubiquitin Proteomics, Control, Virus, 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: In vitro ubiquitylation assay His-tagged recombinant human RAC1 (Cytoskeleton, RC01) and RAC2 (Cytoskeleton, RC02) were preloaded with GTP or GDP as described ( 41 ).

Techniques: Expressing, Mutagenesis, Amplification, Gene Expression, RNA Sequencing

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

Techniques: Activity Assay

Journal: iScience

Article Title: Friendly fungi: Tropical insect families form partnerships with intracellular fungi related to pathogens

doi: 10.1016/j.isci.2024.110674

Figure Lengend Snippet:

Article Snippet: Sordaria fimicola , Carolina Biological Supply , Cat# 156290.

Techniques: Sequencing, Recombinant, Saline, Plasmid Preparation, Wright Stain, Picogreen Assay, Negative Control, Software

Journal: Oncogene

Article Title: KIBRA exhibits MST-independent functional regulation of the Hippo signaling pathway in mammals.

doi: 10.1038/onc.2012.196

Figure Lengend Snippet: Figure 4. KIBRA expression antagonises YAP-induced transcriptional regulation. (a) mRNA expression of the YAP target genes BMP2, FGF1, RASSF4 and PDGFb was assessed by quantitative real-time PCR in MCF10A-YAP cells overexpressing KIBRA, Willin or Merlin. mRNA levels were compared with the empty vector control MCF10A-YAP cells. Willin and KIBRA overexpression increased BMP2 and RASSF4 mRNA levels and decreased FGF1 and PDGFb mRNA levels. Means were calculated from Ct values from two analyses conducted in triplicate. Error bars represent ±s.e. (n ¼ 6). MCF10A-YAP-vector vs MCF10A-YAP-KIBRA or MCF10A-YAP-Willin for all the analysed genes: *Po0.05, **Po0.01, ***Po0.001 Student’s t-test. MCF10A-YAP-vector vs MCF10A-YAP-Merlin: CTGF (P ¼ 0.129), FGF1 (P ¼ 0.005), BMP2 (P ¼ 0.585), RASSF4 (P ¼ 0.307), PDGFb (P ¼ 0.06), Student’s t-test. (b) Immunoblot analysis of KIBRA, Willin or Merlin overexpression in MCF10A-YAP cells, showing 2.7, 1.8 and 8.6-fold increases compared to empty vector control. b-actin was used as a loading control.

Article Snippet: All experiments were carried out at 80–90% confluency. pBABE Nf2 (Johnson et al.45) was obtained from Addgene (plasmid 14116).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Over Expression, Western Blot