Journal: Communications Biology

Article Title: Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins

doi: 10.1038/s42003-025-08222-5

Figure Lengend Snippet: A Schematic representation of the domain structure of DRFs. N-terminal domain (N, black); GTPase binding domain (GBD, red); Diaphanous Inhibitory Domain (DID, orange); Interhelical Domain (IH, yellow); Dimerization Domain (DD, green); Intrinsically Disordered Region (IDR, cyan); Coiled Coil Domain (CC, brown); Formin Homology Domains 1 and 2 (FH1, gray, and FH2, pink); Diaphanous Autoinhibitory Domain (DAD, blue); C-Terminal Domain (C, gold). Diaphanous homologs 1, 2, and 3 (DIAPH1, 2, and 3); Formin Like Protein 1 (FMNL1); Inverted Formin 2 (INF2); Formin Homology Domain Containing Protein 1 (FHOD1); Disheveled Associated Activator of Morphogenesis 1 (DAAM1); Constitutively active DIAPH1 (DIAPH1 ΔDAD ); DIAPH 1 mimic of DIAPH 2 (DIAPH1/2); GBD-DID construct (GDID); DAD construct (DAD). B Ribbon diagram showing two views of a superposition of the DID-DAD structure (PDB entry 8FG1) and the GBD-DID-RhoC structure (PDB entry 1Z2C); RhoC amino acid sequence is 92% identical to RhoA and backbone RMSD between RhoA (PDB entry 1A2B) and RhoC (PDB entry 2GCO) is ~1 Å. The residues of DAD that sterically clash with the GBD because of Rho binding are shown in cyan (box). The color scheme is the same as in panel A and RhoC is in purple. C Thermodynamic cycle of sequential DAD, D, and RhoA, R, binding to GBD-DID, G, and the corresponding equilibrium expressions where [R] and [D] are the concentrations of RhoA and DAD, K d R and K d D , are the intrinsic dissociation constants for RhoA and DAD binding to GDID, and K d R/D and K d D/R are the apparent dissociation constants for RhoA binding to GDID-DAD and DAD binding to RhoA-GDID, respectively. α is a cooperativity factor where α > 1 denotes positive cooperativity and α < 1 denotes negative cooperativity.

Article Snippet: Plasmid pGEX-4T3-RhoA-Q63L, which confers ampicillin resistance and expresses N-terminal GST tagged RhoA Q63L (21,753 Da) was purchased from Addgene (Plasmid #12961).

Techniques: Binding Assay, Construct, Sequencing

Journal: Communications Biology

Article Title: Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins

doi: 10.1038/s42003-025-08222-5

Figure Lengend Snippet: A Superimposed 1 H- 15 N HSQC spectra of 50 µM [ U - 15 N]-DAD in the absence (red) and presence (black) of 60 µM GDID (black). The DAD Q1207 cross peak, which is part of the DID-DAD binding interface, undergoes a large change in chemical shift between the bound and free states. B Superimposed 1 H- 15 N HSQC spectra of 5 µM [ U - 15 N]-DAD with 60 µM of RhoA and 50 µM GDID (black) and 5 µM [ U - 15 N]-DAD with 19 µM of RhoA and 13 µM GDID (red). Free DAD is evident at the lower concentration of the RhoA-GDID complex. C NMR-generated isothermal titration of 5 µM [ U - 15 N]-DAD binding to the RhoA-GDID complex and fit using a ‘total binding, accounting for ligand depletion’ model to estimate K d D/R of 27 ± 5 μM for DAD binding to the RhoA-GDID complex. D ELISA-generated isotherm of RhoA binding to 10 nM GDID fit to a “one site-total and nonspecific binding” model to estimate K d R of 8.9 ± 2.4 nM. E ELISA-generated isotherm of RhoA binding to 10 nM GDID-DAD fit to a ‘one site-total and nonspecific binding’ model to estimate K d R/D of 840 ± 200 nM. All values for ELISA experiments are represented as mean ± SD and were performed as N = 3 independent experiments. Values from individual experiments are shown as gray dots. Note that the decrease in the plateau values observed in the ELISA titrations are due to the Hook or Prozone effect in which high ligand concentrations result in a decrease in signal strength .

Article Snippet: Plasmid pGEX-4T3-RhoA-Q63L, which confers ampicillin resistance and expresses N-terminal GST tagged RhoA Q63L (21,753 Da) was purchased from Addgene (Plasmid #12961).

Techniques: Binding Assay, Concentration Assay, Generated, Titration, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins

doi: 10.1038/s42003-025-08222-5

Figure Lengend Snippet: A Plot of the radius of gyration, R G , versus the number of amino acids in a theoretical polypeptide. The parameter ν characterizes the dimensions of the amino acid chain where 0.60, red, represents an expanded coil and 0.30, maroon, represents the most compact globule state. B Monte-Carlo simulation using ν = 0.60 showing the theoretical persistence length between DID and DAD domains as a function of the number of amino acids: ~109 Å for the 285 amino acid DIAPH1 and ~90 Å for the 140 amino acids of DIAPH1/2. C Rates of actin polymerization for Diaphanous constructs DIAPH1, D1, DIAPH1/2, D1/2 and DIAPH1 ΔDAD , D1 ΔD , in the presence of RhoA, +R, compared to actin alone, A. Initial rates of polymerization, dF/dt, were determined by fitting the first 11 data points to a linear equation yielding slopes of 62 ± 6 AFU*s −1 for A (red), 66 ± 6 AFU*s −1 for D1 (green), 62 ± 7 AFU*s −1 for D1/2 (purple), 85 ± 5 AFU*s −1 for D1 + R (white), 75 ± 4 AFU*s -1 for D1/2 + R (gray) and 104 ± 7 AFU*s −1 for D1 ΔD (orange). D1 ΔD represents 100% activation and D1 or D1/2 represent 0% activation, respectively. Statistical p -values at 95% confidence are shown on the graph and all values are represented as mean ± SD derived from N = 3 independent experiments.

Article Snippet: Plasmid pGEX-4T3-RhoA-Q63L, which confers ampicillin resistance and expresses N-terminal GST tagged RhoA Q63L (21,753 Da) was purchased from Addgene (Plasmid #12961).

Techniques: Construct, Activation Assay, Derivative Assay

Journal: Communications Biology

Article Title: Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins

doi: 10.1038/s42003-025-08222-5

Figure Lengend Snippet: A Membrane to cytosol ratio of CFP-DIAPH1 fluorescence. The sample sizes for DIAPH1 (D1, green circles), DIAPH1 + CN03 (D1 + CN03, red circles), DIAPH1 ΔDAD (D1 ΔD , cyan squares) and DIAPH1 ΔDAD + CN03 (D1 ΔD + CN03, purple squares) are 31, 44, 51 and 105, respectively. B Membrane to cytosol ratio of phalloidin fluorescence, which binds to F-actin. The sample size for D1 (green circles), D1 + CN03 (red cricles), D1 ΔD (cyan squares) and D1 ΔD + CN03 (purple squares) are 31, 44, 51 and 105, respectively. The analysis uses the ratio of intensities of membrane and cytosol, which decreases the bias caused by alternative actin filaments staining . C Confocal microscope images of AD293 cells transfected with CFP-labeled D1 and D1 ΔD . RhoA activator CN03 was at 1 mg/mL. Cells were stained with Alexa Fluor568 Phalloidin to image filamentous actin and Hoechst 33258 to image the nuclei. Scale bar is 10 µm. The significant cytosolic fraction of overexpressed DIAPH1 after CN03 activation of RhoA or of constitutively active DIAPH1 ΔDAD suggests that there are limited membrane binding sites for DIAPH1 as observed previously . The height of the bar represents the mean and the error bars are ± SEM and statistical p -values at 95% confidence are shown on the graph.

Article Snippet: Plasmid pGEX-4T3-RhoA-Q63L, which confers ampicillin resistance and expresses N-terminal GST tagged RhoA Q63L (21,753 Da) was purchased from Addgene (Plasmid #12961).

Techniques: Membrane, Fluorescence, Staining, Microscopy, Transfection, Labeling, Activation Assay, Binding Assay

Journal: Communications Biology

Article Title: Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins

doi: 10.1038/s42003-025-08222-5

Figure Lengend Snippet: A Rates of actin polymerization for Diaphanous constructs DIAPH1, D1, in the presence of 3 µM ctRAGE, 3 C, 3 µM RhoA, 3 R, or 1, 2, or 3 µM of both ctRAGE and RhoA, 1CR, 2CR, 3 CR, respectively, compared to actin alone, A. Initial rates of polymerization, dF/dt, were determined by fitting the first 13 data points to a linear equation yielding slopes of 11.1 ± 0.4 AFU*s -1 for A (red), 11.6 ± 0.3 AFU*s −1 for D1 (beige), 13.1 ± 1.0 AFU*s −1 for D1 + 3C (light blue), 14.6 ± 0.8 AFU*s -1 for D1 + 3 R (dark blue), 17.8 ± 0.7 AFU*s −1 for D1 + 1RC (light green), 20.6 ± 0.8 AFU*s -1 for D1 + 2RC (green), and 22.0 ± 0.9 AFU*s −1 for D1 + 3RC (dark green). Statistical p-values at 95% confidence are shown on the graph. B ELISA-based isotherm of RhoA binding to DIAPH1. The data were fit to “one site-specific binding” curve to estimate the dissociation constant, K d R/D , for the interaction. C ELISA-based binding isotherm of RhoA binding to DIAPH1 in the presence of ctRAGE. The data were fit to a “one site-specific binding” curve to estimate the dissociation constant, K d R/D , for the interaction in the presence of ctRAGE. All values are represented as the mean ± SD and derived from N = 3 independent experiments. Values from individual experiments are shown as gray dots.

Article Snippet: Plasmid pGEX-4T3-RhoA-Q63L, which confers ampicillin resistance and expresses N-terminal GST tagged RhoA Q63L (21,753 Da) was purchased from Addgene (Plasmid #12961).

Techniques: Construct, Enzyme-linked Immunosorbent Assay, Binding Assay, Derivative Assay

Journal: Communications Biology

Article Title: Negative cooperativity regulates ligand activation of DIAPH1 and other diaphanous related formins

doi: 10.1038/s42003-025-08222-5

Figure Lengend Snippet: A Mechanism of DIAPH1-RhoA localization and activation (i-viii). Color scheme of the DIAPH1 domains is as in Fig. . RhoA-GDP is pink and RhoA-GTP is purple. To localize at the membrane RhoA is farnasylated and unphosphorylated. Other cytoskeleton remodeling proteins, such as profilin, cofilin1/ADF, or cross-linkers that contribute to the described mechanism are omitted for clarity; their roles in this model are a subject for future investigations. B Three-dimensional plot showing K d D/R as a function of α and [RhoGTPase] for DRFs. α and [RhoGTPase] are on a logarithmic scale. The line represents α = 1 and has zero curvature in the [RhoGTPase] dimension. The plot was constructed using K d D and K d R derived from the RhoA-DIAPH1 interaction. Derivations are in Supplementary Appendix . C Three-dimensional plot of unbound DAD, 1-F DB , as a function of RhoGTPase concentration and [DAD] eff . [DAD] eff and [RhoGTPase] are on a logarithmic scale. The gradient from deep blue to yellow shows increasing DIAPH1 activation. Derivations are in Supplementary Appendix . The plot was constructed using K d R , K d D and α derived from the RhoA-DIAPH1 interaction.

Article Snippet: Plasmid pGEX-4T3-RhoA-Q63L, which confers ampicillin resistance and expresses N-terminal GST tagged RhoA Q63L (21,753 Da) was purchased from Addgene (Plasmid #12961).

Techniques: Activation Assay, Membrane, Construct, Derivative Assay, Concentration Assay

Journal: Cell death & disease

Article Title: RhoA balances microglial reactivity and survival during neuroinflammation.

doi: 10.1038/s41419-023-06217-w

Figure Lengend Snippet: Fig. 2 RhoA regulates microglial metabolic reprogramming during inflammation. HMC3 microglia expressing the ATP biosensor (A), Glucose biosensor (B), Lactate biosensor (C), or Pyruvate biosensor (D) were transfected with RhoA Q63L (red) or RhoA WT (blue) and exposed to LPS (1 µg/ml; 20 min) (n = 15-30 cells per group from 3 independent experiments for each biosensor). Primary cortical microglia expressing the ATP biosensor (E) or Lactate biosensor (F) were transfected with the RhoA Q63L (red) or RhoA WT (blue) and exposed to LPS (1 µg/ml; 20 min) (n = 6 cells per group from 3 independent experiments for each biosensor). Panels are time-lapse ratio images coded according to the pseudocolor ramps. Graphs (means and SD) display F490/F435 (A and E), FRET/Donor (B), and Donor/FRET (C, D, and F) ratio change at 0 (CT) and 20 min. G Seahorse measurements of bioenergetic parameters in HCM3 microglia expressing RhoA Q63L or RhoA WT. The parameters were calculated based on the OCR following the sequential addition of LPS, oligomycin, FCCP, rotenone, and antimycin A. Results are from at least 3 independent experiments. Graphs show the mean with SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). Scale bars: 20 µm.

Article Snippet: Raichu-RhoA (provided by M. Matsuda [27]), pTriEx-RhoA FLARE.sc Biosensor WT (RRID:Addgene_12150), pTriEx-RhoA FLARE.sc Biosensor Q63L (RRID:Addgene_12151), pTriEx-RhoA FLARE.sc Biosensor T19N (RRID:Addgene_12152), pRK5-myc-RhoA WT (RRID:Addgene_12962), pRK5-mycRhoA Q63L (RRID:Addgene_12964), pRK5-myc-RhoA T19N (RRID:Addgene_12963), EGFP-p65 (RRID:Addgene_111190), GW1-pHRed (RRID:Addgene_31473), GW1CMV-Perceval (RRID:Addgene_21737), Laconic/pcDNA3.1 (+) (RRID:Addgene_118627), Pyronic /pcDNA3.1 (+) (RRID:Addgene_51308), pcDNA3.1 FLII12Pglu-700uDelta6 (RRID:Addgene_17866), Cyto-ABKAR (RRID:Addgene_61510), pLentiEKAR2G2 (RRID:Addgene_40178), Kras-Src FRET biosensor (RRID:Addgene_78302), pFRET-HSP33 cys (RRID:Addgene_16076), pGP-CMV-GCaMP6F (RRID:Addgene_40755), mCherry-Lifeact-7 (RRID:Addgene_54491), pMitoTimer (RRID:Addgene_52659), pCLBW cox8 EGFP mCherry (RRID:Addgene_78520).

Techniques: Expressing, Transfection

Journal: Cell death & disease

Article Title: RhoA balances microglial reactivity and survival during neuroinflammation.

doi: 10.1038/s41419-023-06217-w

Figure Lengend Snippet: Fig. 3 RhoA regulates microglial proinflammatory reactivity. HMC3 microglia expressing a ROS biosensor (A), AMPK biosensor (B), ERK biosensor (C), Src biosensor (D), and GFP-tagged p65 NFkB subunit (E) were transfected with the RhoA Q63L construct or with RhoA WT and then exposed to LPS (1 µg/ml for 20 min) (n = 18–100 cells per group from 3 independent experiments for each biosensor). Panels show time- lapse ratio images or mean fluorescent intensity (MFI) coded according to the pseudocolor ramps. F ELISA (TNF-α or IL-1ß) from culture supernatants of primary cortical microglia transfected with RhoA Q63L or RhoA WT and exposed to LPS (1 µg/ml) for 3 h (n = 4 independent experiments). Graphs are means with SD. *p < 0.05, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). Scale bars: 20 µm.

Article Snippet: Raichu-RhoA (provided by M. Matsuda [27]), pTriEx-RhoA FLARE.sc Biosensor WT (RRID:Addgene_12150), pTriEx-RhoA FLARE.sc Biosensor Q63L (RRID:Addgene_12151), pTriEx-RhoA FLARE.sc Biosensor T19N (RRID:Addgene_12152), pRK5-myc-RhoA WT (RRID:Addgene_12962), pRK5-mycRhoA Q63L (RRID:Addgene_12964), pRK5-myc-RhoA T19N (RRID:Addgene_12963), EGFP-p65 (RRID:Addgene_111190), GW1-pHRed (RRID:Addgene_31473), GW1CMV-Perceval (RRID:Addgene_21737), Laconic/pcDNA3.1 (+) (RRID:Addgene_118627), Pyronic /pcDNA3.1 (+) (RRID:Addgene_51308), pcDNA3.1 FLII12Pglu-700uDelta6 (RRID:Addgene_17866), Cyto-ABKAR (RRID:Addgene_61510), pLentiEKAR2G2 (RRID:Addgene_40178), Kras-Src FRET biosensor (RRID:Addgene_78302), pFRET-HSP33 cys (RRID:Addgene_16076), pGP-CMV-GCaMP6F (RRID:Addgene_40755), mCherry-Lifeact-7 (RRID:Addgene_54491), pMitoTimer (RRID:Addgene_52659), pCLBW cox8 EGFP mCherry (RRID:Addgene_78520).

Techniques: Expressing, Transfection, Construct, Enzyme-linked Immunosorbent Assay

Journal: Cell death & disease

Article Title: RhoA balances microglial reactivity and survival during neuroinflammation.

doi: 10.1038/s41419-023-06217-w

Figure Lengend Snippet: Fig. 5 Inflammation disrupts Ca2+, pH, and mitochondrial homeostasis in RhoA-deficient microglia. A Lifeact fluorescence labeling in RhoA WT, RhoA T19N, or RhoA KO HMC3 microglia (n = 3 independent experiments). B Cell counting in RhoA WT, RhoA Q63L, RhoA T19N, or RhoA KO HMC3 microglia (n = 8 independent experiments). Graph displays mean with SD. ****p < 0.0001 (Two-way ANOVA). RhoA WT, RhoA T19N, or RhoA KO HMC3 microglia expressing a global Ca2+ biosensor (C), mitochondrial Ca2+ biosensor (D), endoplasmic reticulum Ca2+ biosensor (E), pH biosensor (F), MitoTimer biosensor (G), or mitophagy biosensor (H) and exposed to LPS (1 µg/ml; 20 min (C-F) or 60 min (G and H)). Graphs (mean and SD) display fluorescence changes (n = 15 cells per group from 3 independent experiments for each biosensor). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). Scale bars: 20 µm.

Article Snippet: Raichu-RhoA (provided by M. Matsuda [27]), pTriEx-RhoA FLARE.sc Biosensor WT (RRID:Addgene_12150), pTriEx-RhoA FLARE.sc Biosensor Q63L (RRID:Addgene_12151), pTriEx-RhoA FLARE.sc Biosensor T19N (RRID:Addgene_12152), pRK5-myc-RhoA WT (RRID:Addgene_12962), pRK5-mycRhoA Q63L (RRID:Addgene_12964), pRK5-myc-RhoA T19N (RRID:Addgene_12963), EGFP-p65 (RRID:Addgene_111190), GW1-pHRed (RRID:Addgene_31473), GW1CMV-Perceval (RRID:Addgene_21737), Laconic/pcDNA3.1 (+) (RRID:Addgene_118627), Pyronic /pcDNA3.1 (+) (RRID:Addgene_51308), pcDNA3.1 FLII12Pglu-700uDelta6 (RRID:Addgene_17866), Cyto-ABKAR (RRID:Addgene_61510), pLentiEKAR2G2 (RRID:Addgene_40178), Kras-Src FRET biosensor (RRID:Addgene_78302), pFRET-HSP33 cys (RRID:Addgene_16076), pGP-CMV-GCaMP6F (RRID:Addgene_40755), mCherry-Lifeact-7 (RRID:Addgene_54491), pMitoTimer (RRID:Addgene_52659), pCLBW cox8 EGFP mCherry (RRID:Addgene_78520).

Techniques: Labeling, Cell Counting, Expressing

Journal: Cell death & disease

Article Title: RhoA balances microglial reactivity and survival during neuroinflammation.

doi: 10.1038/s41419-023-06217-w

Figure Lengend Snippet: Fig. 2 RhoA regulates microglial metabolic reprogramming during inflammation. HMC3 microglia expressing the ATP biosensor (A), Glucose biosensor (B), Lactate biosensor (C), or Pyruvate biosensor (D) were transfected with RhoA Q63L (red) or RhoA WT (blue) and exposed to LPS (1 µg/ml; 20 min) (n = 15-30 cells per group from 3 independent experiments for each biosensor). Primary cortical microglia expressing the ATP biosensor (E) or Lactate biosensor (F) were transfected with the RhoA Q63L (red) or RhoA WT (blue) and exposed to LPS (1 µg/ml; 20 min) (n = 6 cells per group from 3 independent experiments for each biosensor). Panels are time-lapse ratio images coded according to the pseudocolor ramps. Graphs (means and SD) display F490/F435 (A and E), FRET/Donor (B), and Donor/FRET (C, D, and F) ratio change at 0 (CT) and 20 min. G Seahorse measurements of bioenergetic parameters in HCM3 microglia expressing RhoA Q63L or RhoA WT. The parameters were calculated based on the OCR following the sequential addition of LPS, oligomycin, FCCP, rotenone, and antimycin A. Results are from at least 3 independent experiments. Graphs show the mean with SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). Scale bars: 20 µm.

Article Snippet: Raichu-RhoA (provided by M. Matsuda [27]), pTriEx-RhoA FLARE.sc Biosensor WT (RRID:Addgene_12150), pTriEx-RhoA FLARE.sc Biosensor Q63L (RRID:Addgene_12151), pTriEx-RhoA FLARE.sc Biosensor T19N (RRID:Addgene_12152), pRK5-myc-RhoA WT (RRID:Addgene_12962), pRK5-mycRhoA Q63L (RRID:Addgene_12964), pRK5-myc-RhoA T19N (RRID:Addgene_12963), EGFP-p65 (RRID:Addgene_111190), GW1-pHRed (RRID:Addgene_31473), GW1CMV-Perceval (RRID:Addgene_21737), Laconic/pcDNA3.1 (+) (RRID:Addgene_118627), Pyronic /pcDNA3.1 (+) (RRID:Addgene_51308), pcDNA3.1 FLII12Pglu-700uDelta6 (RRID:Addgene_17866), Cyto-ABKAR (RRID:Addgene_61510), pLentiEKAR2G2 (RRID:Addgene_40178), Kras-Src FRET biosensor (RRID:Addgene_78302), pFRET-HSP33 cys (RRID:Addgene_16076), pGP-CMV-GCaMP6F (RRID:Addgene_40755), mCherry-Lifeact-7 (RRID:Addgene_54491), pMitoTimer (RRID:Addgene_52659), pCLBW cox8 EGFP mCherry (RRID:Addgene_78520).

Techniques: Expressing, Transfection

Journal: Cell death & disease

Article Title: RhoA balances microglial reactivity and survival during neuroinflammation.

doi: 10.1038/s41419-023-06217-w

Figure Lengend Snippet: Fig. 3 RhoA regulates microglial proinflammatory reactivity. HMC3 microglia expressing a ROS biosensor (A), AMPK biosensor (B), ERK biosensor (C), Src biosensor (D), and GFP-tagged p65 NFkB subunit (E) were transfected with the RhoA Q63L construct or with RhoA WT and then exposed to LPS (1 µg/ml for 20 min) (n = 18–100 cells per group from 3 independent experiments for each biosensor). Panels show time- lapse ratio images or mean fluorescent intensity (MFI) coded according to the pseudocolor ramps. F ELISA (TNF-α or IL-1ß) from culture supernatants of primary cortical microglia transfected with RhoA Q63L or RhoA WT and exposed to LPS (1 µg/ml) for 3 h (n = 4 independent experiments). Graphs are means with SD. *p < 0.05, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). Scale bars: 20 µm.

Article Snippet: Raichu-RhoA (provided by M. Matsuda [27]), pTriEx-RhoA FLARE.sc Biosensor WT (RRID:Addgene_12150), pTriEx-RhoA FLARE.sc Biosensor Q63L (RRID:Addgene_12151), pTriEx-RhoA FLARE.sc Biosensor T19N (RRID:Addgene_12152), pRK5-myc-RhoA WT (RRID:Addgene_12962), pRK5-mycRhoA Q63L (RRID:Addgene_12964), pRK5-myc-RhoA T19N (RRID:Addgene_12963), EGFP-p65 (RRID:Addgene_111190), GW1-pHRed (RRID:Addgene_31473), GW1CMV-Perceval (RRID:Addgene_21737), Laconic/pcDNA3.1 (+) (RRID:Addgene_118627), Pyronic /pcDNA3.1 (+) (RRID:Addgene_51308), pcDNA3.1 FLII12Pglu-700uDelta6 (RRID:Addgene_17866), Cyto-ABKAR (RRID:Addgene_61510), pLentiEKAR2G2 (RRID:Addgene_40178), Kras-Src FRET biosensor (RRID:Addgene_78302), pFRET-HSP33 cys (RRID:Addgene_16076), pGP-CMV-GCaMP6F (RRID:Addgene_40755), mCherry-Lifeact-7 (RRID:Addgene_54491), pMitoTimer (RRID:Addgene_52659), pCLBW cox8 EGFP mCherry (RRID:Addgene_78520).

Techniques: Expressing, Transfection, Construct, Enzyme-linked Immunosorbent Assay

Journal: Cell death & disease

Article Title: RhoA balances microglial reactivity and survival during neuroinflammation.

doi: 10.1038/s41419-023-06217-w

Figure Lengend Snippet: Fig. 5 Inflammation disrupts Ca2+, pH, and mitochondrial homeostasis in RhoA-deficient microglia. A Lifeact fluorescence labeling in RhoA WT, RhoA T19N, or RhoA KO HMC3 microglia (n = 3 independent experiments). B Cell counting in RhoA WT, RhoA Q63L, RhoA T19N, or RhoA KO HMC3 microglia (n = 8 independent experiments). Graph displays mean with SD. ****p < 0.0001 (Two-way ANOVA). RhoA WT, RhoA T19N, or RhoA KO HMC3 microglia expressing a global Ca2+ biosensor (C), mitochondrial Ca2+ biosensor (D), endoplasmic reticulum Ca2+ biosensor (E), pH biosensor (F), MitoTimer biosensor (G), or mitophagy biosensor (H) and exposed to LPS (1 µg/ml; 20 min (C-F) or 60 min (G and H)). Graphs (mean and SD) display fluorescence changes (n = 15 cells per group from 3 independent experiments for each biosensor). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). Scale bars: 20 µm.

Article Snippet: Raichu-RhoA (provided by M. Matsuda [27]), pTriEx-RhoA FLARE.sc Biosensor WT (RRID:Addgene_12150), pTriEx-RhoA FLARE.sc Biosensor Q63L (RRID:Addgene_12151), pTriEx-RhoA FLARE.sc Biosensor T19N (RRID:Addgene_12152), pRK5-myc-RhoA WT (RRID:Addgene_12962), pRK5-mycRhoA Q63L (RRID:Addgene_12964), pRK5-myc-RhoA T19N (RRID:Addgene_12963), EGFP-p65 (RRID:Addgene_111190), GW1-pHRed (RRID:Addgene_31473), GW1CMV-Perceval (RRID:Addgene_21737), Laconic/pcDNA3.1 (+) (RRID:Addgene_118627), Pyronic /pcDNA3.1 (+) (RRID:Addgene_51308), pcDNA3.1 FLII12Pglu-700uDelta6 (RRID:Addgene_17866), Cyto-ABKAR (RRID:Addgene_61510), pLentiEKAR2G2 (RRID:Addgene_40178), Kras-Src FRET biosensor (RRID:Addgene_78302), pFRET-HSP33 cys (RRID:Addgene_16076), pGP-CMV-GCaMP6F (RRID:Addgene_40755), mCherry-Lifeact-7 (RRID:Addgene_54491), pMitoTimer (RRID:Addgene_52659), pCLBW cox8 EGFP mCherry (RRID:Addgene_78520).

Techniques: Labeling, Cell Counting, Expressing