Journal: Frontiers in Neuroanatomy

Article Title: Machine learning-based segmentation of the rodent hippocampal CA2 area from Nissl-stained sections

doi: 10.3389/fnana.2023.1172512

Figure Lengend Snippet: Preprocessing of fluorescent images of the CA2 and surrounding areas in the hippocampus. (A) Representative images of a section of the mouse hippocampus immunostained with RGS14 (green, middle) and counterstained with Nissl (blue, left). A merged image is also shown (right). (B) Workflow of denoising the original Nissl-stained image using custom-written scripts. An original image is binarized using a command in ImageJ, which is specified by single quotes (i–ii). The binarized image is despeckled and eroded/dilated (ii–iii). Then, a mask image is created (iii–iv). Using the mask image, the original image is denoised (iv–v). Each arrow in purple indicates transformation from a previous image to the next one using ImageJ, while green arrows signify generation of a denoised image from masked and original images based on a principle of Hadamard product using Python. (C) Workflow of creating a labeled image of the CA2 area. A mask image of the pyramidal cell layer is created from the original image (i–ii) in the similar manner as panel (B) and then manually segmented by skilled experimenters to create a mask image that labels the CA2 area (ii–iii).

Article Snippet: Sections were blocked with 10% goat serum and 0.3% Triton X-100 in PBS for 1 h at room temperature and incubated with a mouse primary antibody against RGS14 (1:500, 75–170, NeuroMab, CA, USA) for 16 h at 4°C.

Techniques: Staining, Transformation Assay, Labeling

Journal: Frontiers in Neuroanatomy

Article Title: Machine learning-based segmentation of the rodent hippocampal CA2 area from Nissl-stained sections

doi: 10.3389/fnana.2023.1172512

Figure Lengend Snippet: Application of CAseg to the prairie vole hippocampus. (A) Representative image of a section of the prairie vole hippocampus immunostained for RGS14 (green) and counterstained with Nissl stain (blue). (B) Representative images of the prairie vole CA2 area predicted by CAseg (green, left). Images labeled as the CA2 area (red, middle), each of which corresponds to the predicted image (green). Images showing the overlap between the predicted and labeled images (yellow, right). (C) The F1-scores of CAseg predicting test images of mice (red) and prairie voles (blue). Each point represents the F1-score of one image.

Article Snippet: Sections were blocked with 10% goat serum and 0.3% Triton X-100 in PBS for 1 h at room temperature and incubated with a mouse primary antibody against RGS14 (1:500, 75–170, NeuroMab, CA, USA) for 16 h at 4°C.

Techniques: Staining, Labeling

Journal: eLife

Article Title: Increased cortical plasticity leads to memory interference and enhanced hippocampal-cortical interactions

doi: 10.7554/eLife.84911

Figure Lengend Snippet: ( A ) Half the animals were injected with a lentivirus for the overexpression of RGS14414 increasing plasticity in the prelimbic cortex, the other half had a control virus. These animals were included in either Experiment 1 (behavioral control), or were implanted with canula to the prelimbic cortex for Experiment 2 (pharmacological controls, total n=65 with n=16–17 per experimental group). ( B ) Immunohistochemistry for RGS14 expression in treated and control animals (purple, cyan DAPI staining). ( C ) Object Space Task training contains five trials with 45 min inter-trial-intervals. In Overlapping one location always contains an object, while the other object moves each trial. In Stable the configuration always remains the same. Discrimination Index (exploration time moved-not moved/sum) over training trials show slowly rising discrimination in Overlapping and not Stable with preference for the less often used locations especially in the 5th trial as expected (rmANOVA condition F 1,65 =16.9 p<0.001, conditionXtrial F 4, 260 =3.3 p=0.012, all other p>0.37, DI interference and exploration times see ). Of note, object locations and configurations were counterbalanced across animals. ( D ) 24 hr after training animals had an interference trial (10 min, different and same configuration as last training trial for Stable and Overlapping, respectively) followed by a test trial another 48 hr later (again different and same configuration as interference trial for Stable and Overlapping, respectively). In the control conditions (right side), for Experiment 1 there was no interference and in Experiment 2 animals were infused with anisomycin after the interference trial. Virus control grey, RGS-overexpressing purple, lighter shades experiment 1 (with or without interference trial), darker shades experiment 2 (with or without anisomycin infusion). There was a significant interaction where interference had the opposite effect in each condition according to virus manipulation (rmANOVA with condition, interference/drug, experiment, virus; cond*int/drug*virus F 1,61 =13.2 p<0.001; stable with interference t 63 =2.1 p=0.039, overlapping with interference t 63 =3.1 p=0.003, other p>0.12). ( E ) One-trial control followed 48 hr later by test, RGS14-overexpressing animals performed better than controls (t 30 =2.2 p=0.037). ( F ) Model-fitting show that RGS14-overexpressing animals have a higher learning rate α (KS-D=0.46 p=0.044). *p<0.05, **p<0.01.

Article Snippet: First, the target sections were selected, rinsed in PBS, and incubated overnight at 4 °C with the rabbit polyclonal anti-RGS14 antibody (Novus biological, NBP1-31174; dilution 1:500).

Techniques: Injection, Over Expression, Control, Virus, Immunohistochemistry, Expressing, Staining

Journal: eLife

Article Title: Increased cortical plasticity leads to memory interference and enhanced hippocampal-cortical interactions

doi: 10.7554/eLife.84911

Figure Lengend Snippet: ( A ) Animals received RGS14 overexpressing (n=4) or control (n=4) virus and were implanted with a hyperdrive (10 tetrode prelimbic, 6 tetrodes hippocampus) three weeks later. ( B ) Animals ran the three conditions of the Object Space Task (OS, Stable and Overlapping as described above, Random with constantly moving objects) and a home cage control (HC, same structure of the day but remained awake in recording box during trial periods). ( C ) Controls had more NonREM sleep (KS-D=0.17 p=0.05) and no change in REM sleep (KS-D=0.15 p=0.12). ( D ) NonREM bout length was longer in controls (MW U=6887019 p<0.0001) but REM length was shorter (MW U=501,849 p=0.048. ( E ) In both groups, hippocampal delta rates were higher than cortical rates (p<0.0001). The rate of delta oscillations in the cortex increased in controls (KS-D=0.11 p=0.007) but not in RGS14 after OS, the rate of hippocampal delta oscillations was overall higher for RGS14 than controls (KS-D=0.31; p<0.0001). ( F ). In controls delta oscillations were larger in the cortex than hippocampus, in contrast in RGS14 the cortex showed the same amplitude delta-waves as the hippocampus (Cortex Con vs RGS14 KS-D=0.34 p<0.0001). In both groups and brain areas delta amplitude increased after OS (KS-D >0.02 p<0.0001)). ( G ) There was a significant interaction in duration of On-Off periods underlying delta-waves with RGS14-overexpressing animals presenting with longer Off-periods (groupXtype interaction ANOVA F 1,164 =9.0 p=0.0031, type F 1,164 =65.0 p<0.0001). But the time course of on-off durations did not differ between groups (PTXtype interaction ANOVA F 3,152 =946.6 p<0.0001, no interaction with group all p>0.8) ( H ) RGS14-overexpresssing animals had more neurons with lower firing rates (KS-D=0.36 p<0.0001, for split by other states see ). ( I ) Division of neurons according to their firing rates (Chi-square 4 =20.13 p=0.0005). ( J ) Spikes were less phase locked to the slow oscillation phase (circ stats p<0.0001 for each neuron group) and less G4-5 neurons led to less spikes during the upstate in RGS14. ( K ) Firing rates during the pre-task period, only including wake since few animals slept (KS-D=0.41 p<0.0001) ( L ) Firing rates on OS days normalized to Pre-Task (wake). From left to right during task (trials, left), post-trial periods wake in recording box, and during NonREM sleep. RGS14 overall showed larger increases in firing rates from pre-task than controls (ANOVA virus F 1, 2545 = 52,43 p<0.0001). In all time periods RGS14 showed an increase to pre-task (one-sample t-test to 1 P =0.003–0001), in Controls during task there was an increase (p=0.0004) during PT wake no change (p=0.3), and during PT NonREM a decrease in firing rates (p=0.0005) Control grey, RGS-overexpressing purple, darker shades home cage (HC), lighter shades Object Space Task (OS), *p<0.05, **p<0.01,****p<0.0001.

Article Snippet: First, the target sections were selected, rinsed in PBS, and incubated overnight at 4 °C with the rabbit polyclonal anti-RGS14 antibody (Novus biological, NBP1-31174; dilution 1:500).

Techniques: Control, Virus

Journal: eLife

Article Title: Increased cortical plasticity leads to memory interference and enhanced hippocampal-cortical interactions

doi: 10.7554/eLife.84911

Figure Lengend Snippet: ( A ) Brain area interactions are known in Task and REM as theta coherence , in NonREM as oscillatory coupling. ( B ) RGS14 showed higher theta coherence (treatment F 1,285 =31.8 p<0.001). Only controls showed higher coherence after learning (controls OS vs HC F 1,144 =5.2 p=0.024, RGS14 OS vs HC F 1,141 =0.6 p=0.44). ( C ) RGS14 showed generally higher rates of oscillations sequences (treatment F 1,856 =18.7 p<0.001), but only in controls could a learning-dependent increase be observed (controls OS vs HC F 1,420 =12.3 p=0.001, RGS OS vs HC F 1,436 =1.3 p=0.25). Control grey, RGS-overexpressing purple, darker shades home cage (HC), lighter shades Object Space Task (OS) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: First, the target sections were selected, rinsed in PBS, and incubated overnight at 4 °C with the rabbit polyclonal anti-RGS14 antibody (Novus biological, NBP1-31174; dilution 1:500).

Techniques: Control

Journal: eLife

Article Title: Increased cortical plasticity leads to memory interference and enhanced hippocampal-cortical interactions

doi: 10.7554/eLife.84911

Figure Lengend Snippet: ( A ) RGS14-overexpressing animals showed changes in 1. ripple rate, 2. amplitude, and 3. frequency (rate KS-D=0.33 p<0.0001, amplitude KS-D=0.13 p<0.0001, frequency KS-D=0.13 p<0.0001). In both groups OS led to a decrease in amplitude and increase in frequency (vehicle KS-D=0.06 p<0.0001, RGS KS-D=0.25 p<0.0001). 4. On the right hippocampal (Hpc) spectral profile 1 s before and after the ripple in the 100–300 Hz range (black Con, purple RGS, dotted Con vs RGS with statistically significant contrast with pixel-based correction for multiple comparison, grey con higher, purple RGS14 higher). 5. Ripples showed less slow oscillation (SO) phase locking in RGS (phase lock circ stat. p<0.0001) ( B ) 1. This was also reflected in decreased delta power around the ripple (Prelimbic PrL spectral profile 1 s before and after the ripple in the 0–20 Hz range). 2. Time-frequency granger analysis showed higher delta Prl→Hpc and 3. higher theta/beta Hpc→Prl directional connectivity (black Con, purple RGS, dotted Con vs RGS with statistically significant contrast with pixel-based correction for multiple comparison). ( C ) Neural firing during ripples in prelimbic cortex. From left to right: 1. individual neuron response (each row one neuron) aligned to ripple (middle); 2. average response across neurons (rmANOVA treat*time F 98,15288 =2.7 p<0.001); 3. neurons were categorized into types with ripple suppressed (blue), ripple neutral (grey) and ripple active (orange, Chi-square 3.46 p=0.18); 4.for each types the response at baseline (200–120ms before ripple), pre-ripple (120 ms-40ms before ripple), during ripple (40ms before – 40ms after ripple peak), and post-ripple (40 ms-120ms after ripple, rmANOVA treat*ripple response F 2,162 =3.2 p=0.043, post-hoc RGS vs. Con active neurons during p=0.015 and after p=0.003). ( D ) Same ripple response as in C.2. but now split for the five firing-rate defined neuron groups. Especially faster firing neurons (veh G2-G5, RGS14 G4 and G5) showed higher ripple responses. Control grey, RGS-overexpressing purple, darker shades home cage (HC), lighter shades Object Space Task (OS), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: First, the target sections were selected, rinsed in PBS, and incubated overnight at 4 °C with the rabbit polyclonal anti-RGS14 antibody (Novus biological, NBP1-31174; dilution 1:500).

Techniques: Comparison, Control

Journal: eLife

Article Title: Increased cortical plasticity leads to memory interference and enhanced hippocampal-cortical interactions

doi: 10.7554/eLife.84911

Figure Lengend Snippet: ( A ) For each condition the percent of neurons that are ripple modulated (suppressed/blue or active/orange) or not (neutral/grey). In controls Overlapping and Random had more ripple-modulated cells, in RGS14 this was already the case for Home Cage and Stable (Chi-square combining Sup and Act to avoid 0, Con Chi-square 3 =17.1 p=0.0007. RGS14 Chi-square 3 =3.8 p=0.28.) ( B ). Ripple response for each condition. There was a significant treatment, condition, time interaction (rmANOVA, Time F 3,450 =24.6 p<0.001, treatment F 1,150 =4.4 p=0.037, TimeXtreatment F 3,450 =4.3 p=0.005, TreatmentXCondition F 3,150 =2.9 p=0.038, timeXtreatmentXcondition F 9,450 =1.1 p=0.005). Baseline (200–120ms before ripple), pre-ripple (120 ms-40ms before ripple), during ripple (40ms before – 40ms after ripple peak), and post-ripple (40 ms-120ms after ripple).( C ). Single and 2-Osc ripple events split for conditions (normalized to count of all ripple events). In vehicles overlapping and random less ripples occurred as single ripples (R) but more followed delta waves (DR), which was not the case for RGS14 (rmANOVA type F 3,681 =2269.9 p<0.0001, TypeXTreatment F 3,681 =4.2 p=0.006, TypeXCondition F 3,681 =2.4 p=0.013, TypeXTreatmentXCondtion F 9,681 =2.3 p=0.018). *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: First, the target sections were selected, rinsed in PBS, and incubated overnight at 4 °C with the rabbit polyclonal anti-RGS14 antibody (Novus biological, NBP1-31174; dilution 1:500).

Techniques:

Journal: eLife

Article Title: Increased cortical plasticity leads to memory interference and enhanced hippocampal-cortical interactions

doi: 10.7554/eLife.84911

Figure Lengend Snippet: Top: effects of experience (black arrow, all OS vs HC) and complex learning (orange arrows, Overlapping/Random) in controls. Experience dependent effects: increased hippocampal-cortical theta-coherence during task and REM, increased delta-amplitude in NonREM (1) and increased spindle and delta rate leading also to more oscillatory coupling events (2). Complex Learning effects: increase number of neurons responsive to ripples (3) and increased fraction of ripples following a delta-wave (4). Bottom: Changes in RGS14. Learning effects already seen in Home Cage (light purple): increased theta coherence, increase oscillatory coupling rate (2) and increased Delta-Ripple fraction (4). But decreased, hippocampal-like firing rates lead to smaller cortical delta-waves (more similar to hippocampal delta-waves) and less slow oscillation (SO) phase locking (1) as well as decreased cortical response to ripples (3).

Article Snippet: First, the target sections were selected, rinsed in PBS, and incubated overnight at 4 °C with the rabbit polyclonal anti-RGS14 antibody (Novus biological, NBP1-31174; dilution 1:500).

Techniques:

Journal: Basic research in cardiology

Article Title: Regulator of G protein signalling 14 attenuates cardiac remodelling through the MEK-ERK1/2 signalling pathway.

doi: 10.1007/s00395-016-0566-1

Figure Lengend Snippet: Fig. 1 RGS14 is down- regulated in the failing heart and in the experimental hypertrophic models. a Western blot analysis of ANP, b-MHC, and RGS14 protein expression in normal donor hearts and failing hearts from patients with dilated cardiomyopathy (n = 4 per group, *P \ 0.05 vs. normal donor heart). b Western blot analysis of ANP, b-MHC, and RGS14 protein expression in hypertrophic hearts from experimental mice undergoing AB (n = 4 mice per group, *P \ 0.05 vs. sham). c Western blot analysis of ANP, b-MHC, and RGS14 in cultured neonatal rat cardiomyocytes stimulated by Angiotensin II (1 lmol/L; n = 4) for 24 or 48 h (*P \ 0.05 vs. PBS). Left Representative western blot. Right Bar graphs: quantitative results. n indicates the number of independent experiments. The data are presented as the mean ± SD

Article Snippet: Cardiac-specific RGS14-overexpressing mice Full-length mouse RGS14 Complementary DNA (cDNA) (OriGene, MC204443) was ligated into the chicken b-actin gene (CAG) promoter expression vector, which was linearized and purified using the QIAquick Gel Extraction Kit (Qiagen, 28704).

Techniques: Western Blot, Expressing, Cell Culture

Journal: The EMBO Journal

Article Title: A unified mechanism for mitochondrial damage sensing in PINK1-Parkin–mediated mitophagy

doi: 10.1038/s44318-025-00604-z

Figure Lengend Snippet: ( A ) Flow cytometry measurements performed as in (Fig. ). Left graph ns P = 0.0988, Right graph from left to right ns P = 0.5132, 0.2695, **** P ≤ 0.0001 (left graph exact P values from left to right P = 7e-15, 1.7e-14, 7e-15, 7e-15; right graph exact P values from left to right 4.62e-11, 7e-15, 7e-15) by two-way ANOVA with Dunnett’s multiple comparisons test. Error bars mean +/− SD. N = 6 independent experiments from two separate transductions (separate transductions denoted by open or closed circles). Error bars mean +/− SD. ( B ) Representative immunoblots of PINK1 stabilization and activity in HeLa dCas9-BFP-ZIM3 cells with indicated sgRNA +/− 10 µM CCCP for 4 h. N ≥ 3 independent experiments. ( C ) Volcano plots of PINK1-YFP (red) interactors measured by AP-MS as in (5I). CCCP treatment, where indicated, was overnight. Other samples were untreated. Two-sided Student’s t tests were performed. Values were corrected for multiple comparisons by calculating a FDR with the permutation method. Proteins were annotated as significant interactors if they had an absolute log2 fold change of >1 (black outline). Proteins associated with TOM (dark blue) and TIM23 (cyan) translocases and cytosolic chaperones (yellow) are indicated. N = 4 replicates/sgRNA on one occasion. The untreated control guide group was the same as used in (Fig. ). ( D ) CN-PAGE separated PINK1-YFP complexes visualized by in-gel fluorescence as in (Fig. ). N = 2 independent experiments with TOMM40 KD, one of which was with antibody gel shift. ( E ) Flow cytometry in HeLa PINK-YFP cells with doxycycline inducible expression of wild-type or C-terminal truncated TOMM5. Demonstrating rescue with TOMM5 WT but not TOMM5 ΔC. N = 6 replicates on at least two occasions. ns P = 0.4357, **** P ≤ 0.0001 (exact P values P < 1e-15 for all comparisons) by ordinary one-way ANOVA with Šídák’s multiple comparisons test. ( F ) Crystal structure yeast TOM complex (PDB: 6JNF (Araiso et al, )) demonstrating the location of TOMM5 and TOMM7 subunits. ( G ) LFQ proteomics of HeLa PINK-YFP whole lysates following the indicated knockdown/treatment. Two-sided Student’s t tests were performed. Values were corrected for multiple comparisons by calculating an FDR with the Benjamini–Hochberg procedure. Proteins were annotated as significant if they had an FDR < 0.05 and an absolute log 2 fold change of >0.5 (black outline). N = 4 replicates/sgRNA on one occasion. .

Article Snippet: TOMM5 WT or TOMM5 1–39 (truncation after residue 39) were cloned into the doxycycline inducible pSBtet-RN, a gift from Eric Kowarz (Addgene plasmid # 60503) (Kowarz et al, ).

Techniques: Flow Cytometry, Western Blot, Activity Assay, Protein-Protein interactions, Control, Clear Native PAGE, Fluorescence, Gel Shift, Expressing, Knockdown

Journal: Journal of Biological Chemistry

Article Title: RGS14 Is a Centrosomal and Nuclear Cytoplasmic Shuttling Protein That Traffics to Promyelocytic Leukemia Nuclear Bodies Following Heat Shock

doi: 10.1074/jbc.m408163200

Figure Lengend Snippet: FIG. 1. Intracellular localization of RGS14. A, constructs. Mouse RGS14-GFP constructs containing various deletions were generated and designated HC1 to HC11. The GenBankTM accession number of the mouse clone used in this study is U85055. RGS domain required for the GAP activity (amino acids 67–184), two Raf-like Ras binding domains (RBDs, amino acids 303–374 and 376–445), Rap-interacting domain (RID, amino acids 300–427), and GoLoco domain required for the GDI activity (Loco, amino acids 500–522) are indicated as filled boxes. The numbers indicate the length of corresponding RGS14 fragment. Lower- case letters on the right column indicate intracellular localization of each construct shown in B. B, localization of GFP fusion proteins. Confocal microscopy was performed with live cells expressing various GFP fusion constructs of RGS14. The constructs exhibited distinct intracellular localization ranging from exclusively cytoplasmic (b) to exclusively nuclear (e). Wild type RGS14 showed intense perinuclear dot-like staining (a). HC6, HC7, and HC9 also localized to the perinu- clear dot-like structures (c). The vector GFP construct showed an evenly distributed GFP expression throughout the cell (f) as the HC8 construct did (d). The centrosomal and nuclear dot-like structures in this figure appear larger because they are overexposed to capture the much less prominent cytoplasmic or nucleoplasmic GFP staining. Confocal zoom factor of 1 was used to capture the images.

Article Snippet: All of the cloned RGS14 constructs were sequenced to confirm proper cloning, and protein lysates from HeLa cells transfected with the constructs were analyzed for proper expression of proteins by immunoblotting with anti-GFP antibody (Santa Cruz Biotechnology).

Techniques: Construct, Generated, Activity Assay, Binding Assay, Confocal Microscopy, Expressing, Staining, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: RGS14 Is a Centrosomal and Nuclear Cytoplasmic Shuttling Protein That Traffics to Promyelocytic Leukemia Nuclear Bodies Following Heat Shock

doi: 10.1074/jbc.m408163200

Figure Lengend Snippet: FIG. 2. Colocalization of RGS14-GFP proteins with the endog- enous pericentrin. HeLa cells were transfected with various RGS14- GFP constructs (HC1, HC6, and HC9) and fixed with 50% acetone and 50% methanol. The fixed cells were indirectly immunostained with anti-pericentrin antibody followed by goat Alexa568-conjugated anti- rabbit antibody. Confocal microscopy was then performed with the immunostained cells. The merged images of green GFP and red Al- exa568 are shown on the right. The 50% acetone, 50% methanol fixation method that is the prerequisite for immunostaining of centrosomes results in a considerable loss of cytoplasmic and nucleoplasmic GFP expression. Confocal zoom factor of 6 was used for capturing the images.

Article Snippet: All of the cloned RGS14 constructs were sequenced to confirm proper cloning, and protein lysates from HeLa cells transfected with the constructs were analyzed for proper expression of proteins by immunoblotting with anti-GFP antibody (Santa Cruz Biotechnology).

Techniques: Transfection, Construct, Confocal Microscopy, Immunostaining, Expressing

Journal: Journal of Biological Chemistry

Article Title: RGS14 Is a Centrosomal and Nuclear Cytoplasmic Shuttling Protein That Traffics to Promyelocytic Leukemia Nuclear Bodies Following Heat Shock

doi: 10.1074/jbc.m408163200

Figure Lengend Snippet: FIG. 3. Endogenous RGS14 localizes to the centrosomes throughout the cell cycle. A, NIH3T3 cells were immu- nostained with anti-centrin antibody fol- lowed by Alexa488-conjugated anti- mouse antibody and then with anti- RGS14 antibody followed by Alexa568- conjugated anti-rabbit antibody. Hoechst 33342 was used to stain DNA. Stained cells were then subjected to confocal mi- croscopy using a 63 oil lens objective. The confocal zoom factor of 6 was used. Left three panels show RGS14, centrin, and the merged image of RGS14 and cen- trin. L and R stand for the centrosomes on the left and right, respectively, as shown with arrows in the middle centrosome panel. The far right panel shows the merged images of centrosomes and DNA. The cell cycle phase of stained cells is indicated as M (mitotic), G1, S, and G2. B, the same experiment described in A was performed with HeLa cells. Only the merged images are shown.

Article Snippet: All of the cloned RGS14 constructs were sequenced to confirm proper cloning, and protein lysates from HeLa cells transfected with the constructs were analyzed for proper expression of proteins by immunoblotting with anti-GFP antibody (Santa Cruz Biotechnology).

Techniques: Staining

Journal: Journal of Biological Chemistry

Article Title: RGS14 Is a Centrosomal and Nuclear Cytoplasmic Shuttling Protein That Traffics to Promyelocytic Leukemia Nuclear Bodies Following Heat Shock

doi: 10.1074/jbc.m408163200

Figure Lengend Snippet: FIG. 4. CRM-1 receptor-dependent nuclear export of RGS14. A, HeLa cells were transfected with either GFP vector or wild type mouse RGS14-GFP and then treated with leptomycin B at 37 °C for 2 h. The nuclear accumulation of RGS14 caused by leptomycin B treatment was observed using confocal microscopy. The merged images (DIC merge) of GFP and differential interference contrast (DIC) are also shown. C stands for the control cells that were not treated with LMB. B, the same experiment was repeated with the wild type human RGS14-GFP. Cells were also incubated with a viable DNA dye, Hoechst 33324 (Ho) to stain the nuclei. The merged image of GFP and Hoechst 33324 clearly dem- onstrates the nuclear accumulation of human RGS14-GFP in the cells treated with leptomycin B.

Article Snippet: All of the cloned RGS14 constructs were sequenced to confirm proper cloning, and protein lysates from HeLa cells transfected with the constructs were analyzed for proper expression of proteins by immunoblotting with anti-GFP antibody (Santa Cruz Biotechnology).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Control, Incubation, Staining

Journal: Journal of Biological Chemistry

Article Title: RGS14 Is a Centrosomal and Nuclear Cytoplasmic Shuttling Protein That Traffics to Promyelocytic Leukemia Nuclear Bodies Following Heat Shock

doi: 10.1074/jbc.m408163200

Figure Lengend Snippet: FIG. 5. Centrosomal localization of wild type RGS14 and RGS14 mutants. A, GFP fusion constructs of mouse RGS14 (GenBankTM

Article Snippet: All of the cloned RGS14 constructs were sequenced to confirm proper cloning, and protein lysates from HeLa cells transfected with the constructs were analyzed for proper expression of proteins by immunoblotting with anti-GFP antibody (Santa Cruz Biotechnology).

Techniques: Construct

Journal: Journal of Biological Chemistry

Article Title: RGS14 Is a Centrosomal and Nuclear Cytoplasmic Shuttling Protein That Traffics to Promyelocytic Leukemia Nuclear Bodies Following Heat Shock

doi: 10.1074/jbc.m408163200

Figure Lengend Snippet: FIG. 6. Putative intracellular targeting motifs of RGS14. A, the amino acid sequences of three putative NLSs (NLS1, bipartite NLS2, and NLS3) in mouse and human RGS14 protein (GenBankTM accession numbers, GI:3914636 for mouse and GI:15559454 for human) are shown. The basic positively charged residues are underlined. The arrow indicates serine 261 of mouse RGS14 that was identified as a PKA phosphorylation site. B, identified NESs in tumor suppressors such as p53 and BRACA1 are shown. The critical hydrophobic residues are underlined. The putative NES of RGS14 overlaps with the GoLoco motif of RGS14. The critical amino acid residues for the GDI activity (two leucine residues and a glutamine residue) marked with two asterisks were mutated to alanine. The hydrophobic residues in putative NES of RGS14 and the triad of DQR essential for the GDI activity were under- lined. The GoLoco motif and the region adjacent to the GoLoco motif are also highly conserved in RGS12 (GenBankTM accession numbers, GI: 2766633 for human). The consensus amino acid sequence of the GoLoco motif is also shown. C, the HC9 construct shown in Fig. 1A contains only the putative NLS3. The five positively charged residues of the putative NLS3 as underlined in A were mutated to alanine. The HC9 construct with the mutated NLS3 was named HC25. In addition, the GFP tag of HC9 and HC25 was replaced with FLAG tag in pCMV10 vector. These are named HC9-FLAG and HC25-FLAG. The top panel shows HeLa cells that were transfected with the HC25 and indirectly immunostained with anti-pericentrin as described previously. The ar- rows indicate the centrosomes where RGS14-GFP and pericentrin co- localize. The middle and lower panels show HeLa cells transfected with HC9-FLAG and HC25-FLAG, respectively. The cells were indirectly immunostained with anti-FLAG antibody followed by goat Alexa488- conjugated anti-mouse antibody. These cells were subsequently immu- nostained with anti-pericentrin as described previously. Confocal zoom factor of 1 or 4 was used to capture the images as indicated.

Article Snippet: All of the cloned RGS14 constructs were sequenced to confirm proper cloning, and protein lysates from HeLa cells transfected with the constructs were analyzed for proper expression of proteins by immunoblotting with anti-GFP antibody (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Activity Assay, Residue, Sequencing, Construct, FLAG-tag, Plasmid Preparation, Transfection

Journal: Journal of Biological Chemistry

Article Title: RGS14 Is a Centrosomal and Nuclear Cytoplasmic Shuttling Protein That Traffics to Promyelocytic Leukemia Nuclear Bodies Following Heat Shock

doi: 10.1074/jbc.m408163200

Figure Lengend Snippet: FIG. 7. Nuclear export-defective RGS14 is colocalized with PML in the nucleus. A, live HeLa cells transfected hRGS14-S-GFP fusion constructs were imaged using confocal microscopy. Cells were incubated with Hoechst 33242 dye prior to confocal microscopy. The merged image of GFP and Hoechst (Ho) demonstrates the nuclear localization of hRGS14-S-GFP. Differential interference contrast (DIC) image is also shown. Confocal zoom factor of 2 was used for capturing the images. B, colocalization of hRGS14-S-GFP with the endogenous PML protein. HeLa cells transfected with hRGS14-S-GFP was indi- rectly immunostained with anti-PML antibody followed by goat Al- exa568-conjugated anti-mouse antibody. Confocal microscopy was then performed with the immunostained cells. Confocal zoom factor of 6 was used for capturing the images. The merged image of green GFP and red Alexa568 is shown on the right. C, colocalization of RGS14-LL/AA-GFP with the endogenous PML protein. HeLa cells transfected with RGS14- LL/AA-GFP was indirectly immunostained with anti-PML antibody, and confocal images were collected as described above. The merged image of green GFP and red Alexa568 is shown on the right.

Article Snippet: All of the cloned RGS14 constructs were sequenced to confirm proper cloning, and protein lysates from HeLa cells transfected with the constructs were analyzed for proper expression of proteins by immunoblotting with anti-GFP antibody (Santa Cruz Biotechnology).

Techniques: Transfection, Construct, Confocal Microscopy, Incubation

Journal: Journal of Biological Chemistry

Article Title: RGS14 Is a Centrosomal and Nuclear Cytoplasmic Shuttling Protein That Traffics to Promyelocytic Leukemia Nuclear Bodies Following Heat Shock

doi: 10.1074/jbc.m408163200

Figure Lengend Snippet: FIG. 8. Heat stress induces the re-localization of cytoplasmic RGS14 protein to the PML nuclear bodies. A, the effect of cellular stresses on the intracellular localization of RGS14 was monitored. HeLa cells transfected with wild type RGS14 were exposed to heat shock (43 °C for 30 min), proteotoxic stress (MG132 for 2–4 h), and transcription-related stress (actinomycin D for 2–4 h). Confocal micros- copy was performed before and after each treatment. B, heat shock- treated cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. Cells were then immunostained with anti-PML antibody as described earlier. The arrows indicate the areas of prominent colocalization. C, HeLa cells were transfected with the GFP cloning vector or various RGS14 constructs (HC1, HC12–14, and HC24) in addition to a construct directing expression of luciferase. Cell lysates were then assayed for luciferase activity. Four independent experiments were performed as duplicate showing similar results. The fold increase represents an increase in luciferase activity compared with the luciferase activity of vector plasmid-transfected cells after normalization as described under “Experimental Procedures.” The filled and open columns represent luciferase activity generated from HSV-TK and CMV promoters, respectively. Significance was deter- mined using Student’s t test. *, p 0.05; **, p 0.01

Article Snippet: All of the cloned RGS14 constructs were sequenced to confirm proper cloning, and protein lysates from HeLa cells transfected with the constructs were analyzed for proper expression of proteins by immunoblotting with anti-GFP antibody (Santa Cruz Biotechnology).

Techniques: Transfection, Cloning, Plasmid Preparation, Construct, Expressing, Luciferase, Activity Assay, Generated