Journal: bioRxiv

Article Title: Differential roles of putative arginine fingers of AAA + ATPases Rvb1 and Rvb2

doi: 10.1101/2024.05.13.593962

Figure Lengend Snippet: A ) Structure of Rvb1/2 heterohexamer (PDB 6GEJ), highlighting the insertion domain and AAA + ATPase domain. Rvb1 is in orange and Rvb2 is in green. B-C) Structure of individual Rvb1 ( B ) and Rvb2 ( C ) protomers and their ATP binding domains (PDB 6GEJ). Key ATPase residues are highlighted, including the conserved trans-arginine fingers (green). Individual subunits colored as in A . ADP molecule shown in grey. D) Sequence conservation map of yeast Rvb1 and Rvb2 showing the conservation status of the sequences between S. cerevisiae and other eukaryotes. Conservation scale indicating level of sequence conservation shown below. Figure made using Consurf. Key ATPase sequence motifs indicated by colored lines or arrows.

Article Snippet: Gels were then transferred to nitrocellulose membranes which were incubated in 3% milk with antibodies against Rvb1 and Rvb2 (1:5000, kind gifts from Dr. Walid Houry), GAPDH (1:10,000, Proteintech, cat# HRP-60004), HA (1:1000, Roche, Cat# 11867423001), and FLAG (1:5000, Sigma, cat# F1804).

Techniques: Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Differential roles of putative arginine fingers of AAA + ATPases Rvb1 and Rvb2

doi: 10.1101/2024.05.13.593962

Figure Lengend Snippet: A ) Cell growth was analyzed via serial dilutions, using a galactose-inducible/glucose-repressible promoter on RVB1 or RVB2 on media containing galactose (bottom panels) or glucose (top panels). B) Western blot analysis of steady-state Rvb1 levels in cells expressing EV or Rvb1/Rvb2 (as indicated) WT, or arginine finger RA or RK mutation. C) Quantification of B . Signal was normalized to total protein (using pre-stained gel) as a loading control and then normalized to cells expressing WT RVB1 or RVB2 , which was set to a value of 1. One-way ANOVA was then used to compare protein levels between the different variants. Error bars represent ± SD; ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; N = 5. D) Western blot analysis of steady-state Rvb2 levels in cells expressing EV or Rvb1/Rvb2 (as indicated) WT, or arginine finger RA or RK mutation. E) Quantification of D . Signal was normalized to total protein (using pre-stained gel) as a loading control, and then normalized to cells expressing WT RVB1 or RVB2 , which was set to a value of 1. One-way ANOVA was then used to compare protein levels between the different variants. Error bars represent ± SD; ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; N = 5.

Article Snippet: Gels were then transferred to nitrocellulose membranes which were incubated in 3% milk with antibodies against Rvb1 and Rvb2 (1:5000, kind gifts from Dr. Walid Houry), GAPDH (1:10,000, Proteintech, cat# HRP-60004), HA (1:1000, Roche, Cat# 11867423001), and FLAG (1:5000, Sigma, cat# F1804).

Techniques: Western Blot, Expressing, Mutagenesis, Staining, Control

Journal: bioRxiv

Article Title: Differential roles of putative arginine fingers of AAA + ATPases Rvb1 and Rvb2

doi: 10.1101/2024.05.13.593962

Figure Lengend Snippet: A ) Steady-state ATPase rate was measured via an NADH-coupled assay. Shown is the calculated ATPase rate plotted at different concentrations of ATP for each Rvb1/2 variant. A no-ATPase control was subtracted from the calculated rates as background. B) The k cat calculated from the curve shown in A of ATP turnover over time at different ATP concentrations for the different Rvb1/2 variants. One-way ANOVA analysis was used to compare k cat between purified Rvb1/2 and arginine finger mutants. Error bars represent ± SD; ns, not significant; **** p < 0.0001; N = 3. C) Single turnover kinetics of Rvb1/2 and arginine finger mutants was measured by analyzing the hydrolysis of ATP using radiolabeled ATP [γP 32 ] over time. * Some radioactivity was detected to migrate between Pi and ATP on the TLC plate, which may be Pi-Pi. D) Cleavage of Pi was calculated by quantifying density of Pi (top band) divided by the density of the total lane from C and multiplying by 100 to obtain a % Pi cleavage. Five replicates, from independent protein preparations, were analyzed. E) Association and dissociation rate constants calculated for Rvb1/2 and variants. For Rvb1/2 and Rvb1/Rvb2-R350A, K D was calculated as K D = k -1 / k 1 . For Rvb1-R366A/Rvb2 and Rvb1-R366A/Rvb2-R350A, K D was calculated after the fraction bound and k -2 = K D * k 2 * k 1 / k -1 . F-G) Association ( F ) and dissociation ( G ) rate constants for binding of Rvb1/2 and variants to mant-ATP. Error bars represent the error in fit of the data ( Figure S4B-C ) to a curve; ns, not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001. H) Comparison of dissociation constant ( K D ) for each Rvb1/2 variant with mant-ATP. One-way ANOVA was performed to compare the dissociation constants. Error bars represent error in the fit to the curve; ns, not significant.

Article Snippet: Gels were then transferred to nitrocellulose membranes which were incubated in 3% milk with antibodies against Rvb1 and Rvb2 (1:5000, kind gifts from Dr. Walid Houry), GAPDH (1:10,000, Proteintech, cat# HRP-60004), HA (1:1000, Roche, Cat# 11867423001), and FLAG (1:5000, Sigma, cat# F1804).

Techniques: Variant Assay, Control, Purification, Radioactivity, Binding Assay, Comparison

Journal: bioRxiv

Article Title: Differential roles of putative arginine fingers of AAA + ATPases Rvb1 and Rvb2

doi: 10.1101/2024.05.13.593962

Figure Lengend Snippet: Mean internal Cα distance differences between A) Rvb1/2 and Rvb1/Rvb2-R350A, B) Rvb1/2 and Rvb1-R366A/Rvb2, C) Rvb1/2 and Rvb1-R366A/Rvb2-R350A variant and D) Rvb1-R366A/Rvb2 and Rvb1/Rvb2-R350A from MD simulations plotted onto the last frame of the simulation. In all panels, ATP is shown as spheres in the active site. Putty thickness and color indicate distance differences where red indicates decreased distances in the mutant variant compared to wildtype and blue indicates increased distances in the variant compared to wildtype control. Boxes and arrows highlight measure changes.

Article Snippet: Gels were then transferred to nitrocellulose membranes which were incubated in 3% milk with antibodies against Rvb1 and Rvb2 (1:5000, kind gifts from Dr. Walid Houry), GAPDH (1:10,000, Proteintech, cat# HRP-60004), HA (1:1000, Roche, Cat# 11867423001), and FLAG (1:5000, Sigma, cat# F1804).

Techniques: Variant Assay, Mutagenesis, Control

Journal: bioRxiv

Article Title: Differential roles of putative arginine fingers of AAA + ATPases Rvb1 and Rvb2

doi: 10.1101/2024.05.13.593962

Figure Lengend Snippet: A ) Western blot analysis of cellular levels of Nop58 (in NOP58-5xFLAG cells) in WT and arginine finger mutant cells. B-C) Quantification of Nop58 from blots shown in A . Signal was normalized to GAPDH loading control, and then normalized to cells expressing WT RVB1 or RVB2 , which was set to a value of 1. Unpaired t-test were then used to compare protein levels between RVB1 and rvb1-R366A ( B ), and between RVB2 and rvb2-R350A ( C ) expressing cells. Error bars represent ± SD; ns, not significant; * p < 0.05; ** p < 0.01; N = 4. D) Serial dilutions of RVB2 or rvb2-R350A cells overexpressing snoRNP core proteins. While overexpression of NOP58 leads to sick growth in WT cells, it rescues the cold sensitive phenotype observed in rvb2-R350A cells.

Article Snippet: Gels were then transferred to nitrocellulose membranes which were incubated in 3% milk with antibodies against Rvb1 and Rvb2 (1:5000, kind gifts from Dr. Walid Houry), GAPDH (1:10,000, Proteintech, cat# HRP-60004), HA (1:1000, Roche, Cat# 11867423001), and FLAG (1:5000, Sigma, cat# F1804).

Techniques: Western Blot, Mutagenesis, Control, Expressing, Over Expression

Journal: bioRxiv

Article Title: Differential roles of putative arginine fingers of AAA + ATPases Rvb1 and Rvb2

doi: 10.1101/2024.05.13.593962

Figure Lengend Snippet: Serial dilutions analyzing the response of Rvb1 and Rvb2 and their variants to glucose starvation via addition of 2-deoxy-D-glucose (2-DG) and sodium azide (NaN 3 ) stress.

Article Snippet: Gels were then transferred to nitrocellulose membranes which were incubated in 3% milk with antibodies against Rvb1 and Rvb2 (1:5000, kind gifts from Dr. Walid Houry), GAPDH (1:10,000, Proteintech, cat# HRP-60004), HA (1:1000, Roche, Cat# 11867423001), and FLAG (1:5000, Sigma, cat# F1804).

Techniques:

Journal: Nature Communications

Article Title: Paraventricular hypothalamic RUVBL2 neurons suppress appetite by enhancing excitatory synaptic transmission in distinct neurocircuits

doi: 10.1038/s41467-024-53258-6

Figure Lengend Snippet: a , b Expression of Ruvbl2 at mRNA ( a ) or protein ( b ) levels in the PVH of 8-week-old male WT mice fed a regular chow diet or fasted for 48 h ( n = 7 per group for mRNA; n = 6 per group for protein). c Schematic of AAV injection into the PVH. AAV2-Cre-GFP or control AAV2-GFP was bilaterally injected into the PVH of 8-week-old Ruvbl2 fl/fl mice. Mouse body weight was recorded weekly for 12 weeks post AAV injection, followed by different analyses including measurement of food intake and body composition, metabolic cages, IPGTT and IPITT. d Representative image of AAV2-Cre-GFP infection in the PVH ( n = 7 mice; scale bar, 100 μm). e Representative images showing PVH RUVBL2 KO efficiency. A monoclonal antibody against RUVBL2 was used to detect RUVBL2 in the PVH by IHC staining ( n = 3 mice; scale bar, 100 μm). f , g Body weight of male ( f ) and female ( g ) mice after AAV injection. For males, AAV2-GFP group ( n = 6 mice); AAV2-Cre-GFP group ( n = 7 mice). For females, n = 7 mice per group. h , i Daily food intake of male ( h ) and female ( i ) mice on chow diet. For males, AAV2-GFP group ( n = 6 mice); AAV2-Cre-GFP group ( n = 7 mice). For females, n = 7 mice per group. j – l Metabolic rate by body weight ( j ), locomotor activity ( k ), and metabolic rate by analysis of covariance (ANCOVA) ( l ) of male mice. ANCOVA was performed using SPSS Version 26 with metabolic rate as dependent variable, genotype as fixed variable and body mass as covariate. AAV2-GFP group ( n = 6 mice); AAV2-Cre-GFP group ( n = 7 mice). m , n IPGTT and AUC ( m ), and IPITT and AUC ( n ) of male mice. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed in mice 12 weeks post AAV injection. AAV2-GFP group ( n = 6 mice); AAV2-Cre-GFP group ( n = 7 mice). Two-tailed unpaired Student’s t -test for ( a , b , h–k ) and ( m, n ) (AUC). Two-way ANOVA followed by Bonferroni’s test for ( f , g , m , n ). ns, no significance. All data are presented as means ± SEM. Source data are provided as a Source Data file.

Article Snippet: 20 μg of total protein was loaded in SDS-PAGE and transferred to PVDF membranes, followed by 1 h of blocking with 5% nonfat milk and incubation with primary antibodies against RUVBL2 (1:10000; proteintech #67851-1-Ig) or HSP90 (1:10000; proteintech #13171-1-AP) at 4 °C overnight.

Techniques: Expressing, Injection, Control, Infection, Immunohistochemistry, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Paraventricular hypothalamic RUVBL2 neurons suppress appetite by enhancing excitatory synaptic transmission in distinct neurocircuits

doi: 10.1038/s41467-024-53258-6

Figure Lengend Snippet: a Schematic of stereotaxic injection of AAV9-DIO-hM3D(Gq)-mCherry or AAV9-EF1α-DIO-mCherry. b Representative images showing AAV9-DIO-hM3D(Gq)-mCherry infection and c-Fos immunoreactivity in the PVH after CNO treatment ( n = 3 mice; scale bar, 100 μm). c , d Food intake of male ( c ) and female ( d ) mice post CNO injection in light cycle. AAV9-DIO-hM3D(Gq)-mCherry or AAV9-EF1α-DIO-mCherry was bilaterally injected into the PVH of chow-fed 8 week-old Ruvbl2-Cre mice and hourly food intake was recorded for 4 h post CNO injection ( n = 5 mice per group). e Schematic of stereotaxic injection of AAV9-DIO-hM4D(Gi)-mCherry or AAV9-EF1α-DIO-mCherry. f Representative images showing AAV9-DIO-hM4D(Gi)-mCherry infection and c-Fos immunoreactivity in the PVH after CNO treatment ( n = 3 mice; scale bar, 100 μm). g , h Food intake of male ( g ) and female ( h ) mice post CNO injection in light cycle. Cre-dependent AAV9-DIO-hM4D(Gi)-mCherry or AAV9-DIO-mCherry was bilaterally injected into the PVH of chow-fed 8 week-old Ruvbl2-Cre mice and hourly food intake was recorded within 4 h post CNO injection ( n = 5 mice per group). i Schematic of stereotaxic injection of AAV9-DIO-hChR2-mCherry or AAV9-DIO-mCherry. j Procedure of optogenetic stimulation. PVH RUVBL2 neurons were stimulated by a 465 nm blue laser with 10 ms pulse duration for 10 min. Food intake in 10 min of laser stimulation, and 10 min before or after laser stimulation was monitored, respectively. Stim, during laser stimulation; Pre, before laser stimulation; Post, after laser stimulation. k Representative image showing AAV9-DIO-hChR2-mCherry infection in the PVH ( n = 5 mice; scale bar, 100 μm). l Food intake of fasted mice before and after optogenetic activation of PVH RUVBL2 neurons. ChR2 group ( n = 5 mice); Control group ( n = 3 mice). The experiment was repeated once using the same mice and all the data were included for quantitation. Two-tailed unpaired Student’s t -test for ( c – l ). ns, no significance. All data are presented as means ± SEM. Source data are provided as a Source Data file.

Article Snippet: 20 μg of total protein was loaded in SDS-PAGE and transferred to PVDF membranes, followed by 1 h of blocking with 5% nonfat milk and incubation with primary antibodies against RUVBL2 (1:10000; proteintech #67851-1-Ig) or HSP90 (1:10000; proteintech #13171-1-AP) at 4 °C overnight.

Techniques: Injection, Infection, Activation Assay, Control, Quantitation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Paraventricular hypothalamic RUVBL2 neurons suppress appetite by enhancing excitatory synaptic transmission in distinct neurocircuits

doi: 10.1038/s41467-024-53258-6

Figure Lengend Snippet: a Schematic of anterograde monosynaptic tracing of PVH RUVBL2 neurons. b Representative images showing PVH RUVBL2 neurons infected with AAV8-EF1α-DIO-EGFP-T2A-TK and HSV-ΔTK-LSL-tdTomato. 72 h after injection of HSV-ΔTK-LSL-tdTomato, the mice were euthanized and their brains were collected for histology analysis ( n = 3 mice; scale bars, 100 μm). c Representative images indicating the brain regions receiving direct projections from PVH RUVBL2 neurons revealed by tdTomato fluorescence signals. These brains regions include the ventral lateral septum nucleus (LSv) ( c1 ), the preoptic area (POA) ( c2 ), the dorsomedial hypothalamus (DMH) ( c3 ), the arcuate hypothalamus (ARC) ( c4 ), the medial amygdala (MeA) ( c5 ), the parabrachial complex (PB) (the parabrachial nucleus (PBN) and the pre-locus coeruleus (pLC)) ( c6 , c7 ), and the nucleus tractus solitarius (NTS) ( c8 ) ( n = 3 mice; scale bars, 100 μm). d Schematic of retrograde monosynaptic tracing of ARC POMC neurons. e Representative image from three mice showing ARC POMC neurons infected with AAV8-EF1α-DIO-EGFP-T2A-TVA, AAV8-EF1α-DIO-oRVG, and RV-EnVA-ΔG-LSL-DsRed as stated in d ; 7 days after injection of RV-EnVA-ΔG-LSL-DsRed, the mice were euthanized and their brains were collected for coronal brain sectioning and subsequent histology analysis. Scale bar, 50 μm. f Representative images showing ARC POMC -projecting neurons in the PVH and PVH RUVBL2 neurons ( n = 3 mice; scale bar, 50 μm). g Schematic of retrograde monosynaptic tracing of ARC AgRP neurons. h Representative image from three mice showing ARC AgRP neurons infected with AAV8-EF1α-DIO-EGFP-T2A-TVA, AAV8-EF1α-DIO-oRVG, and RV-EnVA-ΔG-LSL-DsRed. Scale bar, 50 μm. i Representative images showing ARC AgRP -projecting neurons in the PVH and PVH RUVBL2 neurons ( n = 3 mice; scale bar, 50 μm). Arrows denote PVH RUVBL2 neurons (green) containing DsRed fluorescence signals that are retrogradely derived from ARC POMC or ARC AgRP neurons. Source data are provided as a Source Data file.

Article Snippet: 20 μg of total protein was loaded in SDS-PAGE and transferred to PVDF membranes, followed by 1 h of blocking with 5% nonfat milk and incubation with primary antibodies against RUVBL2 (1:10000; proteintech #67851-1-Ig) or HSP90 (1:10000; proteintech #13171-1-AP) at 4 °C overnight.

Techniques: Infection, Injection, Fluorescence, Derivative Assay

Journal: Nature Communications

Article Title: Paraventricular hypothalamic RUVBL2 neurons suppress appetite by enhancing excitatory synaptic transmission in distinct neurocircuits

doi: 10.1038/s41467-024-53258-6

Figure Lengend Snippet: a Schematic of chemogenetic inhibition of ARC-projecting PVH RUVBL2 neurons. b Representative images from three mice showing viral infection in the PVH ( b1 ) and axonal terminals labeled by EGFP in the ARC ( b2, b3 ) as described in ( a ). c, d Food intake of mice in light ( c ) or dark ( d ) cycle after chemogenetic inhibition of ARC-projecting PVH RUVBL2 neurons. Hourly food intake was measured after CNO injection. n = 3 mice per group. e Schematic of chemogenetic inhibition of DMH-projecting PVH RUVBL2 neurons. f Representative images from three mice showing AAV infection in PVH RUVBL2 neurons ( f1 ) and EGFP-labeled axonal terminals in the DMH ( f2 , f3 ). Arrows denote axonal terminals of PVH RUVBL2 neurons. g , h Food intake of mice in light ( g ) or dark cycle ( h ) after chemogenetic inhibition of DMH-projecting PVH RUVBL2 neurons. Hourly food intake was measured after CNO injection. n = 3 mice per group. i Schematic of optogenetic activation of ARC-projecting PVH RUVBL2 neurons. j Representative images from five mice showing AAV infection in PVH RUVBL2 neurons and ChR2-mCherry-labeled axonal terminals in the ARC. k Food intake of mice after optogenetic activation of ARC-projecting PVH RUVBL2 neurons. ChR2 group ( n = 5 mice); control group ( n = 3 mice). This experiment was repeated once using the same mice and all the data were included for quantitation. l Schematic of optogenetic activation of DMH-projecting PVH RUVBL2 neurons. m Representative images from five mice showing AAV infection in PVH RUVBL2 neurons and ChR2-mCherry-labeled axonal terminals in the DMH from PVH RUVBL2 neurons. n Food intake of mice after optogenetic activation of DMH-projecting PVH RUVBL2 neuron activity. ChR2 group ( n = 5 mice); control group ( n = 3 mice). This experiment was repeated once using the same mice and all the data were included for quantitation. ChR2 group ( n = 5 mice); mCherry group ( n = 3 mice). Scale bars, 100 μm. Two-tailed unpaired Student’s t -test for ( c – n ). ns, no significance. CNO,1 mg/kg BW. All data are presented as means ± SEM. Source data are provided as a Source Data file.

Article Snippet: 20 μg of total protein was loaded in SDS-PAGE and transferred to PVDF membranes, followed by 1 h of blocking with 5% nonfat milk and incubation with primary antibodies against RUVBL2 (1:10000; proteintech #67851-1-Ig) or HSP90 (1:10000; proteintech #13171-1-AP) at 4 °C overnight.

Techniques: Inhibition, Infection, Labeling, Injection, Activation Assay, Control, Quantitation Assay, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Paraventricular hypothalamic RUVBL2 neurons suppress appetite by enhancing excitatory synaptic transmission in distinct neurocircuits

doi: 10.1038/s41467-024-53258-6

Figure Lengend Snippet: a Schematic of AAV injection into the PVH and electrophysiology recordings in ARC POMC neurons. b Representative traces of sEPSCs recorded in ARC POMC neurons in the presence of PTX. c , d Amplitude ( c ) and frequency ( d ) of sEPSCs recorded in ARC POMC neurons. AAV2-mCherry ( n = 30 neurons/3 mice), AAV2-Cre-mCherry ( n = 25 neurons/3 mice). e Representative traces of eEPSCs recorded in ARC POMC neurons in response to paired-pulse stimulations (50 ms interval) in the presence of PTX. f , g Amplitude of the first EPSCs ( f ) and PPR ( g ) recorded in ARC POMC neurons. AAV2-mCherry ( n = 38 neurons/4 mice), AAV2-Cre-mCherry ( n = 23 neurons/3 mice). h Schematic of AAV injection into the PVH and electrophysiology recordings in ARC NPY neurons. i Representative traces of sEPSCs recorded in ARC NPY neurons in the presence of PTX. j , k Amplitude ( j ) and frequency ( k ) of sEPSCs recorded in ARC NPY neurons. AAV2-mCherry ( n = 33 neurons/3 mice), AAV2-Cre-mCherry ( n = 29 neurons/3 mice). l Representative traces of eEPSCs recorded in ARC NPY neurons in response to paired-pulse stimulations (50 ms interval) in the presence of PTX. m , n Amplitude of the first EPSCs ( m ) and PPR ( n ) recorded in ARC NPY neurons. n = 29 neurons/3 mice per group. o Schematic of simultaneously activating ARC-projecting PVH RUVBL2 neurons and ARC NPY neurons. p Food intake of mice within 1 h after different treatments as indicated (as in o ). n = 8 mice per group. q Food intake of mice within 6 h after different treatments as indicated. SHU9119, 0.5 mg/kg. Stim, optical stimulation for 6 h when the laser light (465 nm) was switched on every other 30 min. For SHU9119+Stim, mice were first intraperitoneally injected with SHU9119, followed by optical stimulation for 6 h when the laser light (465 nm) was switched on every other 30 min. n = 6 mice per group. Two-tailed unpaired Student’s t -test for ( c – n ). ns, no significance. One-way ANOVA with Geisser-Greenhouse correction followed by Fisher’s LSD test for ( p ) and ( q ). All data are presented as means ± SEM. Source data are provided as a Source Data file.

Article Snippet: 20 μg of total protein was loaded in SDS-PAGE and transferred to PVDF membranes, followed by 1 h of blocking with 5% nonfat milk and incubation with primary antibodies against RUVBL2 (1:10000; proteintech #67851-1-Ig) or HSP90 (1:10000; proteintech #13171-1-AP) at 4 °C overnight.

Techniques: Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Paraventricular hypothalamic RUVBL2 neurons suppress appetite by enhancing excitatory synaptic transmission in distinct neurocircuits

doi: 10.1038/s41467-024-53258-6

Figure Lengend Snippet: a Representative images for IHC staining of the excitatory synapses in the ARC ( n = 3 mice; scale bar, 100 μm). b Quantitation of VGluT2 + puncta in the ARC. AAV2-GFP ( n = 17 profiles/3 mice), AAV2-Cre-GFP ( n = 20 profiles/3 mice). c Representative images for IHC staining of the excitatory synapses in the DMH. ( n = 3 mice; scale bar, 100 μm). d Quantitation of VGluT2 + puncta in the DMH. AAV2-GFP ( n = 22 profiles/3 mice), AAV2-Cre-GFP ( n = 23 profiles/3 mice). e Representative transmission electron microscopy (TEM) micrographs of the ARC. ( n = 3 mice; scale bar, 200 nm). f Length of the active zone (AZ) in the ARC. AAV2-GFP ( n = 75 profiles/3 mice), AAV2-Cre-GFP ( n = 65 profiles/3 mice). g , h Quantity of synaptic vesicles (SVs) within different distances ( g ) and quantity of SVs within 200 nm ( h ) from the AZ in the ARC. AAV2-GFP ( n = 75 profiles/3 mice), AAV2-Cre-GFP ( n = 65 profiles/3 mice). i Representative TEM micrographs of the DMH ( n = 3 mice; scale bar, 200 nm). j AZ length in the DMH. AAV2-GFP ( n = 70 profiles/2 mice), AAV2-Cre-GFP ( n = 66 profiles/2 mice). k,l Quantity of synaptic vesicles (SVs) within different distances ( k ) and quantity of SVs within 200 nm ( l ) from the AZ in the DMH. AAV2-GFP ( n = 70 profiles/2 mice), AAV2-Cre-GFP ( n = 66 profiles/2 mice). m GO enrichment analysis of the genes based on chromatin immunoprecipitation sequencing (ChIP-Seq). The top canonical pathways associated with the central nervous system are shown ( p < 0.05). n , o Genes related to synaptic vesicle ( n ), or neuron axonogenesis ( o ) identified by ChIP-Seq. p , q Heatmap showing down-regulated genes related to synaptic vesicle ( p ), or neuron axonogenesis ( q ) by RNA sequencing (RNA-Seq). The combined analyses of RNA-Seq and ChIP-Seq (as in n , o ) identify a number of genes related to synaptic vesicle or neuron axonogenesis that are down-regulated after RUVBL2 KO in the PVH. Two-tailed unpaired Student’s t -test for ( b , d , f , h , j , l ). ns, no significance. Two-way ANOVA followed by Bonferroni’s test for ( g ) and ( k ). All data are presented as means ± SEM. Source data are provided as a Source Data file.

Article Snippet: 20 μg of total protein was loaded in SDS-PAGE and transferred to PVDF membranes, followed by 1 h of blocking with 5% nonfat milk and incubation with primary antibodies against RUVBL2 (1:10000; proteintech #67851-1-Ig) or HSP90 (1:10000; proteintech #13171-1-AP) at 4 °C overnight.

Techniques: Immunohistochemistry, Quantitation Assay, Transmission Assay, Electron Microscopy, ChIP-sequencing, RNA Sequencing, Two Tailed Test

Journal: Nature Communications

Article Title: Paraventricular hypothalamic RUVBL2 neurons suppress appetite by enhancing excitatory synaptic transmission in distinct neurocircuits

doi: 10.1038/s41467-024-53258-6

Figure Lengend Snippet: a , b Representative traces of sEPSCs recorded in ARC ( a ) and DMH ( b ) neurons in the presence of PTX. c – f Amplitude and frequency of sEPSCs recorded in ARC ( c , e ) and DMH ( d , f ) neurons. For ARC: WT ( n = 30 neurons/3 mice), Ruvbl2 KI/KI ;Sim1-Cre ( n = 31 neurons/3 mice). For DMH: WT ( n = 28 neurons/4 mice), Ruvbl2 KI/KI ;Sim1-Cre ( n = 25 neurons/3 mice). g , h Representative traces of eEPSCs recorded in ARC ( g ) and DMH ( h ) neurons in response to paired-pulse stimulations (50 ms interval) in the presence of PTX. i – l Amplitude of the first EPSCs recorded in ARC ( i ) and DMH ( j ) neurons. PPR in ARC ( k ) and DMH ( l ) neurons. For ARC: WT ( n = 30 neurons/3 mice), Ruvbl2 KI/KI ;Sim1-Cre ( n = 36 neurons/3 mice). For DMH: WT ( n = 28 neurons/4 mice), Ruvbl2 KI/KI ;Sim1-Cre ( n = 26 neurons/3 mice). m Representative TEM micrographs in the ARC. n-p AZ Length ( n ), SVs within different distances ( o ) and within 10 nm ( p ) from the AZ in the ARC. WT ( n = 36 profiles/2 mice), Ruvbl2 KI/KI ;Sim1-Cre ( n = 66 profiles/2 mice). q Representative TEM micrographs in the DMH. r-t AZ Length ( r ), SVs within different distances ( s ) and within 10 nm ( t ) from the AZ in the DMH. WT ( n = 63 profiles/2 mice), Ruvbl2 KI/KI ;Sim1-Cre ( n = 57 profiles/2 mice). u,v Body weight of male ( u ) and female ( v ) mice on chow. For males: WT ( n = 7); Ruvbl2 KI/KI ;Sim1-Cre ( n = 9). For females: WT ( n =6); Ruvbl2 KI/KI ;Sim1-Cre ( n = 8). w Daily chow intake of male and female mice. For males: WT ( n = 7), Ruvbl2 KI/KI ;Sim1-Cre ( n = 8). For females: n = 6 per group. x,y Body weight of male ( x ) and female ( y ) mice on HFD. For males: n = 8 per group. For females: WT ( n = 8), Ruvbl2 KI/KI ;Sim1-Cre ( n = 9). z Daily HFD intake of male and female mice. For males: n = 8 per group. For females: WT ( n = 7), Ruvbl2 KI/KI ;Sim1-Cre ( n = 8). Scale bar, 200 nm. Two-tailed unpaired Student’s t -test for ( c – n , p , r , t , w , z ). ns, no significance. Two-way ANOVA followed by Bonferroni’s test for ( o , s , u , v , x , y ). All data are presented as means ± SEM. Source data are provided as a Source Data file.

Article Snippet: 20 μg of total protein was loaded in SDS-PAGE and transferred to PVDF membranes, followed by 1 h of blocking with 5% nonfat milk and incubation with primary antibodies against RUVBL2 (1:10000; proteintech #67851-1-Ig) or HSP90 (1:10000; proteintech #13171-1-AP) at 4 °C overnight.

Techniques: Two Tailed Test

Journal: Circulation. Arrhythmia and electrophysiology

Article Title: Loss of microRNA-106b-25 Cluster Promotes Atrial Fibrillation by Enhancing Ryanodine Receptor Type-2 Expression and Calcium Release

doi: 10.1161/CIRCEP.114.001973

Figure Lengend Snippet: Upregulation of RyR2 and downregulation of the miR-106b-25 cluster in pAF patients. A) WB analysis revealed increased RyR2 protein levels in sarcoplasmic reticulum preparation of atrial biopsies from patients with pAF versus control patients in SNR. B) qRT-PCR showing unchanged levels of RYR2 mRNA in atrial samples of pAF patients. C) qRT-PCR showing downregulation of the miR-106b-25 cluster and miR-106a in pAF patients. Numbers in bars indicate number of patients. *P<0.05, **P<0.01 vs. SNR.

Article Snippet: The membranes were then incubated with antibodies in 5% milk against the following proteins: RyR2 (1:5,000; MA3-916, Thermo Fisher Scientific, Houston, TX), Na + /Ca 2+ -exchanger type-1 (NCX1; 1:1,000; R3F1, Swant, Bellinzona, Switzerland), calsequestrin type-2 (CSQ2; 1:1,000; PA1-913, Thermo Fisher Scientific), Ca 2+ /calmodulin protein kinase II phosphorylated RyR2 at Serine2814 (pS2814; 1:1,000; custom-made), 16 and GAPDH (1:10,000; MAB-374, Millipore, Billerica, MA).

Techniques: Quantitative RT-PCR

Journal: Circulation. Arrhythmia and electrophysiology

Article Title: Loss of microRNA-106b-25 Cluster Promotes Atrial Fibrillation by Enhancing Ryanodine Receptor Type-2 Expression and Calcium Release

doi: 10.1161/CIRCEP.114.001973

Figure Lengend Snippet: miR-93 target RYR2-3′UTR. A) Sequences of mature miR-106b, miR-93, miR-25, and miR-106a. B) Predicted interaction between human (h) Ryr2-3′UTR and miR-93 (i) and mutation of the binding site (ii). C) Predicted interaction between mouse (m) Ryr2-3′UTR and miR-93 (i) and mutation of the binding site (ii). D) Dual luciferase assay of 3′UTR constructs in the presence of scramble, miR-106b, miR-93, miR-25, and miR-106a mimics. n=4 or 8 per group. **P<0.01.

Article Snippet: The membranes were then incubated with antibodies in 5% milk against the following proteins: RyR2 (1:5,000; MA3-916, Thermo Fisher Scientific, Houston, TX), Na + /Ca 2+ -exchanger type-1 (NCX1; 1:1,000; R3F1, Swant, Bellinzona, Switzerland), calsequestrin type-2 (CSQ2; 1:1,000; PA1-913, Thermo Fisher Scientific), Ca 2+ /calmodulin protein kinase II phosphorylated RyR2 at Serine2814 (pS2814; 1:1,000; custom-made), 16 and GAPDH (1:10,000; MAB-374, Millipore, Billerica, MA).

Techniques: Mutagenesis, Binding Assay, Luciferase, Construct

Journal: Circulation. Arrhythmia and electrophysiology

Article Title: Loss of microRNA-106b-25 Cluster Promotes Atrial Fibrillation by Enhancing Ryanodine Receptor Type-2 Expression and Calcium Release

doi: 10.1161/CIRCEP.114.001973

Figure Lengend Snippet: Increased RyR2 protein levels in miR-106b-25−/− atria. A) qRT-PCR showing unchanged levels of Ryr2 mRNA in miR-106b-25−/− atria. B) Representative western blots and dot plot showing upregulated RyR2 protein levels in miR-106b-25−/− atrial lysates. *P<0.05. C) Representative western blots and bar graph showing unchanged expression of other major Ca2+-handling proteins and phosphorylation of RyR2 at serine (S)2814 in miR-106b-25−/− atria. N=3–4 animals per group.

Article Snippet: The membranes were then incubated with antibodies in 5% milk against the following proteins: RyR2 (1:5,000; MA3-916, Thermo Fisher Scientific, Houston, TX), Na + /Ca 2+ -exchanger type-1 (NCX1; 1:1,000; R3F1, Swant, Bellinzona, Switzerland), calsequestrin type-2 (CSQ2; 1:1,000; PA1-913, Thermo Fisher Scientific), Ca 2+ /calmodulin protein kinase II phosphorylated RyR2 at Serine2814 (pS2814; 1:1,000; custom-made), 16 and GAPDH (1:10,000; MAB-374, Millipore, Billerica, MA).

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: Circulation. Arrhythmia and electrophysiology

Article Title: Loss of microRNA-106b-25 Cluster Promotes Atrial Fibrillation by Enhancing Ryanodine Receptor Type-2 Expression and Calcium Release

doi: 10.1161/CIRCEP.114.001973

Figure Lengend Snippet: Enhanced RyR2 Ca2+-release activity in miR-106b-25−/− atrial myocytes. A) Representative line-scan images of Ca2+-spark recordings in isolated atrial myocytes. B) Bar graph showing increased CaSF normalized to SR load in miR-106b-25−/− atrial myocytes, normalized by K201. C) Bar graph showing unchanged Ca2+-spark amplitude comparing miR-106b-25−/− and WT atrial myocytes. D) Bar graph showing an increase in the FWHM of Ca2+-sparks in miR-106b-25−/− atrial myocytes, normalized by RyR2 blocker K201. Numbers inside bars indicate number of cells studied from 3 mice per group. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The membranes were then incubated with antibodies in 5% milk against the following proteins: RyR2 (1:5,000; MA3-916, Thermo Fisher Scientific, Houston, TX), Na + /Ca 2+ -exchanger type-1 (NCX1; 1:1,000; R3F1, Swant, Bellinzona, Switzerland), calsequestrin type-2 (CSQ2; 1:1,000; PA1-913, Thermo Fisher Scientific), Ca 2+ /calmodulin protein kinase II phosphorylated RyR2 at Serine2814 (pS2814; 1:1,000; custom-made), 16 and GAPDH (1:10,000; MAB-374, Millipore, Billerica, MA).

Techniques: Activity Assay, Isolation

Journal: Circulation. Arrhythmia and electrophysiology

Article Title: Loss of microRNA-106b-25 Cluster Promotes Atrial Fibrillation by Enhancing Ryanodine Receptor Type-2 Expression and Calcium Release

doi: 10.1161/CIRCEP.114.001973

Figure Lengend Snippet: miR-106b-25 deficiency leads to enhanced AF susceptibility. Representative recordings of surface ECG and intracardiac electrograms in WT (A), miR-106b-25−/− (B), and miR-106b-25−/− mice treated with RyR2 blocker K201 (C). D) Bar graph summarizing the incidence of pacing-induced AF showing an increase in miR-106b-25−/− mice, suppressed by K201. Numbers inside bars indicate numbers of mice studied. *P<0.05.

Article Snippet: The membranes were then incubated with antibodies in 5% milk against the following proteins: RyR2 (1:5,000; MA3-916, Thermo Fisher Scientific, Houston, TX), Na + /Ca 2+ -exchanger type-1 (NCX1; 1:1,000; R3F1, Swant, Bellinzona, Switzerland), calsequestrin type-2 (CSQ2; 1:1,000; PA1-913, Thermo Fisher Scientific), Ca 2+ /calmodulin protein kinase II phosphorylated RyR2 at Serine2814 (pS2814; 1:1,000; custom-made), 16 and GAPDH (1:10,000; MAB-374, Millipore, Billerica, MA).

Techniques: