Journal: Cells

Article Title: Let-7g Upregulation Attenuated the KRAS–PI3K–Rac1–Akt Axis-Mediated Bioenergetic Functions

doi: 10.3390/cells12182313

Figure Lengend Snippet: Suppression of monocyte chemoattractant protein-induced protein-1 (MCPIP1)-mediated degradation of lethal-7g ( let-7g ) by naringenin (NGN) involved the attenuation of glycolysis and mitochondrial mitochondrial oxidative phosphorylation (OXPHOS). ( A , B ) Immunoprecipitated MCPIP1 (IP:MCPIP1) from the nasopharyngeal carcinoma (NPC) or Smulow–Glickman (S-G) cells and recombinant purified MCPIP1 (rMCPIP1) used in the experiments. The inhibition of the let-7g degradation of MCPIP1-mediated by NGN, performed by in vitro RNA cleavage assay and analyzed by Northern blotting (NB). The levels of the IP:MCPIP1 or rMCPIP1 in reactions were determined by Western blot (WB) analysis using specific antibodies. ( C – E ) At 12 h after transfection with FLAG-MCPIP1, FLAG-MCPIP1 (D141N), or MCPIP1 short hairpin RNA (shRNA), cells were treated with vehicle (−) or NGN in the presence of 1 μM oligomycin-A (Oligo-A), carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone (Rot) plus myxothiazol (Myx) or the sequential addition of glucose, Oligo-A, and 2-deoxy-D-glucose (2-DG) at the indicated time points. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse Bioscience XF24 Analyzer. Western blot analysis using specific antibodies determined the levels of the indicated proteins in the total cell lysates. Quantitative real-time polymerase chain reaction (qRT-PCR) determined the relative expression level of let-7g. The let-7g value was normalized to the U6 level. The Y-axis shows the denary logarithm of the normalized let-7g copy number. The values are presented as three independent experiments’ mean ± standard error. * p < 0.05: significantly different from (−)-treated empty vector-transfected, NGN-treated empty vector-transfected, (−)-treated FLAG-MCPIP1-transfected, or (−)-treated MCPIP1 shRNA-transfected cells.

Article Snippet: Monocyte chemoattractant protein-induced protein-1 (MCPIP1) shRNA plasmid and Western blotting luminol reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunoprecipitation, Recombinant, Purification, Inhibition, In Vitro, Cleavage Assay, Northern Blot, Western Blot, Transfection, shRNA, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Plasmid Preparation

Journal: Cells

Article Title: Let-7g Upregulation Attenuated the KRAS–PI3K–Rac1–Akt Axis-Mediated Bioenergetic Functions

doi: 10.3390/cells12182313

Figure Lengend Snippet: A molecular model for the naringenin (NGN)-induced impairment of the mitochondria-mediated bioenergetics in nasopharyngeal carcinoma (NPC) cells. ( A ) The selective interaction of argonaute (AG) protein or an undefined factor with monocyte chemoattractant protein-induced protein-1 (MCPIP1) may accomplish the sequence-specific targeting of pre-lethal-7g ( pre-let-7g ) and promote MCPIP1-mediated pre-let-7g degradation to decrease the biogenesis of let-7g , resulting in the attenuation of the let-7g -mediated translational repression of kirsten rat sarcoma viral oncogene homolog ( KRAS ) mRNA. The resultant elevated KRAS increases the formation of clustered KRAS- phosphatidylinositol 3-kinase (PI3K)-active GTP-binding Ras-related C3 botulinum toxin substrate (GTP-Rac1)–protein kinase B (Akt) signaling molecules in the lipid raft membranes, constituting a central element in the initiation of the coordination of glycolysis with mitochondrial oxidative phosphorylation (OXPHOS) for ATP generation. ( B ) Under the condition of the cellular uptake of NGN, NGN may bind to the AG protein or an undefined cofactor to block the degradation of pre-let-7g by MCPIP1, increasing the level of let-7g , thus inducing the let-7g -mediated translational repression of KRAS mRNA and thereby dismissing the interaction between KRAS and p110α in the lipid raft membrane. The absence of KRAS in the p85α–p110α complexes causes the destabilization of p85α–p110α complexes in the lipid raft membrane. The resultant loss of the KRAS–p85α–p110α complexes in the lipid raft membrane leads to blocking PI3K-GTP-Rac1-mediated Akt activation. Attenuated Akt impaired the aerobic glycolysis and mitochondria-regulated bioenergetic functions in NPC cells.

Article Snippet: Monocyte chemoattractant protein-induced protein-1 (MCPIP1) shRNA plasmid and Western blotting luminol reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Sequencing, Binding Assay, Blocking Assay, Membrane, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: Binding between AKAP79/AKAP150 and the N terminus of AC8. A , GST pull-downs using whole cell lysate from cells transiently expressing tagged AKAP proteins. Lane 1 , GST alone; lanes 2–4 , the following regions of AC8 fused to GST: 1–179 (N terminus), 582–703 (C1b domain), and 1106–1248 (C terminus), respectively. Lane 5 , input control (5%) to confirm expression of each construct. B , top , AKAP79-HA and AKAP150-HA transiently expressed in HEK293 cells were pulled down using GST-fused first half (residues 1–77) or second half (residues 73–179) of the AC8 N terminus. Bottom , immunoblotting of the GST-fused proteins used in the pull-downs in the top . The upper GST bands represent the full-length form of each protein. C , plot of densitometries from 3–6 repeats of blots presented in B to quantify binding of AKAP79/150 to GST-AC8 1–77 versus GST-AC8 73–179. Data are normalized to the intensity of the uppermost GST bands. Error bars , S.E. D , schematic diagram of GST-AC8 constructs used in A–C .

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Binding Assay, Expressing, Control, Construct, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: AKAP disruption enhances CCE-induced AC8 activity. A , CCE-mediated increases in cAMP assessed using the FRET-based sensor Epac2-camps following knockdown of endogenous AKAP79 levels. Data are plotted as relative FRET ratio changes normalized to maximum signal response. Cells were pretreated with 200 n m Tg in Ca 2+ -free conditions, and CCE was induced upon the addition of 2 m m Ca 2+ to the bath solution. B , average Fura-2 traces from HEK-AC8 cells during CCE following transfection with AKAP150-HA, AKAP79-HA, or shRNA AKAP79. All data are normalized to maximal Fura-2 signal obtained upon the addition of 5 μ m ionomycin (Ca 2+ ionophore) and 5 m m Ca 2+ . C , effects of the AKAP/PKA disruptor peptide, St-Ht31 (10 μ m ), on CCE-stimulated AC8 activity assessed using Epac2-camps. Experiments were performed in the presence of 100 μ m IBMX, and CCE was induced upon the addition of 0.5 m m Ca 2+ . St-Ht31P (10 μ m ) was used as a negative control. D , analyses of the effects of AKAP79 knockdown or pharmacological AKAP disruption (St-Ht31) on AC8 activity. Plots of peak CCE-induced cAMP increase relative to control ( left chart ) and peak rate of cAMP production ( right chart ) are presented as mean ± S.E. ( error bars ). **, p < 0.001 using Students' t test.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Disruption, Activity Assay, Knockdown, Transfection, shRNA, Negative Control, Control

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: Co-immunoprecipitation of endogenously expressed AC8 and AKAP150 in MIN6 cells. A , Western blot analysis to confirm the endogenous expression of AKAP150 ( left ) and AC8 ( right ) in MIN6 cells. B , immune complexes for endogenous AKAP150 co-purify with AC8 in MIN6 cell lysate. No AC8 band is seen in IgG controls. Antibodies used for immunoprecipitation ( IP ) and subsequent immunoblotting ( IB ) were specific to AKAP150 and AC8, respectively.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Immunoprecipitation, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: Effects of AKAP150 on Ca 2+ -stimulated AC8 activity in MIN6 cells. A , Fura-2 data showing the standard protocol for inducing CCE in MIN6 cells. Cells were pretreated with 1 μ m Tg in Ca 2+ -free conditions for 3 min prior to the addition of 2 m m external Ca 2+ . The addition of 100 μ m 2-aminoethoxydiphenyl borate ( 2-APB ; a CCE inhibitor when used at high concentrations) from 1 min onward significantly reduced Ca 2+ entry (see bar chart inset ; ***, p < 0.001). Bi , Epac2-camps data showing the effects of AKAP150 overexpression on Ca 2+ -stimulated cAMP production compared with empty vector controls. 20 n m FSK and 100 μ m IBMX were present throughout. Data are plotted as a percentage of maximal FRET signal obtained using saturating cAMP concentrations. Bii , as above, except that the effects of lentiviral shRNA directed against AKAP150 were compared with scrambled shRNA controls. C , bar charts show mean ± S.E. values ( error bars ) for peak amplitude and rate of FRET ratio changes during CCE for data in B . *, p < 0.05 compared with shRNA controls.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Activity Assay, Over Expression, Plasmid Preparation, shRNA

Journal: The Journal of Biological Chemistry

Article Title: AKAP79/150 Interacts with AC8 and Regulates Ca 2+ -dependent cAMP Synthesis in Pancreatic and Neuronal Systems *

doi: 10.1074/jbc.M110.120725

Figure Lengend Snippet: A role for AKAP150 in the regulation of Ca 2+ -stimulated AC activity in hippocampal neurons. A , the basic design of the citrine-Epac2-camps-CFP (Ci/C-Epac2-camps) sensor used for hippocampal experiments and fluorescent images taken from three individual hippocampal neurons showing cytosolic expression of the cAMP sensor. Scale bar , 20 μm. B , in vitro calibrations of the Ci/C-Epac2-camps compared with the original Epac2-camps. Note the reduced pH sensitivity of the citrine-CFP version. C , comparison of Ca 2+ -stimulated AC activity in control, AKAP150-HA-expressing, and AKAP150 knockdown hippocampal neurons assessed using Ci/C-Epac2-camps. 1 μ m FSK was added in Ca 2+ -free conditions at 120 s with the readdition of 2 m m external Ca 2+ at 180 s to monitor Ca 2+ -dependent cAMP production. Maximum FRET ratio change was obtained by the subsequent addition of 10 μ m FSK, 10 μ m isoproterenol, and 100 μ m IBMX. D , overlay of control, AKAP150 overexpression, and shRNA AKAP150 data to compare Ca 2+ -stimulated AC activities. E , data analysis showing significant delay in Ca 2+ stimulation of cAMP production in neurons overexpressing AKAP150. Data represent mean ± S.E. ( error bars ) for each condition. n values are indicated on the graph . *, p < 0.01.

Article Snippet: For knockdown of endogenously expressed AKAP150 in primary cultured hippocampal neurons, shRNA AKAP150 lentiviral particles (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added directly to cultured cells 72 h prior to experiments.

Techniques: Activity Assay, Expressing, In Vitro, Comparison, Control, Knockdown, Over Expression, shRNA

Journal: Cell metabolism

Article Title: Targeting Peripheral CB 1 Receptors Reduces Ethanol Intake via a Gut-Brain Axis

doi: 10.1016/j.cmet.2019.04.012

Figure Lengend Snippet: (A) Time course and rate of increase in oleate-driven respiratory activity in MGN3–1 cells. Cells were treated with 10 or 100 nM JD5037, 2.5 M of FCCP (positive control) or vehicle in the presence of the fluorescent extracellular O2 consumption reagent. Oxidative respiration was recorded every 90 sec and the slope of the initial linear increase was calculated. Points and bars are means ± SEM from n = 11–15 experiments, as indicated. *P < 0.05 compared to vehicle. (B) Verification of Cnr1 knockdown by rtPCR in cells used in panel C. Cellular uptake of the constructs was verified by fluorescent microscopy (20x magnification) and the degree of knockdown was determined by rt-PCR, *P < 0.05, n = 4. (C) Oleate-driven oxidative activity in MGN3–1 cells with shRNA-mediated knockdown of Cnr1 expression and their mock-transfected controls, n = 3–8 experiments, as indicated, *P < 0.05.

Article Snippet: Scramble shRNA with GFP Adenovirus (Ad-scramble-shRNA) , Vector Biolabs , 1122.

Techniques: Activity Assay, Positive Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Construct, Microscopy, shRNA, Expressing, Transfection

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Affinity purification of SirT7-binding factors identified the histone H2A variant mH2A1 by MS analysis (table S2). HA, hemagglutinin. ( B ) Schematic representation of the three mH2A isoforms, of which, only mH2A1.1 binds to ADP-ribose . ( C ) Endogenous SirT7 specifically immunoprecipitates mH2A1.1 in HEK293F cells. IgG, immunoglobulin G. ( D and E ) mH2A1.1 recognizes and binds ADP-ribosylated SirT7. Immunoprecipitation (IP) of bacterially expressed rSirT7 WT or N189A preincubated ± NAD + and added to nuclear extracts of HEK293F SirT7 KO cells (CRISPR-Cas9–mediated KO of SIRT7) expressing mH2A1.1 WT or G224E, a mutant deficient in ADP-ribose binding. Inputs (I) and elutions (E) are shown. A similar experiment with H187Y is shown in fig. S3A. ( F ) Top: Interaction between endogenous SirT7 and mH2A1.1 under high-stringency conditions upon different types of stress in HEK293 cells. C, untreated; IR, 7-gray ionizing irradiation; H 2 O 2 , oxidative stress. Western blot of the input and elution of immunoprecipitation experiments with anti-SirT7 antibody under these conditions. Bottom: Summary of the ADP-ribosylation events detected by MS in SirT7-FLAG expressed in the same cells and purified under the same stress conditions. Further details are shown in table in fig. S3B and in table S1. ( G ) Superose 6 gel filtration chromatography of nuclear endogenous proteins from nuclear extracts of HEK293F cells under normal cell growth (NT) or upon GS. Fraction numbers and approximate molecular weights (MW) are indicated. Western blot of SirT7 and mH2A1.1 are shown. ( H ) High-stringency immunoprecipitation of endogenous mH2A1.1 by WT or N189A SirT7 in HEK293F cells treated under normal conditions or under GS. Inputs (I) and elutions (E) are shown.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Affinity Purification, Binding Assay, Variant Assay, Immunoprecipitation, CRISPR, Expressing, Mutagenesis, Irradiation, Western Blot, Purification, Filtration, Chromatography

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Levels of three mH2A isoforms in WCE of NIH3T3 cells transfected with scramble shRNA or mH2A1 shRNA and cultured under normal or GS conditions. ( B ) Levels of endogenous SirT7 and histone H3 in chromatin and nucleoplasm fractions purified from Wt and Sirt7 −/− MEFs cultured under normal or GS conditions during the indicated times. ( C ) Venn diagrams showing the intersection of SIRT7-associated genes with mH2A1-enriched genes in Wt cells under NT (top) or GS (bottom). SirT7-associated and mH2A1-enriched genes were derived from GREAT analysis (fig. S3B and Materials and Methods). ( D ) Average enrichment of mH2A1 at all genes in Wt (left) or SirT7 −/− MEF (right) cells under NT (black) or GS (red) conditions. Data are expressed as the log 2 ratio of reads per kilobase of transcript per million mapped reads–normalized ChIP/input signals. ( E ) Distribution of sites occupied by SirT7 upon GS around the TSS by GREAT analysis. The values for each bin from the TSS are shown above each bar (TSS: −5 kb, 5 to 50 kb, 50 to 500 kb, and >500 kb). ( F ) KEGG cell signaling pathways for SirT7-associated genes mapped by GREAT analysis under GS in MEF cells. The signaling pathways were ranked by their combined score provided by Enrichr analysis. cGMP-PKG, guanosine 3′,5′-monophosphate–protein kinase G; cAMP, cyclic adenosine 3′,5′-monophosphate; TCA, tricarboxylic acid. ( G ) SirT7 ChIP-qPCR (quantitative polymerase chain reaction) analysis of SirT7 binding sites associated with mH2A1 at distal regions upon shRNA-mediated down-regulation of mH2A1 under normal and GS conditions in NIH3T3 cells. The amplified regions (red) and their distance to each gene are indicated in the upper part of each graph. Each SirT7 ChIP was normalized with respect to its own input. SEM from n = 4. Two-tailed t test (* P < 0.05 and *** P < 0.005).

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Transfection, shRNA, Cell Culture, Purification, Derivative Assay, Protein-Protein interactions, ChIP-qPCR, Real-time Polymerase Chain Reaction, Binding Assay, Amplification, Two Tailed Test

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Heat map showing RNA expression changes relative to NT (as log 2 magnitude of difference between GS and NT) conditions in Wt and SirT7-deficient MEFs. ( B ) Pipeline applied to RNA-seq data to filter genes associated with SirT7/mH2A1 in Wt and Sirt7 −/− MEF cells treated under GS or NT. The analysis was restricted to genes that (i) were associated with SirT7 via GREAT and were mH2A1-enriched, (ii) showed a log 2 fold change (FC) of expression between WT-GS and WT-NT >0.6, and (iii) showed a difference between WT and KO log 2 FC (GS versus NT) of >0.3 (table S3). ( C ) mH2A1 ChIP-seq signals across ctgf , a gene differentially enriched in mH2A1 upon GS in Wt MEFs compared with NT. ( D ) Top: Real-time qPCR (RT-qPCR) analysis of genes regulated by SirT7 upon GS and NT. The expression of SirT7 in SirT7 −/− MEFs was rescued by retroviral-mediated gene transfer of SirT7 WT, H187Y(HY), N189A(NA), and empty vector (−). SEM from n = 4. Two-tailed t tests (* P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.001). Bottom: Relative mH2A1 enrichment (GS versus NT) by ChIP-qPCR analysis at specific regions around the TSS of the indicated genes [ gbp6 , −6 kb; necab1 , −3 kb; lair1 , −27.5 kb; ctgf , +600 base pairs (bp); adra2a , −5 kb; and nrip3 , +7 kb]. SEM from n = 3. One-way analysis of variance (ANOVA) (* P <0.05, ** P < 0.01, and *** P < 0.005). a.u., arbitrary units. ( E ) Chromatin state transitions induced by GS in Wt and SirT7-deficient cells. The colors of the arrows indicate the frequency (%) of the transition as stated in the color scale (right). Bottom right: State map illustrating the specific combination of mH2A1 and/or H3K27me3 in the four chromatin states defined in the analysis. U1, without H3K27me3 or mH2A1; U2, mH2A1; U3, H3K27me3; U4, enriched by H3K27me3 and mH2A1.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: RNA Expression, RNA Sequencing, Expressing, ChIP-sequencing, Quantitative RT-PCR, Retroviral, Plasmid Preparation, Two Tailed Test, ChIP-qPCR

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Model validation studies in WT and Sirt7 −/− mice fed AL or calorie restricted (CR, 30%) for 8 weeks. ( B ) RT-qPCR analysis of the indicated genes in liver samples from Wt and Sirt7 −/− mice fed AL or CR. Three animals were analyzed for each condition. Each quantification was generated from three replicates. Probabilities are those associated with one-way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.005). ( C ) Levels of SirT7 in liver samples from Wt and Sirt7 −/− mice AL and CR after subcellular fractionation. WCE and chromatin fractions are shown. ( D ) SirT7 immunoprecipitation of mH2A1 in the same liver samples. Inputs (I) and elutions (E) are shown. ( E ) Autophagy activity in the Wt and Sirt7 −/− livers under AL or CR monitored by levels of formation of LCIII-2. Left: A representative Western blot of n = 5 replicates used in the quantification shown. Right: Quantification of the relative accumulation of LCIII-2 compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) analyzed with a two-tailed t test (* P < 0.05). GAPDH was used as a loading control, as we did not detect a significant alteration of the levels of the protein in our conditions (data not shown). ( F ) Similar analysis ( n = 5) of Beclin-1 as in (E). ( G ) Model proposed for the dual SirT7/mH2A regulatory axis in GS. On the basis of our data, we speculate that this axis is also involved in CR and aging. Nutrient stress induces SirT7 auto-mADPRT, which leads to mH2A-dependent recruitment of SirT7 to distal regulatory regions and subsequent mH2A enrichment around the associated genes. This axis plays a key role in CR in vivo and possibly in aging by modulating key signaling pathways.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Generated, Fractionation, Immunoprecipitation, Activity Assay, Western Blot, Two Tailed Test, Control, In Vivo, Protein-Protein interactions

Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor-κB-inducing Kinase (NIK) Contains an Amino-terminal Inhibitor of Apoptosis (IAP)-binding Motif (IBM) That Potentiates NIK Degradation by Cellular IAP1 (c-IAP1)

doi: 10.1074/jbc.m114.587808

Figure Lengend Snippet: FIGURE 2. The NIK IBM is important for reinforcing the interactions within the c-IAPTRAF2TRAF3NIK complex and is necessary for proper E3 ubiquitin ligase targeting of NIK by c-IAP1. A, HEK293T cells were transfected with TRAF3 siRNA to suppress endogenous TRAF3 expression along with plasmids encoding NIK-GFP or NIK (A2G/V3G)-GFP and FLAG-cIAP1 or FLAG-cIAP1 (H588A). Cell lysates were prepared after 48 h of incubation and analyzed by Western blot using anti-GFP, anti-TRAF3, and anti-FLAG-HRP. B, FLAG-cIAP1 constructs with either NIK-GFP or NIK (A2G/V3G)-GFP were transfected in HEK293T cells, and co-IPs were conducted using an anti-NIK antibody. Eluates were immunoblotted by anti-FLAG-HRP for detection of the c-IAP1-NIK interaction. In addition, the levels of endogenous TRAF2 and TRAF3 in the c-IAPTRAFNIK complex were also analyzed by Western blot. The expression levels of NIK, cIAP1, TRAF2, and TRAF3 in input lysates were confirmed by immunoblotting with indicated antibodies. CON, control; EV, empty vector.

Article Snippet: Antibodies used for immunoblotting: NIK (H-248), TRAF2 (C-20), TRAF3 (G-6), GST (Z-5) (Santa Cruz Biotechnologies); TRAF2 (BD Pharmingen); FLAG-HRP, HA-HRP, FLAG M2, and -actin (Sigma); GFP (Novus).

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Incubation, Western Blot, Construct, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: A bi-allelic splicing donor site variant in LRRC23 was identified from asthenozoospermia patients. ( A ) A consanguineous pedigree with two infertile males (IV-1 and IV-2). IV-1 was subjected for WES (arrow). Genotypes of the variant (blue) in all attended family members (III-1, III-2, IV-1, IV-2, IV-3, and IV-4) are confirmed by Sanger sequencing. +, wild-type allele. An infertile female sibling (IV-4) is marked in black circle. ( B ) Papanicolaou-stained sperm from the infertile male (IV-2). ( C ) Mapping of the LRRC23 variant. Mutation of G to A at the splicing donor site in the 5 th intron is predicted to prevent LRRC23 mRNA from splicing. ( D ) Sequencing chromatograms presenting the LRRC23 variant in the infertile male (IV-1) and his father (III-2). The variant is underlined and normal splicing donor site (GT) is boxed. ( E-F ) Minigene assay for testing altered splicing of LRRC23 by the variant. ( E ) Minigene constructs expressing LRRC23 ORF containing the 5 th intron (sashed) with wild-type (WT) or mutant (Mut, red) splicing donor site were generated. The constructs are tagged with FLAG and HA at N- and C-termini, respectively. ( F ) RT-PCR of the 293T cells transfected with the minigene constructs reveals the 5 th intron is not spliced out and retained by the variant. Intron-spanning primers, F1 and R1, are used. Three times biological replicated.

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Variant Assay, Sequencing, Staining, Mutagenesis, Mini Gene Assay, Construct, Expressing, Generated, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: Lrrc23 mutant mice mimicking human splice variant phenocopy male infertility and reduced sperm motility. ( A-B ) Immunoblotting of LRRC23 in testis ( A ) and epididymal sperm ( B ) from mutant male mice. Truncated LRRC23 (arrowheads) is detected from testis microsome fraction (filled), but not in mature sperm (empty), of heterozygous (+/Δ) and homozygous (Δ/Δ) males. Acetylated tubulin (AcTub) is a loading control. ( C ) Confocal images of immunostained LRRC23 in Lrrc23 +/Δ and Lrrc23 Δ/Δ epididymal sperm. Samples from WT were used for positive or negative control of normal or truncated LRRC23 ( A, B , and C ). ( D ) Epididymal sperm counts. n.s., not significant. ( E ) Pregnancy rate of Lrrc23 +/Δ and Lrrc23 Δ/Δ males. ( F ) Number of litters from fertile females mated with Lrrc23 +/Δ and Lrrc23 Δ/Δ males. ( G ) Swimming trajectory of Lrrc23 +/Δ and Lrrc23 Δ/Δ sperm in viscous media (0.3% methylcellulose). Swimming trajectory for 2 seconds is overlaid. ( H ) Flagellar waveforms of Lrrc23 +/Δ and Lrrc23 Δ/Δ sperm before (0 minute) and after (90 minutes) inducing capacitation. Flagellar movements for two beat cycles are overlaid and color coded in time. Circles indicate sperm counts from individual males ( D ) and pup numbers from each litter ( F ). Data represented as mean ± SEM ( D and F ). Statistical comparison was perfomed by Mann-whiteny U test (D) or Student’s t-test (F). Experiments were repeated three times with biological replications ( A, B, C, G , and H ).

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Mutagenesis, Variant Assay, Western Blot, Control, Negative Control, Comparison

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: C-terminal truncation of human LRRC23 by the splicing site mutation prevents its interaction with radial spoke (RS) head. ( A ) Sub-tomogram averaging images of RSs from Chlamydomonas reinhardtii ( left ), Trypanosoma brucei ( middle ), and mouse sperm ( right ). Original data from Electron Microscopy Data Bank was rendered. ( B ) Structure of RS in C. reinhardtii . A schematic cartoon shows the RS1 and 2. The structure of RS2 stalk is shown in inset (PDB Id: 7JRJ). ( C - D ) Purification of normal (hLRRC23 WT ) and the mutant human LRRC23 (hLRRC23 Mut ) by the splicing site mutation (c.621+1G>A) in this study. ( C ) Diagrams for the purified recombinant normal and mutant proteins tagged with tagged with GST and HA at N- and C-termini, respectively. ( D ) Purified proteins by Coomassie blue staining ( left ) and immunoblotting with a-HA ( middle ) and a-LRRC23 ( right ). Proteins matched to the predicted size were marked with asterisks. ( E ) A cartoon of the RSPH-trap approach to test LRRC23 interaction with RS proteins. Individual human RS proteins tagged with FLAG (RSPH-FLAG) are expressed in 293T cells and enriched by α-FLAG resin from cell lysates. The recombinant RSPH proteins were incubated with the purified hLRRC23 WT or hLRRC23 Mut and subjected to immunoblotting. ( F ) Interaction of hLRRC23 to a RS head component, RSPH9. The purified hLRRC23 were incubated with the RSPH-Trap (RS head, RSPH6A and RSPH9; stalk, RSPH3 and RSPH22) and subjected to immunoblotting. 5% amount of the hLRRC23s used for the trap assay were loaded as inputs. White lines in individual α-HA blot images indicate marker information (75 kDa, left ; 50 kDa, right ). Experiments were repeated four times. Purified GST was used for negative control ( SI Appendix , Fig. S4 B ). ( G ) A phylogenetic tree constructed by Maximum-likelihood analysis of the protein sequences of the C. reinhardtii RSP15 and the orthologs of LRRC23 and LRRC34. LRR37, the first LRRC23 ortholog identified in Ciona intestinalis is marked in bold. ( H ) Comparison of the reported RSP15 from C. reinhardtii and the predicted structure of LRRC23 and LRRC34 from human. Atomic structure of the C. reinhardtii RS2 containing RSP15 are represented by ribbon (RS2) and surface (RSP15) diagram ( left , PDB Id: 7JU4). Ribbon diagrams of C. reinhardtii RSP15 and AlphaFold-predicted human LRRC23 ( middle ) and LRRC34 ( right ) are shown for structural comparison. Secondary structures are color-coded. Different from C. reinhardtii RSP15 and LRRC34, LRRC23 does not display repeated α-helix (magenta) between β-sheets (gold).

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Mutagenesis, Electron Microscopy, Purification, Recombinant, Staining, Western Blot, Incubation, TRAP Assay, Marker, Negative Control, Construct, Comparison

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: Head of the third radial spoke is absent in Lrrc23 Δ/Δ sperm flagella. ( A - B ) Sub-tomogram averaging (STA) to analyze structural defects at radial spoke (RS) of WT ( A ) and Lrrc23 Δ/Δ sperm ( B ). Shown are STA images resulted from 96-nm doublet repeats from WT and Lrrc23 Δ/Δ sperm. RS2 and 3 are magnified and density to represent RS3 head and the bridge between RS2 and RS3 (red circle) is missed in Lrrc23 Δ/Δ sperm specifically. ( C ) Overwrapped STA images from 96 nm-doublet repeats from WT (gray) and Lrrc23 Δ/Δ (gold) sperm, and Chlamydomonas reinhardtii (cyan). ( D ) A proposed model of impaired sperm motility and male infertility by the LRRC23 loss of function.

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques:

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: LRRC23 mutation disrupts the third radial spoke (RS) in sperm flagellum. ( A ) Immunostaining of flagellar proteins in different compartments. Shown are midpiece (TOM20), annulus (SEPT4 and SEPT12), fibrous sheath (AKAP4), outer dense fiber (ODF2), and axoneme (acetylated tubulin, AcTub) in Lrrc23 +/Δ ( top ) and Lrrc23 Δ/Δ ( bottom ) sperm. Magnified insets are represented for annulus proteins (scale bars in insets = 2μm). Fluorescence and corresponding DIC images are merged. Sperm heads were counter stained with Hoechst. Experiments were performed with three biological replications. (B) Transmission electron microscopy images of Lrrc23 +/Δ ( left ) and Lrrc23 Δ/Δ ( right ) sperm. Shown are longitudinal section of sperm flagella. M, mitochondria; ODF, outer dense fiber; AX, axoneme; CP, central pair; MT, microtubule; FS, fibrous sheath. ( C ) Cryo-electron tomography (cryo-ET) of WT and Lrrc23 Δ/Δ sperm flagella. Shown are representative tomographic slices from WT ( left ) and Lrrc23 Δ/Δ sperm ( right ). The 9+2 axonemal structure are shown in both WT and Lrrc23 Δ/Δ in cross-sectional view ( left ). Axonemal structures are shown with proximal side of the flagellum on the left in longitudinal view ( right ). Magnified insets ( bottom ) reveal that RS1, 2, and 3 are shown in WT sperm ( left , filled arrowheads) but RS3, especially head part, is not clearly visible ( right , red arrowheads) in Lrrc23 Δ/Δ sperm. Lrrc23 +/Δ ( A and B ) or WT ( C ) sperm were used for positive control.

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Mutagenesis, Immunostaining, Fluorescence, Staining, Transmission Assay, Electron Microscopy, Tomography, Positive Control