Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) Schematic of Cas9 fused to the HUH endonuclease PCV with a covalently attached ssODN. ( b ) SDS-PAGE of Cas9 variants reacted with an Alexa 488 fluorescently labelled ssDNA containing the PCV recognition sequence. The top panel is the coomassie stained gel, and the bottom panel is the identical fluorescently imaged gel. PCV is fused to either the carboxyl (Cas9-PCV) or amino (PCV-Cas9) terminus of Cas9. Cas9-PCV(Y96F) represents catalytically inactive PCV(Y96F) fused to Cas9. (C) SDS-PAGE gel shift assay of Cas9 reacting with two ssDNA templates containing the PCV recognition sequence of differing lengths in a 1:1 ssDNA:Cas9 molar ratio.

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: SDS Page, Sequencing, Staining, Electrophoretic Mobility Shift Assay

Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) Schematic of split luciferase insertion. The C-terminus of NanoLuc nanoluciferase (HiBiT) is encoded on the 200bp ssODN along with the 5’ PCV recognition sequence and targeted to the 3’ end of GAPDH . ( b ) Assaying luminescence using different Cas9 variants when inserting HiBiT into GAPDH in HEK-293T cells. PCV is fused to either the amino (PCV-Cas9) or carboxyl (Cas9-PCV) terminus of Cas9. Transfections were performed with ssODN lacking the PCV recognition sequence (PCV- ssODN) or ssODN containing the PCV recognition sequence (PCV+ ssODN). Units are displayed in relative light units (RLU) normalized to Cas9. ( c ) The calculated fold change from (b) between the PCV-ssODN and PCV+ ssODN is shown for each variant. ( d ) Targeting the GAPDH locus in U2-OS cells. ( e ) Targeting a locus in vinculin in HEK-293T cells using an ssODN containing the PCV recognition sequence. ( f ) Fold change in RLU compared to Cas9 when varying the amount of RNP (equimolar ssODN). All graphs represent data from one of multiple independent experiments exhibiting similar results. Data are shown as mean +/− SD (n=3). Significance calculated using 2-tailed Student’s t-test: ** P < 0.01, *** P < 0.001, ns = no significance (P >0.05).

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: Luciferase, Sequencing, Transfection, Variant Assay

Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) HEK-293T cells stably expressing a mutant mCherry-GFP reporter are edited by HDR through a frameshift correction, restoring mCherry activity. ( b ) Representative microscopy images of fluorescent reporter editing. ( c ) The percent of mCherry positive cells determined by flow cytometry at two different RNP concentrations using an ssODN containing the PCV recognition sequence ( d ) RNP transfections at 3 pmol in the presence or absence of ssODN. Data are shown as mean +/− SD (n=3). For (c) and (d), the statistical significance of %mCherry positive cells between PCV-fusions of Cas9 and Cas9 alone was <0.001, calculated using 2-tailed Student’s t-test.

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: Stable Transfection, Expressing, Mutagenesis, Activity Assay, Microscopy, Flow Cytometry, Sequencing, 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: Science advances

Article Title: Paralemmin-1 controls the nanoarchitecture of the neuronal submembrane cytoskeleton.

doi: 10.1126/sciadv.adt3724

Figure Lengend Snippet: Fig. 1. Palm1 is an MPS component. (A) Palm1-interacting prey clones aligned with the βII-spectrin sequence. Each horizontal line represents one of 258 prey clones. Vertical dashed lines indicate the SMO. CH, calponin homology; PH, pleckstrin ho- mology. (B and C) Representative two-color STED images of rat HPN (DIV 19, metha- nol fixation) immunolabeled for Palm1 and the βII-spectrin C terminus (B) or adducin (C). Scale bars, 1 μm. (D) Average autocorrelation (AC) and cross-correlation (CC) analyses of Palm1 and βII-spectrin of n = 27 axons from N = 3 independent neuronal cultures. (E) As (D) for Palm1 and adducin (n = 20, N = 3). (F) Confocal image of a rat HPN (DIV 19, PFA fixation) expressing endogenous Palm1 tagged with mEGFP and detected by using a nanobody against mEGFP. Arrows point at regions displayed in (G) and (H). Scale bar, 25 μm. (G) Close-up STED image of the axon coimmunostained against βII-spectrin. Scale bar, 1 μm. Dashed line on the merged image indicates the region on which the AC and CC analyses shown at the bottom have been performed. (H) Close-up STED images of a dendrite with dendritic spines. Scale bars, 1 μm.

Article Snippet: The specificity of the antibodies was confirmed under the conditions used here, by IF of neuronal cultures, and by Western blotting of brain subcellular fractions (fig. S6, A and B) from WT and Palm1KO animals. mEGFP- Palm1 CRISPR knock- in pORANGE- mEGFP- Palm1 was based on the CRISPR- Cas9 knockin template vector pORANGE (gift from H. MacGillavry, Addgene plasmid #131471; RRID:Addgene_131471) (60).

Techniques: Clone Assay, Sequencing, Immunolabeling, Expressing

Journal: Science advances

Article Title: Paralemmin-1 controls the nanoarchitecture of the neuronal submembrane cytoskeleton.

doi: 10.1126/sciadv.adt3724

Figure Lengend Snippet: Fig. 2. Palm1 populates distal axonal regions before βII-spectrin, but is incorporated into the MPS later. (A) Representative confocal images of rat HPN (DIV 1, 3, 5, 12, and 19, methanol fixation) immunolabeled against Palm1, βII-spectrin, and ankG. Scale bars, 25 μm. Shown are the maximum intensities projection of five z-stacks. Black arrowheads point at the neurite end/growth cone. (B and C) Normalized (A.U., arbitrary units) and smoothed (50 values) fluorescence intensities of Palm1, βII- spectrin, and ankG along the axons indicated by the dashed lines on the representative image at DIV 3 (B) and at DIV 19 (C). Gray area in (B) highlights the typical enrich- ment of Palm1 at the neurite end/growth cone. (D to G) Normalized fluorescence intensities (A.U.) and AC analyses of Palm1 [(D) and (E)] and βII-spectrin [(F) and (G)] along the proximal, middle, and distal axons at different DIVs. Axons analyzed in the same region for (D) and (E) in the proximal/middle/distal region: DIV 3: 35/31/21; DIV 5: 23/29/15; DIV 12: 25/33/26; DIV 19: 17/22/26. Axons analyzed for (F) and (G): DIV3: 24/18/20; DIV 5: 29/26/32; DIV 12: 22/13/23; DIV 19: 12/11/27. All from N = 3. Statistical analyses: One-way ANOVA with post hoc Tukey correction. All P values in file data S1. Histograms show mean ± SEM. (H) Representative image of the growth cone of an HPN (DIV 2, PFA fixation), immunolabeled against Palm1 and βII-spectrin, and phalloidin labeled for F-actin. Scale bar, 5 μm. (I) Ratio of mean fluorescence intensities be- tween the central (CD) and the peripheral (PD) domains of growth cones, segmented according to the phalloidin signal. Growth cones analyzed: n = 56, from N = 3.

Article Snippet: The specificity of the antibodies was confirmed under the conditions used here, by IF of neuronal cultures, and by Western blotting of brain subcellular fractions (fig. S6, A and B) from WT and Palm1KO animals. mEGFP- Palm1 CRISPR knock- in pORANGE- mEGFP- Palm1 was based on the CRISPR- Cas9 knockin template vector pORANGE (gift from H. MacGillavry, Addgene plasmid #131471; RRID:Addgene_131471) (60).

Techniques: Immunolabeling, Fluorescence, Labeling

Journal: Science advances

Article Title: Paralemmin-1 controls the nanoarchitecture of the neuronal submembrane cytoskeleton.

doi: 10.1126/sciadv.adt3724

Figure Lengend Snippet: Fig. 3. Overexpression of both Palm1 splice variants increases the complexity of neuronal morphology and enhances βII-spectrin periodicity. (A) mRNA expres- sion levels of Palm1 and Palm1ΔEx8 during development of HPN. Fold change relative to the housekeeping genes. N = 3, statistical analyses: One-way ANOVA with post hoc Tukey correction. All P values in file data S1. (B) Representative confocal images of rat HPN (DIV 3, PFA fixation) electroporated with plasmids encoding either YFP, YFP-CaaX, YFP-Palm1, or YFP-Palm1ΔEx8. Scale bars, 25 μm. (C) Sholl analysis of neurons overexpressing the indicated constructs. Intersections were counted every 1 μm. Cells analyzed: YFP, n = 34; YFP-CaaX, n = 24; YFP-Palm1, n = 31; YFP-Palm1ΔEx8, n = 27. All from N = 3. (D) STED images of rat HPN at DIV 3 overexpressing YFP, YFP-Palm1, and YFP-Palm1ΔEx8, and endogenous βII-spectrin. Proximal (left) and middle (right) regions of the same axon are shown. YFP was detected using nanobodies. (E) Same as (D) but for DIV 19. Scale bars, 2 μm. Corresponding axons shown in fig. S3. (F and G) AC amplitude analysis of endogenous βII-spectrin along different axonal regions in untransfected (UT) neurons, or after overexpression of YFP, YFP-Palm1, and YFP-Palm1ΔEx8 at DIV 3 (F) and DIV 19 (G). (H and I) Normalized fluorescence intensities (A.U.) of βII-spectrin along the same axonal regions measured in (F) and (G), respectively. (J and K) Correlation scatter plots of the periodicity of βII-spectrin versus Palm1 (J) or Palm1ΔEx8 (K). r, Pearson’s r coefficient; P, P value. Axons analyzed in the proximal/middle region in (F) and (H): WT, 63/62; YFP, 27/27; YFP-Palm1, 31/30; YFP-Palm1ΔEx8, 31/31; and in (G) and (I) to (K): WT, 32/35/30; YFP, 18/17/13; YFP-Palm1, 20/21/20; YFP-Palm1ΔEx8, 19/23/21. All from N = 3. Statistical analyses: one-way ANOVA with post hoc Tukey correction; all P values in file data S1. Histograms show mean ± SEM.

Article Snippet: The specificity of the antibodies was confirmed under the conditions used here, by IF of neuronal cultures, and by Western blotting of brain subcellular fractions (fig. S6, A and B) from WT and Palm1KO animals. mEGFP- Palm1 CRISPR knock- in pORANGE- mEGFP- Palm1 was based on the CRISPR- Cas9 knockin template vector pORANGE (gift from H. MacGillavry, Addgene plasmid #131471; RRID:Addgene_131471) (60).

Techniques: Over Expression, Construct, Fluorescence

Journal: Science advances

Article Title: Paralemmin-1 controls the nanoarchitecture of the neuronal submembrane cytoskeleton.

doi: 10.1126/sciadv.adt3724

Figure Lengend Snippet: Fig. 4. Palm1-KO mature neurons have a disorganized MPS. (A) Representative STED images of βII-spectrin nanoscale organization along the proximal and middle axons in mouse WT and Palm1-KO neurons (DIV 19). Scale bars, 5 μm. (B and C) AC amplitudes calculated from the regions indicated by the dashed lines in (A) (P: proximal; M: middle). (D) AC amplitude analysis and (E) normalized fluorescence intensities (A.U.) of endogenous βII-spectrin in the proximal and middle axons of WT and Palm1-KO neurons. Axons analyzed in the proximal/middle/distal region in (D): WT, 38/43/21; Palm1-KO, 70/58/42; and in (E): WT, 22/23/11; Palm1-KO, 36/27/22. All from N = 3. Sta- tistical analyses: one-way ANOVA with post hoc Tukey correction; all P values in file data S1. Histograms show mean ± SEM.

Article Snippet: The specificity of the antibodies was confirmed under the conditions used here, by IF of neuronal cultures, and by Western blotting of brain subcellular fractions (fig. S6, A and B) from WT and Palm1KO animals. mEGFP- Palm1 CRISPR knock- in pORANGE- mEGFP- Palm1 was based on the CRISPR- Cas9 knockin template vector pORANGE (gift from H. MacGillavry, Addgene plasmid #131471; RRID:Addgene_131471) (60).

Techniques: Fluorescence

Journal: Science advances

Article Title: Paralemmin-1 controls the nanoarchitecture of the neuronal submembrane cytoskeleton.

doi: 10.1126/sciadv.adt3724

Figure Lengend Snippet: Fig. 5. Palm1 reintroduction into Palm1-KO neurons rescues and enhances the periodic organization of the MPS. (A) Representative STED images of recombinant YFP-Palm1 and endogenous βII-spectrin fluorescence periodicity, displaying an untransfected axon lacking Palm1 (top neurite) and an axon rescued by overexpression of YFP-Palm1ΔEx8 (bottom neurite; DIV 13, PFA fixation). AC analysis along the dashed lines shows the enhanced periodic pattern of βII-spectrin after Palm1ΔEx8 overex- pression. Scale bar, 4 μm. (B) AC amplitude analysis of βII-spectrin along different axonal regions in untransfected Palm1-KO neurons, or after overexpression of YFP, YFP- Palm1, or YFP-Palm1ΔEx8 (DIV 13, transfection at DIV 5). (C) Normalized intensities (A.U.) of endogenous βII-spectrin along the same axonal regions analyzed in (B). (D) Correlation scatter plot of βII-spectrin periodicity versus Palm1 or (E) Palm1ΔEx8. r, Pearson’s r coefficient; P, P value. Axons analyzed for (B) to (E) in the proximal/ middle/distal region: Palm1-KO, 23/23/12; YFP, 6/6/6; YFP-Palm1, 6/7/7; YFP-Palm1ΔEx8, 7/7/7. All from N = 1. Statistical analyses: one-way ANOVA with post hoc Tukey correction; all P values in file data S1. Histograms show mean ± SEM.

Article Snippet: The specificity of the antibodies was confirmed under the conditions used here, by IF of neuronal cultures, and by Western blotting of brain subcellular fractions (fig. S6, A and B) from WT and Palm1KO animals. mEGFP- Palm1 CRISPR knock- in pORANGE- mEGFP- Palm1 was based on the CRISPR- Cas9 knockin template vector pORANGE (gift from H. MacGillavry, Addgene plasmid #131471; RRID:Addgene_131471) (60).

Techniques: Recombinant, Fluorescence, Over Expression, Transfection

Journal: Science advances

Article Title: Paralemmin-1 controls the nanoarchitecture of the neuronal submembrane cytoskeleton.

doi: 10.1126/sciadv.adt3724

Figure Lengend Snippet: Fig. 6. Molecular features involved in interactions between the MPS, Palm1, and β-spectrin. (A) The Palm1 W54A mutation abolishes MPS integration and remodel- ing. STED images of rat HPN (DIV 14, PFA fixation) overexpressing YFP-Palm1(W54A), and endogenous βII-spectrin along the proximal (left) and middle axon (right). Scale bar, 1 μm. (B) AC amplitude analyses of YFP, YFP-Palm1, and YFP-Palm1(W54A) and (C) of βII-spectrin along axonal regions in untransfected (UT) neurons, or after electro- poration with the indicated constructs (DIV 13 to 15). Axons analyzed for (B) and (C) in the proximal/middle region: YFP, 10/11; YFP-Palm1, 19/20; YFP-Palm1(W54A), 16/18; Untransfected, 20/21. All from N = 2 to 3. Statistical analyses: one-way ANOVA with post hoc Tukey correction; P values in file data S1. Histograms show mean ± SEM. (D) Scheme of βII-spectrin N-terminal region with Palm1-binding sequence (amino acids 261 to 307) and the interval blocking Palm1/βII-spectrin interaction (amino acids 169 to 184), as well as the collinear PIP2-binding sites in actinin-2. Below, the βII-spectrin prey constructs A to G are aligned, together with the yeast spot colonies detect- ing these interactions (right). (E) Selectivity of Palm1 and phosphomimetic mutant Palm1-E5 for five β-spectrin and three actinin isoforms as preys. (F) Palm1 and Palm1-E5 interact with βII-spectrin even at high 3AT concentrations. Smad/Smurf: positive control. (G) Yeast spot colonies of a C-terminal Palm1 deletion series and Palm1ΔEx8 as baits. (H) Yeast spot colonies of truncated Palm1(1-263), its ΔEx8 splice variant, and several missense mutants as baits. Yeast colony images are shown in the negative for better visualization. In experiments (D), (G), and (H), complete 3AT concentration series (0, 1, 2, 5, 10, 20, 50, 100, and 200 mM) were tested, but only selected concentra- tions with the most informative growth patterns are shown. In experiments (G) and (H), βII-spectrin prey G was used. (I) 3D structure of the βII-spectrin N-terminal domains relevant for Palm1 binding (movie S1), as predicted by AlphaFold2 (Q62261, amino acids 1 to 529).

Article Snippet: The specificity of the antibodies was confirmed under the conditions used here, by IF of neuronal cultures, and by Western blotting of brain subcellular fractions (fig. S6, A and B) from WT and Palm1KO animals. mEGFP- Palm1 CRISPR knock- in pORANGE- mEGFP- Palm1 was based on the CRISPR- Cas9 knockin template vector pORANGE (gift from H. MacGillavry, Addgene plasmid #131471; RRID:Addgene_131471) (60).

Techniques: Mutagenesis, Construct, Binding Assay, Sequencing, Blocking Assay, Positive Control, Variant Assay, Concentration Assay

Journal: Science advances

Article Title: Paralemmin-1 controls the nanoarchitecture of the neuronal submembrane cytoskeleton.

doi: 10.1126/sciadv.adt3724

Figure Lengend Snippet: Fig. 7. Palm1 proximity to adducin at the MPS. (A) Single-color 3D MINFLUX of periodic YFP-Palm1 along the axon of a mature neuron (DIV 18, PFA fixation). Scale bar, 500 nm. YFP-Palm1 was detected by using a nanobody against YFP in combination with DNA-PAINT. (B) YZ projection of the region highlighted in (A) shows a hollow structure, indicating that Palm1 localizes to the plasma membrane. (C) 2D projection of region indicated in (A). (D) First to fourth NN analysis of Palm1 molecules. Data from 6075 trace ID (TID, i.e., individual localization bursts) from n = 4. (E) Two-color 3D MINFLUX (exchange DNA-PAINT) reveals clear periodic patterns (highlighted by black arrowheads) for both YFP-Palm1 (blue) and adducin (magenta). Black boxes indicate Palm1 doublets flanking adducin molecules displayed in the close-ups below. (F) NN distances of Palm1 and adducin obtained from 918 TIDs corresponding to the image shown in (E).

Article Snippet: The specificity of the antibodies was confirmed under the conditions used here, by IF of neuronal cultures, and by Western blotting of brain subcellular fractions (fig. S6, A and B) from WT and Palm1KO animals. mEGFP- Palm1 CRISPR knock- in pORANGE- mEGFP- Palm1 was based on the CRISPR- Cas9 knockin template vector pORANGE (gift from H. MacGillavry, Addgene plasmid #131471; RRID:Addgene_131471) (60).

Techniques: Clinical Proteomics, Membrane

Journal: Science advances

Article Title: Paralemmin-1 controls the nanoarchitecture of the neuronal submembrane cytoskeleton.

doi: 10.1126/sciadv.adt3724

Figure Lengend Snippet: Fig. 8. Visualization of Palm1-induced cellular and molecular manifestations in relation to its expression levels.

Article Snippet: The specificity of the antibodies was confirmed under the conditions used here, by IF of neuronal cultures, and by Western blotting of brain subcellular fractions (fig. S6, A and B) from WT and Palm1KO animals. mEGFP- Palm1 CRISPR knock- in pORANGE- mEGFP- Palm1 was based on the CRISPR- Cas9 knockin template vector pORANGE (gift from H. MacGillavry, Addgene plasmid #131471; RRID:Addgene_131471) (60).

Techniques: Expressing

Journal: Cell reports

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis

doi: 10.1016/j.celrep.2021.110008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Briefly, a guide RNA (5′-GCAGATGTAGTGTTTCCACA-3′) targeting the open reading frame in the immediate vicinity of the stop codon was cloned into the Cas9 expression vector pX330-U6-Chimeric_BB-CBh-hSpCas9 (gift from Feng Zhang; ), Addgene plasmid #42230).

Techniques: Derivative Assay, Recombinant, Electron Microscopy, Transfection, Expressing, Plasmid Preparation, Software, Cell Analysis, Gas Chromatography, Mass Spectrometry

Journal: Antioxidants

Article Title: Combined Activity of the Redox-Modulating Compound Setanaxib (GKT137831) with Cytotoxic Agents in the Killing of Acute Myeloid Leukemia Cells

doi: 10.3390/antiox11030513

Figure Lengend Snippet: Assessment of a potential mechanism for Setanaxib effects. ( A , B ) Effect of Setanaxib on the proliferation of human AML cells with NOX4 or p22-phox knockout. KO human AML cells were obtained using CRISPR/Cas9 technology. The cell lines stably express Cas9 and were either transduced with control sgRNA (sgLuci, designated WT) or sgRNA targeting NOX4 or p22-phox , respectively (designated KO). Absence of the targeted gene was detected as described in Materials and Methods. The experimental setup for the drug treatments and assay of proliferation were as in A,B. Cells were counted using a hemocytometer at day 8. Mean ± SD of cell treatments of two to three independently sgRNA-transduced cell batches is presented. ( C ) Comparison of synergy of Setanaxib with daunorubicin in Ba/F3-FLT3-ITD cells harboring Cas9 and transduced with sgLuci (WT) or sg Nox4 (KO). Treatments were performed as in and proliferation/viability was assessed by Cell Titer Blue assay. The individual experiments were normalized to DMSO controls. Mean ± SD (with technical triplicates) for cells from three independent transductions with sgRNA is shown. ( D , E ) The 32D-FLT3-ITD cells were subjected to single or combined drug treatments with daunorubicin, diphenyleneiodonium (DPI) or N-acetylcysteine (NAC) as indicated for 72 h, and proliferation/viability was assessed by Cell Titer Blue assay. Three independent experiments (in triplicate) were conducted; error bars represent mean ± SD. Statistical analyses were carried out using two-tailed t -test (n.s.—not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: In brief, MOLM13, MV4-11, Ba/F3 cells, and HEK293 cells (with tet-inducible NOX4) with stable Cas9 expression were generated by transduction with lentiviral particles expressing Cas9 (Streptococcus pyogenes gene in lentiCas9-Blast plasmid, #52962, Addgene Cambridge, MA, USA) and cell selection with blasticidin using standard techniques, and subsequent clonal selection was employed for high Cas9 levels, monitored by immunoblotting.

Techniques: Knock-Out, CRISPR, Stable Transfection, Transduction, Control, Comparison, Two Tailed Test

Journal: Antioxidants

Article Title: Combined Activity of the Redox-Modulating Compound Setanaxib (GKT137831) with Cytotoxic Agents in the Killing of Acute Myeloid Leukemia Cells

doi: 10.3390/antiox11030513

Figure Lengend Snippet: Setanaxib promotes ROS formation elicited by daunorubicin. ( A – C ) MV4-11 cells were treated with the indicated concentrations of the FLT3-ITD inhibitor AC220 (quizartinib, positive control), the general NOX inhibitor diphenyleneiodonium (DPI), or Setanaxib for 4 h in serum-free medium. Thereafter, cells were lysed and lysates subjected to immunoblotting for assessment of pathway activation. Blots were probed with activation-specific antibodies to pFLT3 (pY589/591), pSTAT5 (pY694), pAkt (Ser473), or pErk1/2 (Thr202/Tyr204) antibodies. Subsequently, blots were stripped and comparable loading was validated by reprobing of the membranes with antibodies against FLT3, STAT5, Akt, or Erk as indicated. ( A ) Representative result. ( B , C ) Quantification of three independent experiments for the indicated signaling molecules. Setanaxib (GKT137831) is abbreviated as GKT. Error bars represent mean ± SD. Statistical analyses were carried out with two-tailed t -test. ( D ) MOLM-13 cells were treated with 5 mM NAC (positive control), 500 nM of the general NOX inhibitor diphenyleneiodonium (DPI), 40 nM of the protein kinase inhibitor midostaurin, or 10 or 30 µM Setanaxib for 24 h. Thereafter, cells were stained with H 2 DCFDA for 30 min and ROS levels were quantified on the flow cytometer (MFI, mean fluorescence intensity). Error bars represent mean ± SD, ( n = 3). Statistical analyses were carried out using two-tailed t -test. ( E ) The 32D-FLT3-ITD cells were treated with the indicated concentrations of the drugs for 24 h. Thereafter, cells were stained with ROS Deep Red dye for 30 min and ROS levels were quantified by flow cytometry. The experiment was conducted three times independently. Error bars represent mean ± SD, ( n = 3). Statistical analyses were carried out using two-tailed t -test. ( F ) HEK293 cells with tetracycline (tet)-inducible NOX4 overexpression and engineered to constitutively express Cas9 were kept uninduced (−tet) or were induced (+tet) to overexpress NOX4 and were mock-treated with solvent or were treated with Setanaxib as indicated. ROS formation was scored with H 2 DCFDA as in ( D ). Error bars represent mean ± SD, ( n = 3). Statistical analyses were carried out using two-tailed t -test (n.s.—not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: In brief, MOLM13, MV4-11, Ba/F3 cells, and HEK293 cells (with tet-inducible NOX4) with stable Cas9 expression were generated by transduction with lentiviral particles expressing Cas9 (Streptococcus pyogenes gene in lentiCas9-Blast plasmid, #52962, Addgene Cambridge, MA, USA) and cell selection with blasticidin using standard techniques, and subsequent clonal selection was employed for high Cas9 levels, monitored by immunoblotting.

Techniques: Positive Control, Western Blot, Activation Assay, Two Tailed Test, Staining, Flow Cytometry, Fluorescence, Over Expression, Solvent