Journal: eLife

Article Title: Urotensin II-related peptides, Urp1 and Urp2, control zebrafish spine morphology

doi: 10.7554/eLife.83883

Figure Lengend Snippet:

Article Snippet: Commercial assay, kit , Taq Polymerase , New England Biolabs , Cat no: M0273 , .

Techniques: Gel Extraction, Hybridization, Amplification, SYBR Green Assay, Software, Sequencing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: Density of UG nucleotide sequences 100bp upstream and downstream of m6A modifications identified by cross-linking induced mutation sites (CIMS; A ) or cross-linking induced truncation sites (CITS; B ) in relation to random sequences (red line). Grey shading represents 95% confidence regions. ( C ) Schematic of HaloTag immunoprecipitation and dot blot procedure. ( D ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of HaloTag-labeled proteins in HEK293T cells overexpressing HaloTag, TDP43-HaloTag or YTHDF2-HaloTag from 3 biological replicates. ( E ) Diagram illustrating insertion of the HaloTag open reading frame into the endogenous TARDBP locus immediately 5’ to the TDP43 start codon, resulting in a fusion of HaloTag to the N-terminus of TDP43. ( F ) Halo-TDP43 HEK293T cells labeled live with JF646 Halo dye (red), then fixed, permeabilized, and immunostained with anti-TDP43 antibody (green) prior to imaging. DAPI (blue) marks the nucleus of each cell. Scale bar = 10µm. ( G ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of endogenous HaloTag-TDP43 or exogenous HaloTag. Additional replicates shown in Sup. Fig. 1.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Mutagenesis, Immunoprecipitation, Dot Blot, Modification, Isolation, Immunoaffinity Purification, Labeling, Imaging

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) HaloTag-TDP43 immunoprecipitation was followed by DART-seq to delineate m6A sites within TDP43 target RNAs. HaloTag-TDP43 HEK293T cells were transfected with APOBEC1-YTH or APOBEC1-YTHmut and crosslinked before immunoaffinity purification of HaloTag-labeled proteins. Immunoprecipitated RNAs were then sequenced and C-T transitions were identified in the context of DRACH motifs (red shaded box, D=A/G/T, R=A/G, H=A/C/T). Absolute counts ( B ) and relative frequency ( C ) of base pair transitions observed by RNA-seq in each condition. Shaded boxes represent transition types expected from APOBEC1 activity. ( D ) Example m6A sites identified by DART-seq in RPL10A . C-T transitions are highlighted in red, and DRACH motifs in pink. Green arrow, transcription start site; red hexagon, transcription stop site; thick blue bars, coding exons; thin blue bars, untranslated region. ( E ) Absolute count and relative distribution ( F ) of DART-seq reads in cells expressing APOBEC1-YTH and APOBEC1-YTHmut. UTR, untranslated region; CDS, coding sequence. ( G ) Scatter plot of TDP43 targets, determined by fold enrichment in precipitated RNA from HaloTag-TDP43 cells (expressing APOBEC1-YTH and APOBEC10YTHmut) compared to cells transfected with HaloTag. Red dots signify transcripts showing > 2-fold enrichment in both APOBEC1-YTH and APOBEC1-YTHmut expressing cells. TARDBP , yellow dot, identified as high confidence target. ( H ) Stacked bar graph showing percentage of m6A modified RNA in TDP43 targets (red) and non-targets (black). ( I ) Cumulative distribution of RNA methylation in TDP43 targets (red) and non-targets (black). p = 1.87×10 −55 by Kolmogorov Smirnov test. ( J ) Euler diagram depicting overlap between TDP43 targets identified in this study, and those identified by TDP43 cross linking and immunoprecipitation followed by RNA-sequencing (CLIP-seq) in HEK293T cells (Hallegger et al ., 2021) . **p=1.5×10 −117 , hypergeometric test. ( K ) Pie charts demonstrating the percentage of methylated RNA among TDP43 targets (pink) and non-targets (grey). **p<1×10 −5 chi-square test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Immunoprecipitation, Transfection, Immunoaffinity Purification, Labeling, RNA Sequencing, Activity Assay, Expressing, Sequencing, Modification, Methylation

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) TARDBP gene map, illustrating TDP43 binding region (TBR), the location of the DRACH motif (pink square), and the C-T transition (red box) identified by DART-seq within this domain, representing an m6A site. ( B ) Schematic of the TARDBP minigene reporter, consisting of the mCherry ORF upstream of TARDBP exon 6 and 3.4 Kb of the TARDBP 3’ UTR. The A residue adjacent to the detected C-T transition via DART-seq in the WT reporter (mCherry-TBR) was mutated to a G, precluding methylation the mutant reporter (mCherry-mTBR). Red, methylated residue; blue line, DRACH motif; dagger, C-T transition from DART-seq. ( C ) HaloTag-TDP43 was isolated by immunoaffinity purification from HaloTag-TDP43 HEK293T cells expressing mCherry-TBR or mCherry-mTBR, and reporter RNA detected in elution fractions by qRT-PCR. ( D ) Outline of TDP43 autoregulation assay. Excess TDP43 binds to the reporter, triggering reporter splicing, destabilization, and reduced mCherry fluorescence. ( E ) Primary rodent neurons were transfected with WT (mCherry-TBR) or mutant (mCherry-mTBR) reporters, together with EGFP or TDP43-EGFP. After 7d, mCherry expression was assessed by fluorescence microscopy. Scale bar= 20 µm. Normalized RFP (mCherry) intensity in primary neurons expressing WT mCherry-TBR reporter ( F ) or mutant mCherry-mTBR ( G ) reporter together with EGFP or TDP43(WT)-EGFP. Cherry-TBR+GFP n= 160, Cherry-TBR+TDP43(WT)-GFP n= 58, Cherry-mTBR+GFP n= 105, Cherry-mTBR+TDP43(WT)-GFP n= 44. Data in C plotted as mean ± SD, collected from 3 biological replicates. ns= not significant, *p< 0.05, **p< 0.01; one-way ANOVA with Tukey’s test. Data in F and G plotted as mean ± SD, color coded by biological replicate. ns = not significant, *p < 0.05; Welch’s t-test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Binding Assay, Residue, Methylation, Mutagenesis, Isolation, Immunoaffinity Purification, Expressing, Quantitative RT-PCR, Fluorescence, Transfection, Microscopy

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Genome-wide analysis of RNA methylation via epitranscriptomic array. RNA was extracted from control (n= 3) and sporadic ALS (sALS) patient (n= 4) spinal cord samples, prior to m6A RNA immunoprecipitation. The resulting samples were separated into methylated and non-methylated RNA, then labeled with distinct fluorescent dyes (red and green stars) prior to hybridization, allowing relative quantification of methylation at each annotated locus. ( B ) Principal component analysis (PCA) plot comparing methylation levels of control (grey) and ALS (red) patient samples. ( C ) Hierarchical clustering of mRNA methylation profiles from control and ALS mRNA samples. ( D ) Volcano plot depicting fold change in mRNA methylation levels in ALS compared to control spinal cord. ( E ) Hierarchical clustering of lncRNA methylation profiles from control ALS lncRNA samples. ( F ) Volcano plot showing fold change in lncRNA methylation levels in ALS compared to control spinal cord. In D and F , grey horizontal vertical lines represent p= 0.05 and fold change (FC)= 2. ( G ) Euler diagram demonstrating overlap (n= 322, p= 5.09×10 −119 , hypergeometric test) among TDP43 substrates and methylated transcripts identified in HEK293T cells, in additional to hypermethylated transcripts determined via m6A array in sALS spinal cord. Comparisons were limited to the subset of transcripts expressed in both HEK293T cells and human spinal cord (nTPM>2). ( H ) Based on comparisons with the GEO transcription factor loss-of-function database via Enrichr , there was strong enrichment for TDP43-regulated genes not only among the set of 2034 transcripts hypermethylated in sALS spinal cord, but also among the 322 TDP43 targets that were also hypermethylated in sALS (A1 in G ). Combined score = (log 10 p * Z-score). ( I ) Immunohistochemical staining for m6A in control and sALS spinal cord sections. Scale bars= 50 µm. ( J ) Quantification of m6A antibody reactivity in spinal cord neurons from control (n= 110 neurons) and sALS (n= 277 neurons) sections. Plot shows mean +/- SD, color coded by patient. ****p< 0.0001 via Mann-Whitney test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Genome Wide, Methylation, Control, RNA Immunoprecipitation, Labeling, Hybridization, Quantitative Proteomics, Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Representative images of rodent primary neurons transfected with plasmids expressing Cas9-2A-EGFP and sgRNA targeting the neuronal protein NeuN or negative control (LacZ). 5d after transfection, neurons were fixed and immunostained for NeuN (red). White dashed circles indicate nucleus stained with Hoechst (blue). ( B ) NeuN antibody reactivity measured in EGFP-positive neurons expressing sgLacZ (n= 565) or sgNeuN (n= 654), ****p < 0.0001 by Mann-Whitney. ( C ) Schematic depicting m6A writers (green), erasers (red), and readers (orange) targeted by CRISPR/Cas9. ( D ) Primary neurons expressing EGFP and TDP43-mApple were assessed at regular 24h intervals by fluorescence microscopy, and their survival assessed by automated image analysis. Individual neurons are assigned unique identifiers (yellow number) and tracked until their time of death (red), indicated by cellular dissolution, blebbing, or neurite retraction. Scale bar= 20µm. ( E ) Cumulative hazard plot depicting risk of death for neurons expressing TDP43(WT) + non-targeting (NT) (red line), mApple + NT (grey line), or TDP43(WT) + Atxn2 sgRNA (purple line). †p<2.0 ×10 −16 , Hazard ratio (HR)= 3.45; ***p= 5.81 ×10 −4 , HR= 0.80). ( F ) Forest plot showing HR for TDP43-overexpressing neurons upon knockdown of m6A writers (green), erasers (dark red), and readers (orange), in comparison to nontargeting (NT) control. Dashed line indicates HR= 1, representing the survival of the reference condition, neurons expressing TDP43-mApple and NT sgRNA. Values >1 indicate increased toxicity, whereas values <1 denote relative protection. Error bars represent 95% CI. ( G ) Alkbh5 knockout significantly increases TDP43 associated toxicity. †p=3.11 ×10 −5 , HR= 1.59; ***p= 2.65×10 −11 , HR= 2.03. ( H ) Ythdf2 knockout significantly extends survival in TDP43-expressing neurons. ***p <2.0 ×10 −16 , HR= 1.69; †p= 6.2 ×10 −6 , HR= 0.71. ( I ) YTHDF2 overexpression is toxic to neurons. ***p= 3.07×10 −5 , HR= 1.30. ( J ) METTL3/14 overexpression enhances TDP43-dependent toxicity in neurons. †p = 5.53 ×10 −4 , HR= 1.32; ***p =4.16 ×10 −6 , HR= 1.31. p values in E, G-J determined via Cox proportional hazards analysis, with a minimum 3 of biological replicates.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Transfection, Expressing, Negative Control, Staining, MANN-WHITNEY, CRISPR, Fluorescence, Microscopy, Dissolution, Knockdown, Comparison, Control, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Immunostaining of YTHDF2 in control and sALS patient spinal cord samples. Scale bar= 50 µm. ( B ) Quantification of YTHDF2 immunoreactivity in spinal cord neurons from control (n= 117 neurons) and sALS (n= 193 neurons) samples. Plot shows mean +/- SD, color coded by sample. ****p< 0.0001 via Mann-Whitney test. ( C ) Strategy used to create isogenic iPSCs expressing native TDP43(WT)-Dendra2 or TDP43(M337V)-Dendra2. ( D ) Representative images of untransduced (grey) and transduced (green) iNeurons expressing shRNA against YTHDF2 (shYTHDF2) and a GFP reporter. Time of death (red circles) for each cell is used to determine cumulative risk of death, plotted in ( E ) and ( F ). Scale bar= 20µm. shRNA-mediated knockdown of YTHDF2 significantly extended the survival of TDP43(M337V)-Dendra2 iNeurons ( E ; †p= 8.42×10 −12 , HR= 6.25; ***p= 4.82×10 −9 , HR=0.32; #p= 0.08, HR= 1.84) as well as mutant C9ORF72 iNeurons ( F , †p= 1.42×10 −11 , HR= 2.85; ***p= 1.42×10 −16 , HR= 0.32). ns= not significant. Values in ( E , F ) calculated by Cox proportional hazards analysis, with a minimum 3 biological replicates.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Immunostaining, Control, MANN-WHITNEY, Expressing, shRNA, Knockdown, Mutagenesis

Journal: Frontiers in Cell and Developmental Biology

Article Title: Pak1ip1 Loss-of-Function Leads to Cell Cycle Arrest, Loss of Neural Crest Cells, and Craniofacial Abnormalities

doi: 10.3389/fcell.2020.510063

Figure Lengend Snippet: Cell cycle progression is arrested in Pak1ip1 mray/mray embryos. Representative histograms displaying cell cycle profiles of WT control and Pak1ip1 mray/mray embryos obtained by flow cytometry of propidium iodide labeled cells. Three different stages were analyzed, E10.5 (A,D,G) , E12.5 (B,E,H) , and E14.5 (C,F,I) to obtain a stage-dependent developmental delineation of cell cycle progression. (G) At all stages, a significant decrease in the proportion of cells in G 2 /M phase can be observed in Pak1ip1 mray/mray embryos with this fraction decreasing as development proceeds. In contrast, the proportion of cells in G 0 /G 1 stage are significantly increased at E10.5 and E14.5 with this increase being highest at E14.5. * p < 0.05 and ** p < 0.01. (J,K) Lateral views of Ccng1 RNA in situ hybridization analysis at E9.5 demonstrates upregulated expression in Pak1ip1 mray/mray embryos compared to WT with greatest differences observed at the ventral forebrain (FB) and along the body axis (asterisks). (L,M) Frontal views of E11.5 embryos confirm Ccng1 upregulation in the mutant with greatest increase observed in the frontonasal prominences (FNP) (asterisks). (N) The results of real time qRT-PCR confirm a significant upregulation of Ccng1 transcripts in E10.5 Pak1ip1 mray/mray embryos. BA: branchial arch, FB: forebrain, HB: hindbrain, MB: midbrain, MdP: mandibular process, MxP: maxillary process.

Article Snippet: Lastly, cells were resuspended in 500 μl of propidium iodide/RNase staining solution (Cell Signaling Technologies, Danvers, MA, United States) before running them again through Life Technologies Attune NxT Flow Cytometer.

Techniques: Control, Flow Cytometry, Labeling, RNA In Situ Hybridization, Expressing, Mutagenesis, Quantitative RT-PCR

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: Lz expansion is associated with increasing levels of circulating and endothelial IGF2 (A) Weights of micro-dissected Lz. (B) Linear correlation analyses between fetal and Lz weights: p = 0.002 (E14), p < 0.0001 (E16), and p < 0.0001 (E19) (n = 46–189 placentae from n > 10 L per group in [A] and [B]). (C) Levels of IGF2 (ng/mL) in plasma of wild-type fetuses. (D) Linear correlation analyses between fetal weights and circulating IGF2: p < 0.0001 (E16 and E19) (n = 70–79 per group in [C] and [D]). (E) Igf2 mRNA in situ hybridization (blue) in E14 wild-type Lz (red arrows—FPEC, feto-placental endothelial cells; AS, antisense probe; inset with S, sense probe; scale bars, 20 μm). (F) Relative Igf2 mRNA expression levels measured by qRT-PCR in FPEC from wild-type Lz (n = 6–7 per group). (G) Imprinted genes that rank within top 100 expressed genes in E16 wild-type FPEC (FPKM, fragments per kilobase million; n = 4). (H) Double immunostaining for IGF2 and CD31 in E19 wild-type placenta. Endothelial cells are very thin and hard to detect except where the cytoplasm is more voluminous around the nucleus, with intense IGF2 stain (white arrows). Transmembrane glycoprotein CD31 immunostaining is in the membrane and largely marks endothelial intercellular junctions (scale bars, 20 μm). (I) Semi-quantitative measurement of IGF2 protein in FPEC versus trophoblast cells (E19 wild-type Lz, n = 60 cells per group from two placentae). White arrows—endothelial cells; scale bars, 50 μm. For (E), (H), and (I): FC, fetal capillaries; MBS, maternal blood spaces; LT, labyrinthine trophoblast cells; S-TGC, sinusoidal trophoblast giant cells. Data in (A), (C), (F), (G), and (I) are presented as averages ± standard deviation (SD); ∗∗∗ p < 0.001 calculated by one-way ANOVA plus Tukey’s multiple comparisons test in (A) and (F) or by unpaired t test with Welch’s correction in (C) and (I). See also .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Clinical Proteomics, In Situ Hybridization, Expressing, Quantitative RT-PCR, Double Immunostaining, Staining, Immunostaining, Membrane, Standard Deviation

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: Deletion of Igf2 in the epiblast or endothelium impairs Lz expansion (A) Left: schematic of Igf2 expression in conceptuses with conditional deletion driven by Meox2 Cre . Right: immunostaining for YFP (green) in a representative fetus and placenta paraffin section at E12 of gestation, double transgenic for Meox2 Cre and Rosa26 fl STOP fl YFP 10 reporter. YFP expression in the placenta is localized to the Lz and Cp (high magnification, inset). Blue—DAPI stain for nuclei; scale bars: 1 mm (low magnification) and 100 μm (high magnification). (B) Fetal and placental growth kinetics, measured as average wet-weights for each genotype per litter (E12: n = 10 L [n = 41 controls {C} and n = 32 Igf2 EpiKO ]; E14: n = 25 L [n = 114 C and n = 88 Igf2 EpiKO ]; E16: n = 37 L [n = 154 C and n = 127 Igf2 EpiKO ]; E19: n = 37 L [n = 164 C and n = 121 Igf2 EpiKO ]). (C) Absolute volumes of the placental layers (Db, decidua basalis; Jz, junctional zone; Lz, labyrinthine zone; Cp, chorionic plate), measured by stereology (n = 6 per group). (D) Absolute volumes of Lz components, measured by stereology (LT, labyrinthine trophoblast; MBSs, maternal blood spaces; FCs, fetal capillaries) (n = 6 per group). (E) Left: schematic representation of Igf2 expression in conceptuses with conditional deletion driven by Tek Cre . Right: representative confocal microscopy of frozen sections from a fetus and its corresponding placenta, double transgenic for TeK Cre and Ai9(RCL-tdT) reporter at E16 of gestation. Scale bars: 2 mm (fetus) and 1 mm (placenta). (F) Fetal and placental growth kinetics (E12: n = 5 L [n = 17 C and n = 16 Igf2 ECKO ]; E14: n = 8 L [n = 26 C and n = 34 Igf2 ECKO ]; E16: n = 13 L [n = 60 C and n = 46 Igf2 ECKO ]; E19: n = 7 L [n = 31 C and n = 27 Igf2 ECKO ]). (G) Absolute volumes of the placental layers measured by stereology (n = 5–7 per group). (H) Absolute volumes of Lz components, measured by stereology (n = 5–7 per group). (I) Double immunostaining for EPCAM (epithelial cell adhesion molecule) (red) and MCT1 (monocarboxylate transporter 1) (green) in a representative frozen placental section at E12 of gestation. EPCAM expression is observed as clusters of positive cells within the Lz placenta. Blue—DAPI (4′,6-diamidino-2-phenylindole) stain for nuclei; scale bars: 500 μm (left panel) and 20 μm (right panel). (J) Analysis of EPCAM high -positive cells by flow cytometry. Left panel: example of gating used to identify EPCAM high -positive cells (the viability dye 7-aminoactinomycin D [7-AAD] was used to exclude dead cells). Right: quantification of placental EPCAM high -positive cells at E12 in conceptuses with conditional Igf2 deletion driven by Meox2 Cre (n = 10 C and n = 9 Igf2 EpiKO from 2 L) or Tek Cre (n = 8 C and n = 8 Igf2 ECKO from 2 L). For all graphs data are shown as averages; error bars represent SD in (C), (D), (G), (H), and (J) or 95% confidence intervals (95% CI) in (B) and (F); N.S.—statistically not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 calculated by a mixed effects model in (B) and (F) (see STAR Methods), two-way ANOVA plus Sidak’s multiple comparisons tests in (D) and (H) or unpaired t tests in (C), (G), and (J). See also .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Expressing, Immunostaining, Paraffin Section, Transgenic Assay, Staining, Confocal Microscopy, Double Immunostaining, Flow Cytometry

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: Lack of fetus-derived IGF2 reduces the expansion of feto-placental microvasculature in late gestation (A) Functions enriched in DEGs at E19. (B) qRT-PCR analysis of angiopoietin-Tie2/TEK signaling components in Lz (n = 6–8 per group). (C) TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining in E16 Lz (arrows point to apoptotic cells) and data quantification (n = 6 samples per group); scale bars, 50 μm. (D) Left: representative double immunostaining for TUNEL (red) and laminin (green, marker of feto-placental capillaries) in the Lz of an E16 Igf2 EpiKO mutant placenta (DAPI, blue marks the nuclei; white and red arrows indicate TUNEL + FPECs and LT, respectively; scale bars, 25 μm). Right: quantification of TUNEL + cells that are positive or negative for laminin (n = 6 Igf2 EpiKO mutant placentae). (E) Feto-placental endothelial cell (FPEC) proliferation measured by flow cytometry (left—representative histograms at E16; right—data quantification; n = 4–11 per group). (F) qRT-PCR analysis of Adgre1 in Lz. (G) Representative F4/80 immunostainings in E16 Lz (arrows indicate macrophages). Scale bars, 100 μm. Right: percentage of macrophages/Lz at E16 (n = 6–8 samples per group). (H) Representative CD31 immunostaining in Lz (scale bars, 100 μm). (I) qRT-PCR analysis for SynT-II (syncytiotrophoblast layer II) marker genes. For all graphs, data are presented as averages or individual values; error bars are SD; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by two-way ANOVA plus Sidak’s multiple comparisons tests in (B), (C), (E), (F), and (I) or Mann-Whitney tests in (G). See also Figure S4 and .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Derivative Assay, Quantitative RT-PCR, TUNEL Assay, Staining, Double Immunostaining, Marker, Mutagenesis, Flow Cytometry, Immunostaining, MANN-WHITNEY

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: Genetic models of mismatched placental and fetal growth reveal circulating IGF2 as a major endocrine regulator of FPEC and Lz expansion (A–E) Column 1: schematic diagrams of the genetic models: Igf2 EpiKO (A), Igf2 ECKO (B), Igf2 TrKO (C), Igf2 UbKO (D), and H19 -DMD EpiKO (E). Columns 2 and 3: total numbers (column 2) and proportion of FPEC/Lz (column 3), measured by flow cytometry (n conceptuses per group: Igf2 EpiKO : n = 9–18; Igf2 ECKO : n = 5–11; Igf2 TrKO : n = 6–17; Igf2 UbKO : n = 3–26; H19 -DMD EpiKO : n = 9–15). Column 4: Lz growth kinetics ( Igf2 EpiKO : n = 9–20 L; Igf2 ECKO : n = 3–9 L; Igf2 TrKO : n = 4–9 L; Igf2 UbKO : n = 3–8 L; H19 -DMD EpiKO : n = 3–4 L). Column 5: IGF2 levels (ng/mL) in plasma (n per group: Igf2 EpiKO : n = 12; Igf2 ECKO : n = 9; Igf2 TrKO : n = 6–7; Igf2 UbKO : n = 7–11; H19 -DMD EpiKO : n = 9). Data are shown as averages or individual values and error bars are SD (columns 2, 3, and 5) and 95% CI (column 4). N.S.—not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 calculated by two-way ANOVA plus Sidak’s multiple comparisons tests (second and third columns), mixed effects model (fourth column) or Mann-Whitney tests (fifth column). See also A, S5B, and .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Flow Cytometry, Clinical Proteomics, MANN-WHITNEY

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: IGF2 signaling regulates angiogenic properties of endothelial cells (A) Volcano plot representation of DEGs identified by RNA-seq in E16 FPEC ( Igf2 EpiKO versus controls). Significant upregulated and downregulated DEGs (false discovery rate [FDR] < 0.05) are shown with red and blue, respectively. (B) Top scoring biological processes enriched in DEGs. Biologically validated DEGS are listed in parentheses. The dotted line corresponds to FDR-corrected p value of 0.05. (C) Biological validation. Data are shown as averages (n = 11–12 samples per group); error bars are SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 calculated by Mann-Whitney tests. (D) Volcano plot representation of DEGs identified by RNA-seq in E16 FPEC ( Igf2 ECKO versus controls). Significant upregulated and downregulated DEGs (FDR < 0.05) are shown with red and blue, respectively. (E) Transcription factors (TFs) identified by analysis of motif enrichment (AME). (F) IPA regulatory network built with the four TFs identified using AME analysis. Proteins labeled with a star are known regulators of angiogenesis (angiostatic or pro-angiogenic factors) and key references are listed in . See also C and S5D and and .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: RNA Sequencing, Biomarker Discovery, MANN-WHITNEY, Labeling

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: IGF2 Acts on FPECs via IGF2R-ERK signaling ex vivo (A) Primary FPEC isolated from E16 Lz: D0—freshly isolated cells; D10—FPEC at passage one (P1, 10 days of culture). (B) Confocal imaging of passage one FPEC, stained for CD31 (scale bars, 20 μm). (C) Flow cytometry analysis of P1 FPEC stained for CD31, demonstrating that these are almost exclusively CD31 + . (D) qRT-PCR analysis for Igf1r , Igf2r , and Insr in FPECs isolated by FACS (n = 6–7 per group). (E) Relative expression of the three IGF receptors in P1 FPEC. (F) qRT-PCR analysis of Igf2 mRNA levels in P1 FPEC cultured in 5% O 2 versus primary FPEC isolated from E16 Lz by FACS. (G) Schematic representation of IGF2 and IGF receptors. IGF2 Leu27 analog acts specifically on IGF2R and picropodophyllin (PPP) inhibits phosphorylation of IGF1R. (H) Representative images of capillary-like tube formation assay in primary FPEC seeded on matrigel and exposed to exogenous IGF2, IGF2 Leu27 , PPP, or PPP+IGF2 (equal seeding of cell numbers at 30 min and tube formation at 8 h), and quantification of number of nodes, branches, and total length (n = 5–6 independent experiments). (I) qRT-PCR analysis of Igf2r mRNA levels in primary FPECs upon knockdown by siRNA (n = 8 samples/group). (J) Proliferation assay of primary FPEC with or without IGF2R siRNA knockdown, in presence or absence of IGF2, on 4 consecutive days after plating. Cells with IGF2R siRNA knockdown exhibit significant proliferation defects that are further accentuated upon IGF2 treatment (n = 5 biological replicates per group). (K) qRT-PCR analysis of Angpt2 mRNA levels in primary FPECs transfected with scrambled siRNA or IGF2R siRNA, upon 4 days of treatment with 50 ng/mL mouse recombinant IGF2 (n = 8 samples/group). (L) Left side: identification of delayed ERK1/2 phosphorylation in FPECs with IGF2R siRNA knockdown upon acute treatment with 50 ng/mL mouse recombinant IGF2. HSP90 was used as internal control for protein loading. Right side: quantification of ratios pERK1/2 to total ERK1/2 for n = 3 independent biological replicates. For all graphs, data are presented as averages or individual values and error bars represent SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 calculated by a Mann-Whitney test in (F), two-way ANOVA tests with Sidak’s multiple comparisons test in (H), (J), and (L), Wilcoxon matched-pairs signed rank test in (I) and paired Student’s t test in (K).

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Ex Vivo, Isolation, Imaging, Staining, Flow Cytometry, Quantitative RT-PCR, Expressing, Cell Culture, Phospho-proteomics, Capillary Tube Formation Assay, Knockdown, Proliferation Assay, Transfection, Recombinant, Control, MANN-WHITNEY

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: IGF2 acts on FPECs via IGF2R in vivo (A) Representative double immunostaining for IGF2R (red) and CD31 (green) in Igf2r ECKO mutant and control Lz at E16 (DAPI, blue; scale bars, 25 μm). (B) Flow cytometry analysis showing that the majority (>80%) of Igf2r ECKO mutant feto-placental endothelial cells (FPECs) express YFP (n = 6–14 per genotype). (C) Fetal and placental growth kinetics in Igf2r ECKO ( Igf2r fl/+ ; Tek +/Cre ) mutants compared with Igf2r fl/+ controls (n = 8–28 conceptuses from n = 3–8 L for each developmental stage). (D) Proportion and total numbers of FPEC/Lz measured by flow cytometry (n = 6–14 per group). (E) Representative CD31 staining in E16 Lz (scale bars, 100 μm). (F) Lz growth kinetics: Igf2r ECKO (n = 8–16 conceptuses per group). (G) IGF2 levels (ng/mL) in plasma at E16 (n = 9 per group). (H) Model summarizing the proposed actions of fetus-, endothelial-, and trophoblast-derived IGF2. For all graphs, data are presented as averages or individual values and error bars represent SD in (B), (D), and (G), or 95% CI in (C) and (F). N.S.— not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 calculated by two-way ANOVA tests in (B) and (D), mixed effects model in (C) and (F) and Mann-Whitney tests in (G). See also Figure S7 and .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: In Vivo, Double Immunostaining, Mutagenesis, Control, Flow Cytometry, Staining, Clinical Proteomics, Derivative Assay, MANN-WHITNEY

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet:

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Plasmid Preparation, Recombinant, Blocking Assay, BIA-KA, Western Blot, Stripping Membranes, Reverse Transcription, SYBR Green Assay, Red Blood Cell Lysis, Staining, Enzyme-linked Immunosorbent Assay, In Situ, TUNEL Assay, Imaging, Flow Cytometry, Transfection, Gene Expression, Expressing, Microarray, Isolation, Software

Journal: Sensors (Basel, Switzerland)

Article Title: COVID-19 Testing and Diagnostics: A Review of Commercialized Technologies for Cost, Convenience and Quality of Tests

doi: 10.3390/s21196581

Figure Lengend Snippet: At-Home Molecular Diagnostic (RT-PCR) Tests using Respiratory Specimens and granted FDA Emergency Use Authorization.

Article Snippet: Bio-Rad Laboratories , Bio-Rad SARS-CoV-2 ddPCR Test (05/01/2020) , Reverese transcriptase droplet digital PCR test, for prescription use only , Supermix, reverse transcriptase (RT), and 300 mM dithiothreitol (DTT) solution.

Techniques: Diagnostic Assay, Microarray, Hybridization, Digital PCR, Quantitative RT-PCR, Mass Spectrometry, Amplification, Lysis, Negative Control, Positive Control

Journal: Sensors (Basel, Switzerland)

Article Title: COVID-19 Testing and Diagnostics: A Review of Commercialized Technologies for Cost, Convenience and Quality of Tests

doi: 10.3390/s21196581

Figure Lengend Snippet: At-Home Molecular Diagnostic (RT-PCR) Tests using Saliva Specimens and granted FDA Emergency Use Authorization.

Article Snippet: Bio-Rad Laboratories , Bio-Rad SARS-CoV-2 ddPCR Test (05/01/2020) , Reverese transcriptase droplet digital PCR test, for prescription use only , Supermix, reverse transcriptase (RT), and 300 mM dithiothreitol (DTT) solution.

Techniques: Diagnostic Assay, Quantitative RT-PCR, Digital PCR, Labeling, High Throughput Screening Assay, Transferring

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: Maximal distance of dendritic staining

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: Control

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: Subcellular distribution of BDNF and TrkB mRNAs in cultured hippocampal neurons. Staining by nonradioactive in situ hybridization with digoxigenin-labeled riboprobes and oligonucleotides. Fields are viewed with Nomarski optics, except immunofluorescence in B and F.A, The BDNF riboprobe labels the cell soma and a process identified as a dendrite in B by a double-labeling with an anti-MAP2 monoclonal antibody. C, No staining is observed with a BDNF sense riboprobe. D, The BDNF antisense oligonucleotide probe shows somatodendritic labeling.E, The TrkB riboprobe stains the cell soma and a dendritic process identified as a dendrite, in F, by the anti-MAP2 antibody. G, No labeling is detected with the TrkB sense riboprobe. H, The TrkB antisense oligonucleotide probe labels the cell soma and a dendrite.Arrowheads indicate labeling at dendritic branchings or varicosities. Scale bar (shown in H): 20 μm forA–H.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: Cell Culture, Staining, In Situ Hybridization, Labeling, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: High potassium increases the dendritic localization of TrkB, but not of β-actin, mRNAs in cultured hippocampal neurons. Nonradioactive in situhybridization on cultured hippocampal neurons viewed with Nomarski optics. A, The TrkB riboprobe stains both cell bodies and dendrites. B, On depolarization with 10 mm KCl for 3 hr, the dendritic labeling is detectable at a much greater distance from the cell body. C, The β-actin probe labels the cell bodies under control conditions and (D) after 3 hr depolarization in 10 mm KCl. Scale bar (shown in D): 20 μm forA–D.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: Cell Culture, Labeling, Control

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: Quantitative analysis of dendritic localization of BDNF and TrkB mRNAs after depolarization of hippocampal neurons in culture. Bars in A and Bindicate the fold increase, with respect to controls, of the mean maximal distance at which the in situ labeling in dendrites was detectable (MDDL). A, BDNF mRNA. Depolarization with either 10 (K) or 20 mm KCl (KK) increases the mean maximal distance of dendritic labeling (MDDL) at 3 hr (3 h K and 3 h KK) but not at 30 min (30′ K and 30′ KK). The average MDDL measured with 10 mm KCl (3 h K) is larger than with 20 mm KCl (3 h KK). B, TrkB mRNA. Both KCl concentrations induce a significant increase in MDDL of TrkB mRNA after 30 min (30′ K, 30′ KK) and a stronger increase at 3 hr (3 h K, 3 h KK). ○, Significantly different with respect to the 3 h K-stimulated; ⋄, significantly different with respect to the 3 h KK stimulated; and *, significantly different with respect to the control (no stimulation). Error bars represent SE. The corresponding numerical values, number of dendrites measured, and significance values are shown in Table ​Table11.C, D, E, F, Correlation plots of the MDDL versus dendritic length. Each point refers to one MDDL determination, together with the length of the corresponding dendrite, under the experimental conditions indicated. The scatter plots were fitted by linear regression lines through the origin. C, Slope = 0.30, correlation coefficient r = 0.57; D, slope = 0.68; r = 0.80; E, slope = 0.26; r = 0.31; F, slope = 0.67; r = 0.72.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: In Situ, Labeling, Control

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: The effects of KCl depolarization do not depend on new mRNA synthesis. Nonradioactive in situ hybridization on cultured hippocampal neurons viewed with Nomarski optics. In control conditions, cells were kept in the presence of actinomycin-d for 3.5 hr and then stained with (A) BDNF or (C) TrkB riboprobes. After a pretreatment of 30 min with actinomycin-d, cultures were depolarized for 3 hr in 10 mm KCl in the continuous presence of actinomycin-d and stained with (B) BDNF or (D) TrkB riboprobes. Scale bar (shown inD): 20 μm for A–D. E, Quantitative analysis of the MDDL for BDNF and TrkB mRNAs with actinomycin-d. Bars indicate the fold increase, with respect to controls, of the mean maximal distance at which the in situ labeling was detectable (MDDL). Results are similar for both BDNF and TrkB mRNAs: treatment of control cultures with actinomycin-d for 3.5 hr (C act.D) reduces the MDDL with respect to untreated controls (C), whereas a strong increase of MDDL induced by 3 hr in 10 mm KCl also occurs in the presence of actinomycin-d (3h K act.D). Error bars represent SE. *, Significantly different from controls. The corresponding numerical values, the number of dendrites measured, and the significance values are shown in Table ​Table11.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: In Situ Hybridization, Cell Culture, Control, Staining, In Situ, Labeling

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: Effects of Ca2+-free medium on the KCl-induced increase in dendritic localization of BDNF and TrkB mRNAs. Quantitative analysis of the MDDL for BDNF and TrkB mRNAs in nominally Ca2+-free medium supplemented with 10 μm EGTA or BAPTA-AM. Bars indicate the fold increase, with respect to controls, of the mean maximal distance at which the in situ labeling was detectable (MDDL) for 80–150 dendrites. A, In nominally Ca2+-free medium the MDDL for BDNF mRNA, induced by depolarization with 10 mm KCl for 3 hr, does not change with respect to controls. The effects are identical in the presence of either EGTA or BAPTA-AM. B, MDDL for TrkB mRNA. At the level of p ≤ 0.05 (Student’st test), no significant variation in MDDL with respect to controls can be observed when cultures are depolarized under Ca2+-free conditions. Identical effects are seen with either EGTA or BAPTA-AM. C, Unstimulated controls in normal medium. CEGTA, Unstimulated controls maintained 3 hr in Ca2+-free medium containing EGTA;CBAPTA-AM, unstimulated controls maintained 3 hr in Ca2+-free medium containing BAPTA-AM; 3h K EGTA, neurons stimulated with 10 mm KCl in Ca2+-free medium containing EGTA; 3h K BAPTA-AM, neurons stimulated with 10 mm KCl in Ca2+-free medium containing BAPTA-AM. Error bars represent SE.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: In Situ, Labeling

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: Effects of TTX, kinurenic acid, and nifedipine on the KCl-induced increase in dendritic localization of BDNF and TrkB mRNAs. Quantitative analysis of the MDDL for BDNF and TrkB mRNAs in the presence of TTX, kinurenic acid, and nifedipine. Barsindicate the fold increase, with respect to controls, of the mean maximal distance at which the in situ labeling was detectable (MDDL). A, Continuous presence of tetrodotoxin (TTX) inhibits the increase in MDDL for BDNF mRNA, induced by depolarization with 10 mmKCl for 3 hr (3h K TTX). The glutamate receptor antagonist kinurenic acid partially counteracts the KCl-induced increase by either 10 (3h K kinu) or 20 mmKCl (3h KK kinu). B, The L-type Ca2+ channel blocker nifedipine has distinct effects at different KCl concentrations, with a partial inhibition at 10 mm KCl (3h K nife) and an almost complete inhibition at 20 mm KCl (3h KK nife).C, TTX strongly inhibits the MDDL increase for TrkB mRNA in 10 mm KCl (3h K TTX). The glutamate receptor antagonist kinurenic acid reduced the 10 mm KCl depolarization effects (3h K kinu) more effectively than at 20 mm KCl (3h KK kinu). D, In contrast, nifedipine almost completely abolishes the effects induced by 20 mm KCl (3h KK nife) and only partially inhibits those induced by 10 mm KCl depolarization (3h K nife). Error bars represent SE. ○, Significantly different with respect to the 3 h K-stimulated; ⋄, significantly different with respect to the 3 h KK stimulated; *, significantly different with respect to the control (no stimulation). Also see Table ​Table11.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: In Situ, Labeling, Inhibition, Control

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: A short incubation in high potassium increases BDNF and TrkB protein levels in the dendritic compartment. Immunohistochemistry on cultured hippocampal neurons. Each picture represents the integration in a single projection of a series of five optical sections obtained with a confocal microscope, as described in Materials and Methods. A, Anti-BDNF immunostaining of control cultures. B, Anti-BDNF immunostaining after 10 mm KCl depolarization for 10 min. C, Anti-BDNF staining after a preincubation with nocodazole for 6 hr followed by 10 min of 10 mm KCl depolarization in the continuous presence of nocodazole. D, Staining with anti-TrkB antibody in control conditions. E, Staining with anti-TrkB antibodies after 10 min depolarization in 10 mm KCl. F, Anti-TrkB staining after a preincubation with nocodazole for 6 hr followed by 10 min of 10 mm KCl depolarization in the continuous presence of nocodazole. Scale bar (shown in F): 20 μm forA–F.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: Incubation, Immunohistochemistry, Cell Culture, Microscopy, Immunostaining, Control, Staining

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: Quantitative analysis of BDNF and TrkB protein levels in proximal and distal regions of the dendrites. Immunofluorescence for BDNF or TrkB was acquired by five confocal sections and integrated in a single projection, in control conditions and after 10 min depolarization with 10 mm KCl, in the presence or absence of cycloheximide (A, C) and of nocodazole (B, D). Fluorescent density of BDNF (A, B) and TrkB (C, D) was determined in proximal and distal regions of the projections of labeled dendrites as described in Materials and Methods. Bars represent the mean fold increase of the fluorescence density of 45 dendrites, with respect to the controls (=1.0). Error bars represent SE.A, Incubation of cells in 10 mm KCl for 10 min leads to a strong increase of BDNF fluorescence density in both proximal and distal regions (10′K). Incubation of control cells with the protein synthesis inhibitor cycloheximide does not alter the basal levels of fluorescence density for BDNF in proximal and distal regions (C + Cyclo). Cycloheximide completely inhibits the increase in fluorescence density induced by the 10 mm KCl stimulus (10′K + Cyclo).B, After pretreatment of cells with nocodazole for 6 hr the levels of BDNF fluorescence density in the proximal dendrites depolarized for 10 min with 10 mm KCl in the continuous presence of nocodazole (10′K + Noco) were comparable to control (C + Noco), whereas a significant fluorescence density increase could be detected in the distal dendrites (10′K + Noco). C, Incubation of cells in 10 mm KCl for 10 min led to a strong increase of fluorescence density for TrkB in the distal region (10′K) but not in the proximal region, and this effect was abolished by cycloheximide (10′K + Cyclo). Incubation of control cells with cycloheximide reduces the basal levels of TrkB fluorescence density in the proximal but not in the distal regions (C + Cyclo). D, After pretreatment of cells with nocodazole for 6 hr, followed by a depolarization for 10 min with 10 mm KCl in continuous presence of nocodazole, the TrkB fluorescence density in the proximal dendrites (10′K + Noco) was comparable to the controls (C + Noco), whereas a fluorescence density increase could be detected in the distal dendrites (10′K + Noco). Significance with respect to controls: op ≤ 0.05; *p ≤ 0.01; **p ≤ 0.001.E, Western blot for the soluble and microtubule cellular pools of tubulin in hippocampal neurons in culture. M1, Soluble tubulin fraction; M2, polymerized tubulin fraction. After 6 hr incubation with nocodazole, the soluble, unpolymerized tubulin fraction is doubled.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: Immunofluorescence, Control, Labeling, Fluorescence, Incubation, Western Blot

Journal: Journal of Hematology & Oncology

Article Title: A cytoplasmic long noncoding RNA LINC00470 as a new AKT activator to mediate glioblastoma cell autophagy

doi: 10.1186/s13045-018-0619-z

Figure Lengend Snippet: FUS interacted with LINC00470 and AKT to form a ternary complex in the cytoplasm. a The interaction of LINC00470 and FUS was detected through RIP assays in U251 cells. Data are presented as the mean ± S.E.M. of three independent experiments. ** p < 0.01. b RNA pulldown showed binding between LINC00470 and FUS. c RIP assays showed that there was no interaction between LINC00470 and AKT in U251 cells. Data are presented as the mean ± S.E.M. of three independent experiments. d HEK293 cells were transfected with HA-AKT, Flag-FUS, and pcDNA3.1-LINC00470. Two-step co-immunoprecipitation verified their interaction. The expression levels of LINC00470, AKT, and FUS were measured with RT-qPCR and Western blotting, respectively. e The localization of AKT and FUS was detected by immunofluorescence staining in HEK293 cells. f The co-localization of AKT and FUS was detected by immunofluorescence staining in U251 cells. g Left, the interactions between endogenous FUS and AKT in the cytoplasm and nucleus were measured by co-immunoprecipitation; right, an RNA pulldown assay showed binding between endogenous LINC00470 and AKT in the cytoplasm and nucleus of U251 cells transfected by si-FUS. h Western blotting detected the expression of FUS in GBM cells transfected by si-FUS. Expression levels of AKT and p-AKT S473 were measured by Western blotting in GBM cells that re-expressed LINC00470 in FUS-KD GBM cells. i Western blotting detected the expression levels of p-AKT S473 in the cytoplasm and nucleus of U251 cells transfected by si-LINC00470

Article Snippet: The following primary antibodies were used: AKT (rabbit, Proteintech, 10176-2-AP, WB1:1500, IP:1:250, RIP:1:100); FUS (rabbit, Abcam, ab23439, WB1:2000, IP1: 200, RIP1:100); phospho-Akt (Ser473) (rabbit, Cell Signaling, #4060, WB1:1500); phospho-Akt (Thr308) (rabbit, Cell Signaling, #13038, WB1:1500); hexokinase I (rabbit, Cell Signaling, #2024, WB1:1000); hexokinase II (rabbit, Cell Signaling, #2867, WB1:1000); Flag (mouse, Sigma-Aldrich, F1804, IP 1:200); GAPDH (mouse, Sangon, D190090, WB 1:5000); H3 (rabbit, Beyotime, AH433, WB 1:500); and p53 (mouse, Active Motif, 39739, WB 1:1000, RIP 1:150).

Techniques: Binding Assay, Transfection, Immunoprecipitation, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: Journal of Hematology & Oncology

Article Title: A cytoplasmic long noncoding RNA LINC00470 as a new AKT activator to mediate glioblastoma cell autophagy

doi: 10.1186/s13045-018-0619-z

Figure Lengend Snippet: FUS bound to AKT and promoted AKT activation. a Co-IP analysis measured the exogenous interaction between FUS and AKT in HEK293 cells. b Representative immunofluorescence staining displayed the co-localization of FUS and AKT in the nucleus of HEK293 cells. c Co-IP analysis measured the endogenous interaction between FUS and AKT in U251 cells. d Representative immunofluorescence staining displayed the endogenous co-localization of FUS and AKT in the cytoplasm of U251 cells. e Left, GST pulldown assays showed that the GGR domain of FUS pulled down AKT; right, GST pulldown assays showed that the N-terminal region of AKT mainly pulled down FUS. f Upper, Western blotting measured the expression levels of FUS and AKT in GBM cells transfected by si-FUS; lower, Western blotting measured the expression levels of AKT and FUS in GBM cells transfected with si-AKT. g Western blotting measured the expression levels of AKT and pAKT in the whole lysis, cytoplasm, and nucleus of U251 cells transfected by pcDNA3.1-FUS

Article Snippet: The following primary antibodies were used: AKT (rabbit, Proteintech, 10176-2-AP, WB1:1500, IP:1:250, RIP:1:100); FUS (rabbit, Abcam, ab23439, WB1:2000, IP1: 200, RIP1:100); phospho-Akt (Ser473) (rabbit, Cell Signaling, #4060, WB1:1500); phospho-Akt (Thr308) (rabbit, Cell Signaling, #13038, WB1:1500); hexokinase I (rabbit, Cell Signaling, #2024, WB1:1000); hexokinase II (rabbit, Cell Signaling, #2867, WB1:1000); Flag (mouse, Sigma-Aldrich, F1804, IP 1:200); GAPDH (mouse, Sangon, D190090, WB 1:5000); H3 (rabbit, Beyotime, AH433, WB 1:500); and p53 (mouse, Active Motif, 39739, WB 1:1000, RIP 1:150).

Techniques: Activation Assay, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Western Blot, Expressing, Transfection, Lysis

Journal: Journal of Hematology & Oncology

Article Title: A cytoplasmic long noncoding RNA LINC00470 as a new AKT activator to mediate glioblastoma cell autophagy

doi: 10.1186/s13045-018-0619-z

Figure Lengend Snippet: LINC00470 promoted the tumorigenesis of GBM cells. a Expression levels of LINC00470 were measured by RT-qPCR in primary cultured GBM cells (LINC00470 had relatively low expression in PG-1 and PG-2; LINC00470 had relatively high expression in PG-3 and PG-4). Primary cultured GBM cells were transfected with si-LINC00470 or pcDNA3.1-LINC00470. Data are presented as the mean ± S.E.M. of three independent experiments; ** p < 0.01, *** p < 0.001. b An EDU assay was applied to assess cell proliferation of primary cultured GBM cells. Primary cultured GBM cells were transfected with pcDNA3.1-LINC00470 or si-LINC00470. c Western blotting measured the expression levels of autophagy marker LC3, beclin-1, ATG7, and ATG5 in PG-1 and PG-3 cells. The cells were transfected with pcDNA3.1-LINC00470 or si-LINC00470. d Electron microscopy detected the autophagy of U251 cells transfected with pcDNA3.1-LINC00470. e Western blotting measured the expression levels of autophagy marker LC3, beclin-1, ATG7, and ATG5 in PG-1 and PG-3 cells. The cells were transfected with si-HK1, si-FUS or si-AKT. f Survival analysis showed that Sprague Dawley rats transplanted with U251-sh-LINC00470 cells have longer overall survival. g Tumor growth for U251-sh-control and U251-sh-LINC00470 in Sprague Dawley rats.* p < 0.05, ** p < 0.01. h H&E staining showed the volume and morphology of tumors in mice transplanted with U251-sh-LINC00470 cells. The white circle represents the size of the tumor. i Western blotting measured the expression levels of the autophagy marker LC3, beclin-1, ATG7, and ATG5 in intracranial transplanted tumors. j Expression of Ki-67 and LINC00470 in intracranial transplanted tumors was detected by immunohistochemical staining or in situ hybridization, respectively

Article Snippet: The following primary antibodies were used: AKT (rabbit, Proteintech, 10176-2-AP, WB1:1500, IP:1:250, RIP:1:100); FUS (rabbit, Abcam, ab23439, WB1:2000, IP1: 200, RIP1:100); phospho-Akt (Ser473) (rabbit, Cell Signaling, #4060, WB1:1500); phospho-Akt (Thr308) (rabbit, Cell Signaling, #13038, WB1:1500); hexokinase I (rabbit, Cell Signaling, #2024, WB1:1000); hexokinase II (rabbit, Cell Signaling, #2867, WB1:1000); Flag (mouse, Sigma-Aldrich, F1804, IP 1:200); GAPDH (mouse, Sangon, D190090, WB 1:5000); H3 (rabbit, Beyotime, AH433, WB 1:500); and p53 (mouse, Active Motif, 39739, WB 1:1000, RIP 1:150).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Transfection, EdU Assay, Western Blot, Marker, Electron Microscopy, Control, Staining, Immunohistochemical staining, In Situ Hybridization

Journal: EMBO Molecular Medicine

Article Title: HSD17B7 gene in self‐renewal and oncogenicity of keratinocytes from Black versus White populations

doi: 10.15252/emmm.202114133

Figure Lengend Snippet: Differentially expressed genes (DEGs) in Black versus White HKC strains identified in the present study were compared with those in a publicly available data set of HNSCCs (TCGA Firehose Legacy, 520 patients) selected on the basis of the same criteria (|log 2 (Foldchange)|> 0.5 and P value < 0.05) as for analysis of the HKC profiles in Table . Shown is the list of the overlapping DEGs together with a heatmap indication of average log2 fold difference and corresponding statistical significance in both HKC and TCGA (HNSCC) data sets (within brackets). Kaplan–Meier plots of long‐term overall survival of HNSCC patients from the TCGA dataset divided on the basis of high versus low HSD17B7 expression levels relative to its median expression value among males (darkgreen and lightgreen lines) and females (red and orange lines), showing statistically significant association of elevated HSD17B7 expression with poorer patient survival. Statistical significance was calculated by the log‐rank test between the groups. High_female: n = 57 patients. Low_female: n = 79 patients. High_male: n = 203 patients. Low_male: n = 181 patients. Single and multiple COX regression analysis of HNSCC data sets from the TCGA for high risk of patients’ death as a function of levels of HSD17B7 expression, either as a single variable or after adjusting for gender, ethnicity, age, or all three together. Only HNSCC samples from patients of White (Caucasian) versus Black ancestries were used for this analysis. Cox proportional hazards regression model was fitted with the R function coxph. Gene Ontology (GO) analysis of the list of genes that positively correlate with HSD17B7 expression in HKC strains and HNSCCs, with corresponding Benjamini scores (left and right columns, respectively). GO categories are divided into Biological Processes and Cellular Components and the most significant categories in both HKC strains and TCGA (HNSCC) are reported. Correlation analysis between the holoclone‐forming capability of HKC strains (as determined in Fig ) and HSD17B7 expression levels in the corresponding transcriptomic profiles. Values for individual HKC strains are indicated by dots. n (HKC strains) = 44. The linear regression R (0.53) and P ‐value (0.0004) are indicated. The graph shows absolute expression values (probe intensity after global normalization by TAC software) obtained from human Clariom D microarrays. The statistical test is a simple linear regression. RT‐qPCR analysis of HSD17B7 expression in the indicated HKC strains derived from White versus Black donors (white and black bars), as a validation of the corresponding transcriptomic profiles of Fig .** P < 0.01. Immunofluorescence (IF) analysis of HSD17B7 protein expression in White and Black HKCs. Shown are representative images as well as IF signal quantification at the level of individual cells (dots) for the indicated strains. 19–46 cells were counted in each case. n (HKC strains per ancestry) = 5, *** P < 0.0005, horizontal line: median, 2‐tailed unpaired t ‐test. Unit of y‐axis: pixels. Scale bar: 20 µm. Map of eQTL position within a 1 MB region encompassing the HSD17B7 gene, with corresponding exons shown as navy blue bars, and the additional indicated genes. Shown are all eQTLs associated with HSD17B7 expression in the HKC transcriptomic profiles, with the ones with differential distribution in the Black versus White HKC strains indicated in colors. Position of the eQTLs affecting recognition of the indicated transcription factors is also shown. Plots with allele frequency and associated levels of HSD17B7 expression in Black versus White samples for two of the identified eQTLs (rs4542196 and rs10737487). Log2 intensity values are reported on the y ‐axis, while genotypes (AA, GA, GG) are reported on the x ‐axis. Rs4542196 (AA: 19 White, 2 Black; GA: 3 White, 14 Black; GG: 1 White, 12 Black). Rs10737487 (AA: 18 White, 1 Black; GA: 5 White, 15 Black; GG: 1 White, 12 Black). Boxes represent the first (lower) quartile, the median (central band) quartile, and the third (upper) quartile. Whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the box. Summary table of eQTLs with statistically significant association with HSD17B7 expression in experimentally determined HKC transcription profiles (52 strains) and differential distribution in Black versus White populations by analysis of 1,000 Genomes Phase I May 2011 [AFRICA ( N = 246), EUROPE ( N = 380)] with Fixation index ( F ST ) value > 0.3. The effect of the genotype was modeled as additive linear and tested for its significance ( P ‐values) using t‐statistic. Linkage analysis on the six reported SNPs using the LDmatrix tool ( https://ldlink.nci.nih.gov/?tab=ldmatrix ). Pairwise linkage disequilibrium statistics, calculated on all populations (AFR, AMR, EAS, EUR, SAS), are reported as heat maps of shaded colors corresponding to the indicated gradient of values (red: R 2 ; blue: D′). The specific R 2 and D′ numbers are provided in Appendix Table .

Article Snippet: Equal protein amounts—determined by quantification with BCA assay (Pierce BCA protein assay kit—Thermo Fisher Scientific #23225)—were subjected to 10% SDS–PAGE followed by immunoblot analysis, with sequential probing with antibodies against HSD17B7 (Santa Cruz, sc‐393936, diluted 1:500 in TBS 5%BSA solution) and vinculin (HRP conjugate; Cell Signaling Technology, #18799, diluted 1:2,000 in TBS 5% BSA solution) and corresponding secondary antibody (anti‐mouse HRP diluted 1:2,000 in TBS 5% milk solution), with Super Signal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, cat. # 34580) for detection.

Techniques: Expressing, Software, Quantitative RT-PCR, Derivative Assay, Biomarker Discovery, Immunofluorescence

Journal: EMBO Molecular Medicine

Article Title: HSD17B7 gene in self‐renewal and oncogenicity of keratinocytes from Black versus White populations

doi: 10.15252/emmm.202114133

Figure Lengend Snippet: A Colony‐forming assays of multiple HKC strains from White and Black donors (passages 2,3) infected with an HSD17B7 ‐expressing lentivirus (HSD‐oe) versus empty vector control (ctrl), plated in triplicate dishes at limited density (1,000 cells per 60‐mm dish) at 5 days after infection. A week later cells were fixed and stained by crystal violet, and number of colonies (> 0.149 mm ) was determined. Shown are numbers of colonies ± SD. n (dishes per condition) = 3. * P < 0.05. 2‐tailed unpaired t ‐test. B Colony‐forming assays of the indicated SCC cell lines stably infected with an HSD17B7 ‐expressing lentivirus (HSD‐oe) versus empty vector control (ctrl), utilizing the same conditions as in the previous panel. Shown are numbers of colonies ± SD. n (dishes per condition) = 3. * P < 0.05; **** P < 0.0001. Two‐tailed unpaired t ‐test. C Proliferation live‐cell imaging assays of SCC cell lines plus/minus HSD17B7 overexpression as in the previous panel. Cells were plated in triplicate wells in 96‐well plates followed by cell density measurements (IncuCyte), taking four images per well every 2 h for 150–160 h. n (wells per condition) = 3. **** P < 0.0001. Pearson r correlation. D–F Intradermal tumorigenicity assays with HKCs (269W) stably infected with an HSD17B7 overexpressing lentivirus (HSD17B7‐oe) versus control (Ctrl). Experimental conditions were the same as in Fig . Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cell density assessed by immunohistochemical staining with anti‐pan‐keratin antibodies (D), as well as % of Ki67‐positive cells (E) and K1 positivity (F) of pan‐keratin positive areas. For (D), n (tumors per condition) = 9; for (E and F) n (3 fields per tumor, 5 tumors per condition) = 15. * P < 0.05, **** P < 0.0001. Horizontal line: median. 2‐tail unpaired t ‐test. Scale bar: 500 µm (D), 100 µm (F). G, H Intradermal tumorigenicity assays with SCC13 cells overexpressing HSD17B7 (HSD17B7‐oe) versus control (Ctrl). Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cells density, assessed by immunohistochemical staining with anti‐pan‐keratin antibodies (G) as well as % of Ki67‐positive cells in pan‐keratin‐positive areas (H). For (G), n (tumors per condition) = 4; for (H), n (3–5 fields per tumor, 4 tumors per condition) 15, 19. * P < 0.05; horizontal line: median. One‐tailed paired t ‐test. Scale bar in (G, H): 100 µm.

Article Snippet: Equal protein amounts—determined by quantification with BCA assay (Pierce BCA protein assay kit—Thermo Fisher Scientific #23225)—were subjected to 10% SDS–PAGE followed by immunoblot analysis, with sequential probing with antibodies against HSD17B7 (Santa Cruz, sc‐393936, diluted 1:500 in TBS 5%BSA solution) and vinculin (HRP conjugate; Cell Signaling Technology, #18799, diluted 1:2,000 in TBS 5% BSA solution) and corresponding secondary antibody (anti‐mouse HRP diluted 1:2,000 in TBS 5% milk solution), with Super Signal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, cat. # 34580) for detection.

Techniques: Infection, Expressing, Plasmid Preparation, Control, Staining, Stable Transfection, Two Tailed Test, Live Cell Imaging, Over Expression, Injection, Immunohistochemical staining, One-tailed Test

Journal: EMBO Molecular Medicine

Article Title: HSD17B7 gene in self‐renewal and oncogenicity of keratinocytes from Black versus White populations

doi: 10.15252/emmm.202114133

Figure Lengend Snippet: Colony‐forming assays of multiple HKC strains from donors of White (213W) and Black (246B, 261B, 285B) origin, which were infected with two HSD17B7 ‐silencing lentiviruses (shHSD2, shHSD4) versus vector control (shCtrl). Gene silencing efficiency is shown in Appendix Fig . Cells were plated in triplicate dishes at limited density (1,000 cells per 60‐mm dish) at 5 days after infection and fixed a week later for determination of colony formation as in Fig . Shown are representative dishes together with data quantification, expressed as numbers of colonies ± SD. n (dishes per condition) = 3. ** P < 0.01. 2‐tailed unpaired t ‐test. Sphere‐forming assays of multiple HKC strains plus/minus HSD17B7 silencing as in the previous panel. Cells were cultured in Matrigel in triplicate dishes, utilizing the same conditions as for the experiments of Fig . Data are expressed as ratio of large spheres (> 2,000 pixels ≥ 0.0095 mm 2 ) versus total number of spheres (> 100 pixels ≥ 0.00047 mm 2 ). ± SD. n (dishes per condition) = 3. ** P < 0.01; **** P < 0.0001, 2‐tailed unpaired t ‐test. EdU labeling assays of multiple HKC strains plus/minus HSD17B7 silencing as in the previous panels. Cells were plated in triplicate dishes 5 days after lentiviral vector infection and selection and pulse labeled with EdU for 3 h. Shown are % of EdU‐positive cells ± SD. n (dishes per condition) = 3. * P < 0.05, ** P < 0.01; *** P < 0.0005; **** P < 0.0001, 2‐tailed unpaired t ‐test. Colony‐forming assays of the indicated SCC cell lines plus/minus HSD17B7 silencing as in the previous panels. Cells plated in triplicate dishes 5 days after lentiviral vector infection and selection were tested for clonogenic capability as in (A). Data are represented as number of colonies ± SD. n (dishes per condition) = 3. * P < 0.05, ** P < 0.01; **** P < 0.0001, 2‐tailed unpaired t ‐test. EdU labeling assays of the indicated SCC cell lines plus/minus HSD17B7 silencing. Experimental conditions were as in (C). n (dishes per condition) = 3. **** P < 0.0001, 2‐tailed unpaired t ‐test. Data are represented as % of EdU‐positive cells ± SD. Colony‐forming assays of SCC13 cells plus/minus lentivirally mediated HSD17B7 overexpression and subsequent infection with two shRNA silencing vectors as indicated. Experimental conditions were as in (D). n (dishes per condition) = 3. * P < 0.05, ** P < 0.01; *** P < 0.0005, 2‐tailed unpaired t ‐test. Data are represented as number of colonies ± SD.

Article Snippet: Equal protein amounts—determined by quantification with BCA assay (Pierce BCA protein assay kit—Thermo Fisher Scientific #23225)—were subjected to 10% SDS–PAGE followed by immunoblot analysis, with sequential probing with antibodies against HSD17B7 (Santa Cruz, sc‐393936, diluted 1:500 in TBS 5%BSA solution) and vinculin (HRP conjugate; Cell Signaling Technology, #18799, diluted 1:2,000 in TBS 5% BSA solution) and corresponding secondary antibody (anti‐mouse HRP diluted 1:2,000 in TBS 5% milk solution), with Super Signal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, cat. # 34580) for detection.

Techniques: Infection, Plasmid Preparation, Control, Cell Culture, Labeling, Selection, Over Expression, shRNA

Journal: EMBO Molecular Medicine

Article Title: HSD17B7 gene in self‐renewal and oncogenicity of keratinocytes from Black versus White populations

doi: 10.15252/emmm.202114133

Figure Lengend Snippet: Top bar: gradient of population admixture of the indicated HKC strains of Black versus White individuals, on the basis of the SNP genotype analysis shown in Fig , with the simplest assumption of two ancestries ( K = 2). Gradient ranges from black to white colors for Black and White individuals with no population admixture, respectively. Individuals with increasing proportions of White population admixture are represented with degrading shades of gray. Left: heatmap of expression levels of the indicated genes from the OXPHOS gene signature of Fig , in Black versus White HKC strains ordered according to genome admixture calculations and in three strains of White HKCs with HSD17B7 overexpression. Modified Z scores of the individual genes, as median‐centered log2 intensity values divided by standard deviation, are shown by color gradient variations. Right: heatmap of expression levels of the same genes in three White HKC strains infected with an HSD17B7 overexpressing lentivirus versus empty vector control as in Fig (strains: 260, 275, 249). Expression levels are derived from global transcriptomic analysis (Clariom D hybridization; GEO accession number GSE172288) and expressed as logFold change values of treated versus control samples. Gene Ontology (GO) analysis of genes analyzed in the previous panel. Shown is a list of process networks with statistical significance of enrichment in Black HKCs. The complete list is provided in Dataset .

Article Snippet: Equal protein amounts—determined by quantification with BCA assay (Pierce BCA protein assay kit—Thermo Fisher Scientific #23225)—were subjected to 10% SDS–PAGE followed by immunoblot analysis, with sequential probing with antibodies against HSD17B7 (Santa Cruz, sc‐393936, diluted 1:500 in TBS 5%BSA solution) and vinculin (HRP conjugate; Cell Signaling Technology, #18799, diluted 1:2,000 in TBS 5% BSA solution) and corresponding secondary antibody (anti‐mouse HRP diluted 1:2,000 in TBS 5% milk solution), with Super Signal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, cat. # 34580) for detection.

Techniques: Expressing, Over Expression, Modification, Standard Deviation, Infection, Plasmid Preparation, Control, Derivative Assay, Hybridization

Journal: EMBO Molecular Medicine

Article Title: HSD17B7 gene in self‐renewal and oncogenicity of keratinocytes from Black versus White populations

doi: 10.15252/emmm.202114133

Figure Lengend Snippet: A Purified mitochondrial preparation from multiple HKC strains from White and Black donors (12 per ancestry; passage 2,3; triplicate cultures) were analyzed for levels of electron transport chain activity (ETC, nmol of reduced cytochrome c/min/mg protein), ATP (nmol/mg protein), and ROS (MtROS (nmol/mg mitochondrial protein). In parallel, acid‐soluble metabolites (ASM, pmol/h/mg protein) levels were measured as a readout of β‐oxidation. Similar measurements of HKCs strains at a later passage are shown in Appendix Fig . Data are displayed as average values for all tested HKC strains (3 cultures per strain for ETC, ATP, and FAO, 1 culture per strain for mtROS, white and black dots, depending on ancestry) together with mean ± SD. n (HKC strains per ancestry) = 12. *** P < 0.0005; **** P < 0.0001, 2‐tailed unpaired t ‐test. Individual experimental values are provided in Dataset . B Multiple HKC strains (249W, 260W, 275W) stably transduced with an HSD17B7 ‐expressing lentivirus (HSD‐oe) versus empty vector control (Ctrl) were analyzed as in (A). Data are shown as individual values for 3 parallel cultures per strain together with mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.0005; 2‐tailed unpaired t ‐test. Individual experimental values are provided in Dataset . C, D SCC13 cells stably infected with an HSD17B7 ‐expressing lentivirus (HSD‐oe) versus empty vector control (Ctrl) (C) or two HSD17B7 ‐silencing lentivirus (shHSD2, shHSD4) versus vector control (shCtrl) (D) were analyzed as in the previous panels. Shown are individual values for 3 parallel cultures per condition together with mean ± SD. ** P < 0.005; *** P < 0.0005; **** P < 0.0001. 2‐tailed unpaired t ‐test. Additional metabolic measurement experiments of SCC cells plus/minus HSD17B7 overexpression and silencing, in the context of additional manipulations, are shown in Fig .

Article Snippet: Equal protein amounts—determined by quantification with BCA assay (Pierce BCA protein assay kit—Thermo Fisher Scientific #23225)—were subjected to 10% SDS–PAGE followed by immunoblot analysis, with sequential probing with antibodies against HSD17B7 (Santa Cruz, sc‐393936, diluted 1:500 in TBS 5%BSA solution) and vinculin (HRP conjugate; Cell Signaling Technology, #18799, diluted 1:2,000 in TBS 5% BSA solution) and corresponding secondary antibody (anti‐mouse HRP diluted 1:2,000 in TBS 5% milk solution), with Super Signal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, cat. # 34580) for detection.

Techniques: Purification, Activity Assay, Stable Transfection, Transduction, Expressing, Plasmid Preparation, Control, Infection, Over Expression

Journal: EMBO Molecular Medicine

Article Title: HSD17B7 gene in self‐renewal and oncogenicity of keratinocytes from Black versus White populations

doi: 10.15252/emmm.202114133

Figure Lengend Snippet: Upper panel: schematic representation of the 17‐beta‐dehydrogenase domain shared between the HSD17B1 and HSD17B7 enzymes with positions of the NADP and steroid‐binding sites and the active site (Breton et al , ). Two conserved charged residues in these domains, Tyrosine (Y) 193 and Lysine (K) 197, were changed into Glycine (G). Lower panel: close‐up views of active site of HSD17B7 showing the NADP and steroid‐binding pocket and position of the Y193G and K197G amino acid substitutions. Clonogenicity assays with two different SCC cell lines stably infected with lentiviruses overexpressing HSD17B7 in wild type (HSD‐wt) or Y193G and K197G mutated (HSD‐mut) forms versus empty vector control (Ctrl). SCC13 cells were cultured in either DMEM + 10% FBS (*) or HKC growth media. HSD17B7 overexpression was verified by RT‐qPCR and immunoblot analysis shown in Appendix Fig . Experimental conditions are as in Fig . Data are represented as number of colonies (> 0.149mm 2 ) ± SD. n (dishes per condition) = 3. * P < 0.05, ** P < 0.005; *** P < 0.0005, **** P < 0.0001. 2‐tailed unpaired t ‐test. Purified mitochondrial preparation from SCC13 cells infected with lentiviruses expressing wild‐type (HSD‐wt) and Y193G and K197G mutated (HSD‐mut) forms versus empty vector control (Ctrl) as in (B) were assayed for levels of electron transport chain activity, ATP and mtROS production as in Fig . Data are displayed as average values of triplicate measurements together with mean ± SD. n (dishes per condition) = 3. ** P < 0.005; *** P < 0.0005. 2‐tailed unpaired t ‐test. Individual experimental values are provided in Dataset . SCC13 cells were infected with two HSD17B7 ‐silencing lentiviruses (shHSD2, shHSD4) versus vector control (shCtrl) followed, 3 days after infection and antibiotic selection, by treatment with zymosterol (2.5 µM) or ethanol vehicle alone for 48 h. Purified mitochondrial preparation was assayed for levels of electron transport chain activity, ATP, and mtROS production as in Fig . Data are displayed as average values of triplicate measurements together with mean ± SD. n (dishes per condition) = 3. **** P < 0.0001, 2‐tailed unpaired t ‐test. Individual experimental values are provided in Dataset .

Article Snippet: Equal protein amounts—determined by quantification with BCA assay (Pierce BCA protein assay kit—Thermo Fisher Scientific #23225)—were subjected to 10% SDS–PAGE followed by immunoblot analysis, with sequential probing with antibodies against HSD17B7 (Santa Cruz, sc‐393936, diluted 1:500 in TBS 5%BSA solution) and vinculin (HRP conjugate; Cell Signaling Technology, #18799, diluted 1:2,000 in TBS 5% BSA solution) and corresponding secondary antibody (anti‐mouse HRP diluted 1:2,000 in TBS 5% milk solution), with Super Signal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, cat. # 34580) for detection.

Techniques: Binding Assay, Stable Transfection, Infection, Plasmid Preparation, Control, Cell Culture, Over Expression, Quantitative RT-PCR, Western Blot, Purification, Expressing, Activity Assay, Selection