Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Immunofluorescence staining revealed EGFL7 expression in granule cells and larger blood vessels of the human dentate gyrus as well as larger neurons of the hilus. Scale bars represent 50 μm or 10 µm (magnification). ( b ) Furthermore, EGFL7 was expressed in the dentate gyrus of mice as detected by fluorescence in situ hybridization (FISH). Scale bars represent 50 µm or 10 µm (magnification). ( c ) Cells of murine hippocampi were isolated by fluorescence-activated cell sorting using a combination of the following markers: GFAP + /CD133 + /EGFR - for quiescent neural stem cells (qNSCs), GFAP + /CD133 + /EGFR + for active NSCs (aNSC), GLAST - /CD133 - /EGFR + for neural progenitor cells (NPCs), CD24 + for neuroblasts (NBs), Thy-GFP1 + for neurons and CD31 + for endothelial cells (ECs). Expression of Egfl7 was measured by quantitative reverse transcriptase-polymerase chain reaction using two housekeeping genes and data was plotted as normalized to unsorted hippocampus tissue (HC). Data are presented as mean values with 95% confidence interval (CI). ( d ) EGFL7-specific FISH probes in combination with cell type-specific markers verified EGFL7-expression in aNSCs/NPCs (Stmn1; arrowheads) and neurons (NeuN; arrowheads). Scale bars represent 10 µm.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Immunofluorescence, Staining, Expressing, Fluorescence, In Situ Hybridization, Isolation, FACS, Reverse Transcription, Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Representative images of neural stem and progenitor cells isolated from mouse hippocampus (HC) and cultured as spheres. Scale bars represent 25 µm. ( b ) Size of EGFL7 -/- neurospheres was increased (58.95 ± 11.24 µm (n = 5) versus 43.85 ± 2.25 µm in wild-type control (WT); n = 4; p = 0.0317). ( c ) Fluorescence-activated cell sorting revealed an increase in activated neural stem cells (aNSCs) in EGFL7 -/- mice (5.56 ± 1.65% versus 2.20 ± 0.53% in WT, n = 3; p = 0.0499) but about equal amounts of quiescent qNSCs. ( d ) Flow cytometry-based cell cycle analysis of EGFL7 -/- and WT neurospheres yielded an increased amount of cells in the G2/M phase in EGFL7 -/- mice (33.65 ± 6.10% versus 25.10 ± 2.03%; n = 3; p = 0.0286). ( e ) The paradigm used for cell cycle analysis in vivo . ( f ) Representative images of the dentate gyrus 1 h post administration of CldU. Quantification of cells yielded an increased amount of cells in S phase in EGFL7 -/- mice (11.00 ± 2.71 versus 5.25 ± 1.71 cells per section in WT; n = 4; p = 0.0571). ( g ) Representative images of the dentate gyrus 24 h post administration of IdU and double-stained for Ki67/IdU. Quantification revealed sustained proliferation in EGFL7 -/- mice (17.25 ± 2.06 versus 10.00 ± 2.45 cells per section in WT; n = 4; p = 0.0286). Scale bars represent 90 µm. Statistical analysis was performed by Mann–Whitney U -test. Data are represented as mean ± SEM; * p < 0.05.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Isolation, Cell Culture, Control, Fluorescence, FACS, Flow Cytometry, Cell Cycle Assay, In Vivo, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Markers used in immunofluorescence analyses (IF) to label specific cell types of neural stem cell (NSC) differentiation in the dentate gyrus (DG): GFAP/Nestin for NSCs (type 1), Stmn1 for activated aNSCs and neural precursor cells (NPCs, type 1, type 2), Mash1 for aNSCs/type 2a cells, DCX for type 2b/type 3 and immature neurons, NeuN for mature neurons and granule cells. ( b ) Experimental paradigms of applied neurogenesis assays. ( c ) Quantitative analyses revealed that NSCs (GFAP + /Nestin + /BrdU + ) remained unchanged in EGFL7 -/- mice (10.50 ± 3.51 versus 11.00 ± 1.41 cells per section in wild-type controls (WT); n = 4; p > 0.9999). ( d ) The number of type 2a (BrdU + /Mash1 + ) cells was increased (11.25 ± 1.71 versus 3.00 ± 0.82 in WT; n = 4; p = 0.0286). ( e ) Type 2b/type 3 (BrdU + /DCX + ) cells were decreased (7.00 ± 2.16 versus 14.75 ± 5.56 cells per section in WT; n = 4; p = 0.0286). ( f ) The amount of aNSCs/NPCs (BrdU + /Stmn1 + ) was strongly increased in the DG of EGFL7 -/- animals (18.25 ± 4.99 versus 5.25 ± 3.59 cells per section in WT; n = 4; p = 0.0286). ( g ) Last, more newborn neurons (BrdU + /NeuN + ) were formed in the DG of EGFL7 -/- mice (21.50 ± 4.12 versus 5.75 ± 4.35 cells per section in WT; n = 4; p = 0.0286). Statistical analysis was performed by Mann-Whitney U -test. Data are represented as mean ± SEM; * p < 0.05. Scale bars represent 60 µm.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Immunofluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Description of the EGFL7 fl/fl;Ascl1-CreERT2 mouse model allowing for a tamoxifen-inducible, type 2a cell-specific knock-out of EGFL7. ( b ) Experimental paradigms of applied neurogenesis assays. ( c ) The amount of aNSCs/NPCs (BrdU + /Mash1 + ) were found to be increased (5.25 ± 2.63 versus 1.00 ± 1.15 cells per section in EGFL7 fl/fl ; n = 4; p = 0.0286). ( d ) The number of type 2b/type 3 cells (BrdU + /DCX + ) was decreased in EGFL7 del;Ascl1-CreERT2 mice (8.50 ± 5.51 versus 18.75 ± 2.22 in EGFL7 fl/fl ; n = 4; p = 0.0286), ( e ) while quantification of BrdU + /Stmn1 + activated neural stem and precursor cells (aNSCs/NPCs) revealed a significant upregulation in EGFL7 del ;Ascl1-CreERT2 mice (20.25 ± 3.10 versus 8.00 ± 4.24 cells per section in EGFL7 fl/fl control; n = 4; p = 0.0286). ( f ) Futhermore, the amount of adult-born neurons (BrdU + /NeuN + ) was increased (18.25 ± 6.80 versus 5.50 ± 2.65 cells per section in EGFL7 fl/fl ; n = 4; p = 0.0286). Statistical analysis was performed by Mann-Whitney U -test. Data are represented as mean ± SEM; * p < 0.05; Scale bars represent 60 µm.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Knock-Out, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Volcano and ( b ) MA plots of RNA-sequencing-based transcriptomics in neural stem and precursor cells confirmed EGFL7 knock-out and identified an upregulation of the cytokine VEGF-D (n = 4 for each genotype; two datasets). Volcano plots illustrate the Log2 difference (i.e., fold change) versus -logP of the t-test. MA plots display the LOG2 difference versus the mean expression level (LOG2 RPKM). The VENN diagrams show the agreement of experiment 1 and 2 and the combined analysis of regulated candidates, based on P value and fold change. ( c ) Upregulation of VEGF-D upon EGFL7 knock-out was confirmed by quantitative reverse transcriptase-polymerase chain reaction (6.56 ± 2.85 versus 1.47 ± 0.61 in wild-type control (WT); n = 3; p = 0.039). ( d ) Expression of VEGF-D in the dentate gyrus of the hippocampus was visualized by immunofluorescent staining using a VEGF-D-specific antibody. Statistical analysis was performed by Student’s t-test. Data are represented as mean ± SEM; * p < 0.05. Scale bars represent 90 µm or 45 µm (magnifications).

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: RNA Sequencing, Knock-Out, Expressing, Reverse Transcription, Polymerase Chain Reaction, Control, Staining

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Representative pictures of analyzed spines of 10-week-old EGFL7 -/- and WT mice. ( b ) The analysis of DG spines of 10-week-old mice showed increased number of spines in EGFL7 -/- mice and ( c ) significantly more thin spines. ( d ) Representative pictures of analyzed spines of 20-week-old EGFL7 -/- and WT mice. ( e ) After 20 weeks the number of spines is unaffected. ( e) The numbers of thin, stubby and mushroom spines were also not affected. ( g ) The number of dendrites and their total length was found increased in EGFL7 -/- primary neuron cultures as detected by Map2 IF staining ( h ). ( i ) Timeline for analysis of neurogenesis in aged mice and quantification of BrdU + /NeuN + newborn neurons across different ages. Statistical analysis was performed by Mann–Whitney U -test. Data are represented as mean ± SD; * p < 0.05; n = 3. Scale bar represents 5 µm.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) In the Morris water maze (MWM), the latency to reach the visible platform, the proportion of time spent in the platform quarter and the swimming velocity were similar in both genotypes. ( b ) Body weights were similar throughout experiments. Exemplary cohort is shown as mean ± SD. ( c ) EGFL7 -/- mice reached the hidden platform faster. Escape latency was reduced considering the full-time course (AUCs) and late trials. ( d ) The proportion of time spent in the platform quarter after its removal was increased. For MWM sample sizes were n = 19 for EGFL7 -/- mice and n = 33 for wild-type littermate controls (WT). Data were compared by paired (visible platform) and unpaired two-sided Student’s t-tests, or 2-way ANOVA for time courses. ( e ) In the IntelliCage, EGFL7 -/- mice maintained longer avoidance of a previously punished corner (air puff), as revealed by a reduced proportion of nosepoke errors (n = 16 per genotype). ( f ) During preference learning in active module times (11-13:00 and 16-18:00 h each day), EGFL7 -/- mice showed faster relearning to prefer a specified corner upon switching to the opposite side (reversal learning), indicated by a higher accuracy of nosepokes (n = 16 per genotype). Overall activity is shown in ( Supplementary Fig. 8_2 ). ( g,h ) During periods in which the learning modules were inactive (doors remain closed, “default-times”), EGFL7 -/- mice retained higher preference of rewarding corners and the proportion of “memorizers” was higher, the latter defined as 35% accuracy of corner visits (random = 25%). Time course data are represented as mean ± SEM, summarizing data as mean ± SD. The boxes show the interquartile range, the line is the median, and whiskers are plotted minimum to maximum. Scatters represent individual mice. Time courses were compared by 2-way ANOVA and subsequent posthoc t-tests to assess genotype differences at specific time points without multiplicity adjustment for between group comparisons, AUCs were compared with 2-sided unpaired t-tests; * p < 0.05.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Activity Assay

Journal: Blood Advances

Article Title: The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

doi: 10.1182/bloodadvances.2019001002

Figure Lengend Snippet: EGFL7 is a survival factor in myeloma cells (A) Fold change in EGFL7 gene expression of the human stromal cell line HS-5 and the MM cell lines U-266, RPMI8226, (abbreviated RPMI), MM.1S, H929, and KMS11, as determined by RT-PCR when compared with the EGFL7 expression in the human myeloid leukemia HL60 cells. (B) A representative western blot is shown for EGFL7, with b-ACTIN as a control, in tumor lysates from mice injected with indicated RPMI cells. (C) Representative images of RPMI8226 cells stained for EGFL7 (green fluorescence) and 4′,6-diamidino-2-phenylindole (DAPI; blue nuclear staining) by immunofluorescent staining. (D) Fold change in EGFL7 gene expression of MACS-isolated CD138+ cells from normal donors when compared with the EGFL7 expression in CD138− cells. (E) Fold change in EGFL7 gene expression as determined by RT-PCR in human cell samples of patients with MM (patients #1 and #2 MM patient sample at diagnosis and patient #3 MM patient sample at refractory stage of the disease; for more clinical details, see supplemental Figure 1) when compared with EGFL7 gene expression found in human BMMCs of healthy donors (n = 3/condition). (F) Western blot analysis of EGFL7 and b-ACTIN as a control in indicated cell population from healthy volunteers and patients with MM. (Upper) Band quantification using the ImageJ program. (Lower) Representative western blots of the same samples. (G-H) EGFL7 (EGFL OE), EGFL7 knockdown (KD), or Mock MM cells (RPMI8226, MM.1S, and U-266) were generated. (G) Fold change in EGFL7 gene expression when compared with Mock cells, as determined by RT-PCR (n = 3/condition). (H) Cells were counted 24 hours after cell seeding after trypan blue exclusion (n = 6/group). (I-J) RPMI8226 cells (OE, KD, Mock) were stained with Annexin V-FITC and PI and analyzed 48 hours after cell plating by FACS. (I) A representative FACS blot is shown for each condition (n = 6/group). (J) Percentage of Annexin V+ and PI− cells (as a measure of early and late apoptosis) by FACS after 48 hours (n = 3/group). (K) Representative western blot analysis of the expression of phosphorylated AKT, BAX, and b-ACTIN (control) in cell lysates of Mock, EGFL7 OE, and EGFL7 KD RPMI8226 cells. (L) Fold change in caspase 3/7 activity of EGFL7 OE or KD cells when compared with Mock RPMI8226 cells 48 hours after cell plating, as determined by Versa Max (n = 6/condition). (M-N) EGFL7 OE and wild-type (RPMI) cells were treated with various concentrations of the AKT1/2 inhibitor. (M) Proliferation was determined using the CCK-8 kit after 24 hours in culture. % Absorbance indicates the rate of viable cells (n = 6/condition). (N) Fold increase in EGFL7 expression after AKT1/2 inhibitor treatment when compared with EGFL7 expression in nontreated cells. (O-P) MM cells were cocultured with a confluent layer of GFPpos-EGFL7 Mock, EGFL7 OE, or EGFL7 KD ECs in direct contact, and the percentage of GFP-MM cells was determined after 24 hours of coculture (n = 3/condition). (O) Percentage of GFP− MM cells in the adherent fraction. (P) Absolute number of nonadherent suspension cells retrieved from the cocultures. As for RT-PCR data, transcripts were normalized to b-ACTIN. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice with similar results. Data are represented as mean ± SEM. * P ≤ .05; ** P ≤ .01; *** P ≤ .001. P values were determined using a Student t test.

Article Snippet: Immunohistochemical staining for EGFL7 on human patient BM sections was performed using the EGFL7 polyclonal Ab (cat# 19291-1-AP; Proteintech) followed by 4′,6-diamidino-2-phenylindole development and hematoxylin counterstaining.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Control, Injection, Staining, Fluorescence, Isolation, Biomarker Discovery, Knockdown, Generated, Activity Assay, CCK-8 Assay, Suspension

Journal: Blood Advances

Article Title: The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

doi: 10.1182/bloodadvances.2019001002

Figure Lengend Snippet: EGFL7 augments MM growth in vivo. (A) Human RPMI8226 wild-type cells were injected s.c. into NOD/SCID mice (n = 6/group). (B) Human EGFL7 plasma levels were measured in murine plasma samples at indicated times by enzyme-linked immunosorbent assay in (n = 6). (C) Representative western blotting for EGFL7 and b-ACTIN in tumors extracted from tumors injected with Mock, EGFL7 OE, or KD RPMI8226 cells. (D-E) The size of tumors that formed after s.c. injection of Mock, EGFL7 OE, or KD cells after day 53. (D) Tumor weight was measured (left panel; n = 6/group). Representative macroscopic tumor images were taken on day 53 postinoculation (right panel). (E) Representative hematoxylin and eosin-stained tumor sections demonstrating increased necrotic areas (yellow lines) in tumors established with EGFL7 KD cells (scale bar, 200 µm). (F-G) RPMI8226 wild-type tumor-bearing NOD/SCID mice were injected with/without neutralizing Abs against EGFL7 (anti-EGFL7) starting after visible tumors had been established by day 25 (n = 6/group). (F) Tumor weight on day 53 after initial tumor inoculation (left panel). Representative macroscopic images of tumors (right panels). (G) Histological images of hematoxylin and eosin–stained tumor sections (scale bar of the insert, 100 µm). Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

Article Snippet: Immunohistochemical staining for EGFL7 on human patient BM sections was performed using the EGFL7 polyclonal Ab (cat# 19291-1-AP; Proteintech) followed by 4′,6-diamidino-2-phenylindole development and hematoxylin counterstaining.

Techniques: In Vivo, Injection, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Western Blot, Staining

Journal: Blood Advances

Article Title: The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

doi: 10.1182/bloodadvances.2019001002

Figure Lengend Snippet: Increased circulating and tissue-bound EGFL7 in the murine Vk*MYC model of MM. (A-F) MACS-isolated CD138+ Vk*MYC BM and spleen cells were injected into C57BL/6J mice via the tail vein. C57BL/6J mice transplanted with Vk*MYC tumor cells (Vk*MYC mice) and control mice (WT) that did not receive tumor cells were analyzed 25 days after cell inoculation. (A) Representative macroscopic images of whole spleens (left panel) and total spleen weight (right panel; n = 6/group) of WT and Vk*MYC mice. (B) Spleen and BM cells from transplanted Vk*MYC recipient or WT mice were stained for the plasma cell marker CD138 and the B-cell marker B220. Percentage of CD138+ cells per total BM or spleen cells and percentage of B220+ cells per total BM cells are given in the left panels. Representative FACS blots of CD138+ cells in BM and spleen are shown in the right panels. (C) Immunohistochemistry analysis of EGFL7 in BM and spleen sections of WT and VK*MYC mice (scale bar of the insert, 10 µm). Hematoxylin and eosin–stained sections (H&E; scale bar of the insert, 20 µm) show massive infiltration of MM cells in the tissue section of Vk*MYC mice. (D) Representative western blot for EGFL7 and b-ACTIN in BM and spleen cell lysates of WT and Vk*MYC mice. Two independent experiments were performed. (E) Fold change of EGFL7 expression in total BMNCs, MACS-isolated CD31+ BM cells, and splenocytes of Vk*MYC mice when compared with the EGFL7 expression in spleen and BM of nontumor-bearing mice (n = 2/group). Transcripts normalized to β-ACTIN. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice, with similar results. (F) Plasma mouse EGFL7 as determined by enzyme-linked immunosorbent assay (n = 6/group). (G-J) Vk*MYC BM and spleen cells of donor mice with a tumor load of 30% in the BM were injected into C57BL/6J mice via the tail vein. VK*MYC transplanted mice were left untreated for 21 days to ensure sufficient tumor load. Then, 3 mice were treated with neutralizing Abs against EGFL7 (anti-EGFL7) or with isotype (nonbinding) control Ab at 1.5 mg/kg IV. On day 42, mice were euthanized. (G) Spleen weight was determined. (H) Frequency of B220−CD138+ MM cells in splenocytes as determined by FACS. (J) Hematoxylin and eosin–stained sections (scale bar of the insert, 50 µm) showing mild infiltration of MM cells in the BM. (I) Frequency of B220−CD138+ MM cells in BM of Vk*MYC mice treated with anti-EGFL7 Ab or isotype controls (n = 3/group). The data represent the data of 1 experiment. A similar experiment was performed independently, showing similar results. Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

Article Snippet: Immunohistochemical staining for EGFL7 on human patient BM sections was performed using the EGFL7 polyclonal Ab (cat# 19291-1-AP; Proteintech) followed by 4′,6-diamidino-2-phenylindole development and hematoxylin counterstaining.

Techniques: Isolation, Injection, Control, Staining, Clinical Proteomics, Marker, Immunohistochemistry, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Blood Advances

Article Title: The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

doi: 10.1182/bloodadvances.2019001002

Figure Lengend Snippet: ITGB3+ MM cells adhere to immobilized EGFL7 via its RGD region (A) Fold change in ITGB3 expression of HUVEC, HEL, RPMI8226, U-266, MM.1S, and HS-5 cells when compared with ITGB3 expression in HL60 cells using RT-PCR. Transcripts normalized to b-ACTIN. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice with similar results. (B) Representative FACS plots showing gating for PI negative cells. Histogram showing ITGB3 levels on indicated MM cell lines. (C) Percentage of adhesion of RPMI8226 cells after 4 hours on precoated plates with deposited ECM from cells infected with AdNull (Mock), AdEGFL7 full-form (EGFL7), or Addeleted RGD (delRGD), or precoated with rec. murine EGFL7 (rec. EGFL7) or with rec. FN. The percentage of adherent cells was quantified after cellular staining with crystal violet and absorbance measurement at 550 nm. Cumulative data of 2 independent experiments are shown (n = 5/condition per experiment). (D) Representative FACS histogram of ITGB3 expression using RPMI8226 cells stably transduced with ITGB3 (ITGB3 OE) in the left panel. The right panel shows the percentage of ITGB3 expression on ITGB3 OE cells, as determined by FACS (n = 6/group). (E) Representative western blot image of Tyr747, FAK EGFL7, and b-ACTIN proteins in EGFL7 OE/KD, and ITGB3 OE/KD in RPMI8226 cells. Two independent experiments were performed. (F) Human ITGB3 OE or ITGB3 KD cells were cultured on EGFL7- and FN-coated plates for 4 hours with/without the ITGB3 inhibitor Cilengitide (Celin). Quantification of adherence using crystal violet absorbance of adherent cells (n = 5 wells/condition). (G) Cell proliferation rate was determined after 24 hours by counting Trypan blue-negative ITGB3 OE and Mock MM cells (n = 3/wells per condition). (H) EGFL7 OE or EGFL7 KD MM cells were coinfected with Mock vector (−ITGB3) or ITGB3 OE (+ITGB3) vectors. Cells were counted after 24 hours in culture (n = 6/condition). Experiments were repeated at least twice. Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

Article Snippet: Immunohistochemical staining for EGFL7 on human patient BM sections was performed using the EGFL7 polyclonal Ab (cat# 19291-1-AP; Proteintech) followed by 4′,6-diamidino-2-phenylindole development and hematoxylin counterstaining.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Staining, Stable Transfection, Transduction, Western Blot, Cell Culture, Plasmid Preparation

Journal: Blood Advances

Article Title: The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

doi: 10.1182/bloodadvances.2019001002

Figure Lengend Snippet: EGFL7 loss in MM cells overrides BTZ resistance (A-B) RPMI8226, U-266, and MM.1S cells were cultured for 24 hours in the presence of BTZ at indicated concentrations. (A) Fold change in EGFL7 gene expression of BTZ-treated MM cells when compared with non-BTZ-treated cultures, as determined by RT-PCR. Transcripts normalized to β-ACTIN. Graphs represent averages from independently prepared templates per condition. (B) Viable MM cells were counted 24 hours after BTZ treatment, using Trypan blue (n = 6/condition). (C) RPMI8226 cells were treated with rec. EGFL7 in the presence/absence of 5 nM BTZ. Cells were counted after 24 hours in culture (n = 6/group). (D) EGFL7 OE and Mock RPMI8226 cells were treated with/without BTZ. Cells were counted after 24 hours (n = 6/group). (E) RPMI Mock cells were cultured with/without neutralizing Abs against EGFL7 in the presence or absence of 10 nM BTZ. Cell proliferation was assessed using the CCK-8 kit after 24 hours in culture (n = 6/condition). (F-G) RPMI8226, U-266, and MM.1S cells were treated with/without BTZ for 24 hours. (F) Fold change in ITGB3 expression in BTZ-treated cells when compared with non-BTZ-treated cells after 24 hours determined by RT-PCR. Gene expression was normalized to β-ACTIN expression. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice with similar results. (G) Representative FACS histograms showing ITGB3 expression in PI-negative gated cells after being cultured with or without BTZ for 24 hours. (H) Percentage of ITGB3-positive cells in BTZ or non-BTZ cultures, as determined by FACS (n = 6/condition). (I) Mock, EGFL7 KD, and EGFL7 OE RPMI cells were treated with/without BTZ for 24 hours in vitro. Washed RPMI8226 OE, Mock, or KD cells were allowed to adhere to plastic for 4 hours. Adherent cells were quantified using a fluorescence plate reader. Results are shown as percentage adhesion over the input. Experiment was run twice in triplicate (n = 6/condition). (J) Absolute number of GFP+ RPMI8226 cells per well after 4 hours of culture on EGFL7 OE or EGFL7 KD BMEC1 cells. (K) RPMI8226 cells were cultured in the presence or absence of BTZ with/without the integrin inhibitor Cilengitide (Celin; n = 6/group). The number of viable cells was counted after 24 hours. The experiment was run once in triplicate (n = 6/condition). (L) Mock, ITGB3 OE, and ITGB3 KD RPMI cells were treated with/without BTZ. Viable cells were counted using the Trypan exclusion assay (n = 6/group). The experiment was repeated twice. (M) Mock, EGFL7 OE, and EGFL7 KD RPMI cells concomitantly infected with ITGB3 OE or KD plasmids were treated for 24 hours with/without BTZ (n = 6/group). The number of viable cells was determined. The experiment was performed twice. (N) Study treatment scheme. Mock, EGFL7 OE, and EGFL7 KD RPMI MM cells concomitantly infected with/without ITGB3 OE or KD plasmids were injected s.c. into NOD/SCID mice, and tumors were established. Starting on the 25th day (treatment day 1), tumor-carrying mice were randomized according to tumor size. A total of 5 injections of PBS/dimethyl sulfoxide carrier, or BTZ (1 mg/kg) were given twice per week (n = 5 mice per group). Tumor weight was determined on day 36 (n = 5-7/group). Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

Article Snippet: Immunohistochemical staining for EGFL7 on human patient BM sections was performed using the EGFL7 polyclonal Ab (cat# 19291-1-AP; Proteintech) followed by 4′,6-diamidino-2-phenylindole development and hematoxylin counterstaining.

Techniques: Cell Culture, Gene Expression, Reverse Transcription Polymerase Chain Reaction, CCK-8 Assay, Expressing, In Vitro, Fluorescence, Exclusion Assay, Infection, Injection

Journal: Blood Advances

Article Title: The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

doi: 10.1182/bloodadvances.2019001002

Figure Lengend Snippet: EGFL7, a KLF2 downstream target, promotes primary MM survival. (A) Fold change in ITGB3, EGFL7, and KLF2 expression in ITGB3 OE RPMI cells when compared with Mock cells, as determined by RT-PCR (n = 3/group). (B) Fold change in KLF2 expression in Mock, si-KLF2, and KLF2 OE RPMI cells as determined by RT-PCR (n = 3/group). (C) Fold change in EGFL7 expression in KLF2 OE or si-KLF2 RPMI cells when compared with KLF2 Mock or si-KLF2 cells, respectively, as determined by RT-PCR. (D) Representative western blot for indicated proteins from lysates of si-Ctrl, si-KLF2, Mock, and KLF2 OE cells. (E) Viable cell quantification of KLF2 OE and Mock cells after 24 hours in culture (n = 6/group). (F) KLF2 OE, Mock, si-Ctrl, and si-KLF2 cells were cultured in the presence or absence of rec. EGFL7. Cells were counted after 24 hours (n = 6/group). (G) Fold change in KLF2 expression in BTZ-treated MM cells at the indicated concentration for 24 hours when compared with non-BTZ-treated controls, as evaluated by RT-PCR. (H) MM cells (EGFL7 OE/EGFL7 KD, KLF2 OE, KLF2 KD, or KLF2 OE + EGFL7 KD) were cultured with/without 10 µM BTZ. After 24 hours, viable cells were enumerated (n = 3/group). (I) Immunohistochemical staining for EGFL7 (background panel; scale bars, 50 µm) and hematoxylin and eosin (insert; scale bars, 10 µm) in BM sections of patients with MM. For patient details, see supplemental Figure 1. (J) KLF2 expression determined by RT-PCR in total BMMCs, MACS-isolated BM CD138+ and CD138− cells derived from MM patients #1, #2, and #3. KLF2 expression was given as a fold change to the expression found in MACS-isolated CD138− cells from a healthy donor as the comparator. (K) EGFL7 KD or OE was achieved in human primary MACS-isolated CD138+ MM cells using a lentiviral virus–containing GFP. Cell proliferation was monitored by GFP positivity using FACS after 24 hours (n = 3/group). (L) EGFL7 OE, KD, KLF2 OE or KD was achieved in primary PCR in CD138+ MM patient cell samples (#1-#3). Cell proliferation of transduced cells was determined after 24 hours (n = 6/group). (M) Fold change in ITGB3 expression, as determined by RT-PCR. For all RT-PCR results, transcripts normalized to b-ACTIN. Experiments were repeated twice with similar results. (N) Model of the EGFL7-ITGB3-KLF2-EGFL7 axis in MM cells. EGFL7 partially through binding to ITGB3 on MM cells upregulates the transcription factor KLF2. In turn, KLF2 augments EGFL7 expression. This leads to a positive forward amplification loop that promotes MM survival, a mechanism that seems especially active under the pressure of chemotherapeutic drugs such as BTZ. We propose that EGFL7 contributes to BTZ-induced drug resistance. Data are represented as mean ± SEM. * P ≤ .05; ** P ≤ .01; *** P ≤ .001. P values were determined using a Student t test.

Article Snippet: Immunohistochemical staining for EGFL7 on human patient BM sections was performed using the EGFL7 polyclonal Ab (cat# 19291-1-AP; Proteintech) followed by 4′,6-diamidino-2-phenylindole development and hematoxylin counterstaining.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Concentration Assay, Immunohistochemical staining, Staining, Isolation, Derivative Assay, Virus, Binding Assay, Amplification