Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) E2012 and E2012-BPyne structures. (b) Cell membranes were photolabeled with E2012-BPyne and then clicked with TAMRA-azide and analyzed (left: Coomassie blue; right: in-gel fluorescence). (c) Biotinylated proteins were captured by E2012-BPyne and analyzed by WB. (d) IFITM3 co-immunoprecipitated with γ-secretase subunits using anti-PS1. (e) IFITM3 was co-purified with γ-secretase subunits by GY6 or 163-BP-L-biotin. (f) An interaction (red) between PS1 and IFITM3 was determined by in situ PLA in mouse primary neurons (bottom panel). Negative controls using PS1 or IFITM3 alone (top two panels), dapi (blue), F-actin (green), scale bar = 50μm. (g) Labeling of IFITM3 by E2012-Bpyne in WT and dKO MEF cells. All WB and IF images are representative of three independent experiments (except b: 2 replicates).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Fluorescence, Immunoprecipitation, Purification, In Situ, Labeling

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) LC-MS/MS analysis of the 15 kDa band identified four peptides that match with human IFITM3. (b) WB analysis of E2012-BPyne (500 nM) labeled PS1-NTF protein. (c) Structures of imidazole GSMs, acid GSM and GSIs. (d) WB analysis of E2012-BPyne labeled proteins in the absence or presence of imidazole GSMs, acid GSM and GSIs. Labeled proteins were captured and analyzed by WB for IFITM3. (e) Structures of GY6 and 163-BP-L-biotin. (f) IFITM3 co-immunoprecipitates with γ-secretase subunits. CHAPSO solubilized cell membranes were immunoprecipitated with anti-IFITM3 antibody and probed with antibodies against PS2-CTF and Pen-2. Rabbit IgG was used as a negative control. (g) IFITM3 does not co-immunoprecipitate with SPP. CHAPSO solubilized cell membranes were immunoprecipitated with a monoclonal anti-IFITM3 antibody (9D11) and probed with antibodies against SPP and IFITM3. Mouse IgG was used as a negative control. (h) Analysis of the total protein level in WT MEF or PS1/2 double KO MEF cells. The same amount of membrane proteins was loaded and analyzed by Western blotting. ( i ) IFITM3 mRNA expression levels were measured by RT-PCR in WT MEF or PS1/2 double KO MEF cells (n=6). All WB images and graphs are representative of three independent experiments (except; a/d: 2 replicates). Graphs are mean ± SD. ns, not significant, two-sided Student’s t-test.

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Immunoprecipitation, Negative Control, Membrane, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) IFITM3 KD by siRNA (n=3) in HEK-APP WT cells was confirmed by WB. Scramble siRNA (SC, n=3), a negative control. (b) IFITM3 KD reduces secreted Aβ40 (n=6, ****p<0.0001) and 42 (n=6, ****p<0.0001). Aβ levels were calculated as % of SC. (c) IFITM3 was KO of U138 cells, and empty vector (EV) was used as control. IFITM3 was reintroduced by transient transfection in both EV and KO cell lines. IFITM3 expression was confirmed by WB. (d) Effect of KO and rescue of IFITM3 on γ-secretase activity for Aβ40 (n=3, *p= 0.0249, ****p<0.0001) and 42 (n=3, *p=0.0359, **p=0.0025) cleavage. (e) Comparison IC 50 of GSM E2012-BPyne against γ-secretase for Aβ40 (n=6, **p= 0.0017) and Aβ42 (n=6, ns) cleavages in U138 EV and KO cells. All WB images and graphs are representative of three independent experiments (except; e: contains data from 2 experimental replicates of n=3). Graphs are mean ± SD. Ns, not significant, two-sided Student’s t-test (except; d: one-way ANOVA and Fishers LSD test).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Negative Control, Plasmid Preparation, Control, Transfection, Expressing, Activity Assay, Comparison

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Quantification of WB showed that IFITM3 KD did not change protein expression levels of APP, Nct and PS1-NTF in HEK-APP WT cells (n=3). (b) Schematic representation of cell-free γ-secretase assay. γ-Secretase is incubated with a recombinant APP substrate in the presence of 0.25% CHAPSO. Cleaved Aβ40 and 42 species are measured with cleavage specific antibodies and AlphaLISA technology. ( c ) Schematic model showing different GSM and GSI binding sites in γ-secretase: E2012 (imidazole GSM), GSM-1 (acid GSM), and L458 (transition state analogue inhibitor, GSI). (d-f) Comparison of IC 50 of (d) GSM-25 (EV: n=9, KO: n= 8) for Aβ40 (****p<0.0001) and Aβ42 (ns), (e) GSM-1 (n=6) for Aβ40 (**p=0.0039) and Aβ42 (ns), and (f) L458 (n=3) for Aβ40 (ns) and Aβ42 (ns) cleavages in the U138 EV or KO cell lines (n≥3). (g) IFITM3 knockdown (KD) does not affect expression of γ-secretase subunits. IFITM3 was knocked-down by siRNA (6 pmol, n=3) in HEK-NotchΔE cells and scramble siRNA (SC, n=3) was used as a negative control. Cell lysates were probed by antibodies against Nct, PS1-NTF and IFITM3. β-Actin was used as a loading control. (h) Effect of IFITM3 KD on γ-secretase activity. IFITM3 KD increased γ-secretase cleaved product NICD, analyzed by WB. Cell lysates were probed by antibodies against c-myc (NotchΔE) and NICD and a representative quantification of NICD (n=8, ***p=0.001) is shown (lower panel). (i) Cell based NICD AlphaLISA assay (left panel) revealed an increase in NICD production with IFITM3 KD. Quantification of NICD (n=8, ***p=0.001) is shown in the right panel. (j) Effect of IFITM3 KO on γ-secretase activity. KO cells lines have increased γ-secretase activity as compared to the EV cell line. The NICD cleavage in vitro was measured by AlphaLISA assay (n=3,**p=0.0096). All WB images and graphs are representative of three independent experiments Graphs are mean ± SD. ns, not significant, two-sided Student’s t-test.

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Expressing, Incubation, Recombinant, Binding Assay, Comparison, Knockdown, Negative Control, Control, Activity Assay, In Vitro

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Protein levels of γ-secretase subunits and IFITM3 in pooled membranes from 4- and 28-month-old WT mouse brains (n=5 mice per age and sex group except n=4 for 28 female, female: **p=0.0014, male: **p=0.0065). Quantified as percent of 4-month female level. (b) γ-Secretase activity for Aβ40 (female: p=***0.0002, male: **p=0.0095) and Aβ42 (female: p=**0.0024, male: **p=0.0088) production in vitro from pooled membrane. (c) Solubilized membranes were captured by 163-BP-L-biotin and then analyzed for γ-secretase and IFITM3 levels. (d) WB for PS1-NTF and IFITM3 (left panel, representative mice shown) in membranes of 18-month-old WT (n=4) and IFITM3−/− (n=5) mouse brains. γ-Secretase activity for Aβ40 (***p=0.0004) and 42 (****p=<0.0001) cleavage (right panels). (e) APP, Nct, PS1-NTF and IFITM3 in membranes from 3- and 12-month-old WT and 5xFAD mice. Quantified as percent of 3-months-old WT (n=5 per group except n=4 for WT at 12 months)(3moWT-12mo5X: ****p<0.0001, 12moWT-12mo5X: ***p=0.004, 3mo5X-12mo5X: ***p=0.0003). (f) WB for PS1-NTF and IFITM3 (left panel) in membranes from 4-month-old 5xFAD (n=3) and IFITM3−/−; 5xFAD (n=3) mouse brains. γ-Secretase activity in vitro for Aβ40 (***p=0.0003) and 42 (**p=<0.0011) cleavage (right panels). (g) Fluorescence microscopy of amyloid plaques in cortex and hippocampus (Thioflavin-S, green) in 4-month-old PFA perfused mice (5xFAD: n=4, IFITM3−/−; 5xFAD: n=5). Scale bar = 300μm (left), 200μm (right). Number of plaques per mm 2 of tissue was calculated throughout the brain and averaged (cor (cortex): *p=0.0109, hip (hippocampus): **p=0.0026). All WB and IF images and graphs are representative of three independent experiments (except; c and quantifications in e: 2 replicates and g: every 1/6 th section throughout the brain). Bar graphs are mean ± SD. Violin plots represent median (middle line) and interquartile range (outer lines). Ns, not significant, two-sided Student’s t-test (except; e: two-way ANOVA followed by Tukey).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Activity Assay, In Vitro, Membrane, Fluorescence, Microscopy

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) WBs for Nct and PS1-NTF were quantified by Odyssey imaging (n=5 mice pooled per group, except n=4 for 28F, all=ns). (b) Effect of aging on subcellular localizations of IFITM3. A hemibrain from male wild-type C57BL/6 mouse at 4 and 28 months (n=1 per group) were homogenized and layered on iodixanol gradient (2.5 – 30 %). Fractions were collected from the top and resolved by WBs for γ-secretase, IFITM3 and different subcellular markers. (c) WBs for APP, Nct, and PS1-NTF were quantified by Odyssey imaging (n=5 mice per group except n=4 for WT at 12 months). APP: 3moWT-3mo5X: ****p<0.001, 3mo5X-12mo5X: ****p<0.0001, 12moWT-12mo5X: ****p<0.0001). Nct: 3moWT-12moWT: ****p<0.001, 3mo5X-12mo5X: ****p<0.001, 12moWT-12mo5X: ****p<0.001. PS1-NTF: 3moWT-12mo5X: ***p=0.0004, 12moWT-12mo5X: ****p<0.001. ( d ) Immunostaining of IFITM3 in mouse brains. Fluorescence microscopy of IFITM3 expression in 12-month-old PFA perfused mice (WT, upper panel, 5XFAD, lower panel). Representative images of cortex, hippocampus and subiculum (left to right) show IFITM3 (green) and DAPI (blue). Scale bar = 1000μm, 200μm, 100μm (left to right). Total IFITM3 fluorescence area within the hippocampus and cortex of WT and 5XFAD was quantified using FIJI. Total IFITM3 was divided by tissue area and 5XFAD expression was normalized to average of WT (WT: n=7, 5XFAD: n=9)(cor (cortex): **p=0.0035, hip (hippocampus): ****p<0.0001 ). (e) IFITM3 expression in astrocytes and microglia is upregulated in 5XFAD mice compared to WT mice. Fluorescence microscopy of IFITM3, GFAP (top) and Iba1 (bottom) expression in 12-month-old PFA perfused mice (WT, upper panel, 5XFAD, lower panel). Representative images of the hippocampus and cortex show IFITM3 (red), GFAP (green – top), Iba1 (green - bottom), and DAPI (blue), scale bar = 500μm. Inset panels (left to right) show GFAP or Iba1 (green), IFITM3 (red) and merge. Scale bar = 50μm. All WB images and graphs are representative of three independent experiments (except; b: 2 replicates). Graphs are mean ± SD. ns, not significant, , two-sided Student’s t-test, (except; c: one-way ANOVA followed by Tukey).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Imaging, Immunostaining, Fluorescence, Microscopy, Expressing

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) The expression profiles of IFITM3 in human control (n=76) and LOAD (n=80) from temporal cortex (**p=0.002)(Mayo Clinic cohort). (b) IFITM3 mRNA expression levels in control and LOAD samples (LOAD: n=18 and control: n=10, **p=0.0056). (c) The protein levels of γ-secretase subunits and IFITM3 in human brain membranes (LOAD: n=18 and control: n=10, *p=0.0127). IFITM3 quantified as relative intensity. (d) IFITM3 expression (left) between control (n=10), LOAD-L (n=10) and LOAD-H (n=8)(****p<0.0001). γ-Secretase activity for Aβ40 (**p=0.0037, ***p=0.0007) and 42 (**p=0.0036, ***p=0.0002) cleavage between groups. (e) Primary mouse neurons were treated with control (n=2), 10ng/mL (n=3, ***p=0.0005) or 100 ng/mL (n=3, ***p=0.0003) of IFN-γ, membranes were probed for γ-secretase and IFITM3 (left panel, quantified: right panel). (f) Quantification of secreted Aβ40 from 10ng/mL (n=8, **p=0.0010) and 100 ng/mL (n=8, **p=0.0026) IFN-γ treated neurons Aβ42 from 10ng/mL (n=8, ***p=0.0006) and 100 ng/mL (n=8, ***p=0.0003) IFN-γ (n=8). (g) γ-Secretase activity for Aβ40 (n=6, ***p=0.0007) and 42 (n=6, ***p=0.006) cleavage from IFN-γ treated neuron membranes. (h) Photolabeled PS-NTF in neuronal membranes and quantified as % of control (control: n=10, 10ng/ml: n=5, **p=0.0023, 100ng/ml: n=7,****p<0.0001). (i) WB for IFITM3 and PS1-NTF primary human astrocytes treated with PBS, IL-6, or IL-1β (control: n=6, IL-6: n=4, **p=0.0014, IL-1β: n=3, *p=0.0103), data normalized to PBS. (j) γ-Secretase activity for Aβ40 (control: n=8 IL-6: n=7, **p=0.0098, IL-1β: n=9,****p<0.0001) and 42 (control: n= IL-6: n=7, *p=0.0467, IL-1β: n=9, ****p<0.0001) in astrocyte membrane. All WB images and graphs are representative of three independent experiments (except; b: 1 replicate, c/g: 2 replicates, d: γ-secretase activity graph contains data from two independent replicates, h: 5 replicates). Graphs are mean ± SD. Ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-sided Student’s t-test.

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Expressing, Control, Activity Assay, Membrane

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Spearman’s correlation of mRNA expression of human IFITM3 gene with age was analyzed in the cortex (n=158) and hippocampus (n=123) of normal human brains using the Genotype-Tissue Expression (GTEx) cohort.. (b) mRNA expression in non-demented subject control (n=10) and LOAD samples (n=18) of MAP2 (ns), GFAP (**p=0.0046), and AIF1 (ns) were measured, which were used in – . (c) Expression profiles of MAP2 (ns), GFAP (****p<0.0001), and AIF1 (ns) in the temporal cortex of human control (n=76) and LOAD samples (n=80) using the Mayo Clinic cohort data. Correlation analyses were carried out and p values were calculated. The protein levels of Nct (****p<0.0001) and PS1-NTF (**p=0.0042) in human brain membranes (control and LOAD). The samples were analyzed by WB and quantified (n=10 and 18, respectively). Signal was normalized to HeLa cell membrane. (e-f) IFITM3 SNP Genotypes. (e) Allelic discrimination plot depicting rs34481144 genotype calls for control (n=9), LOAD-L (n=10), and LOAD-H (n=8) brain samples. The axes show delta Rn values obtained from TaqMan SNP genotyping analysis. Samples without genomic DNA were used as non-template controls (shown as black squares in the left lower quadrant, n=2). (f) Allele frequency of rs34481144 genotype in control (n=9) and LOAD (n=18). ( g ) mRNA level of IFITM3 gene in four types of EGFP/L10a-expressing mouse hippocampal neurons (GAD2 (glutamate decarboxylase 2), CCK (cholecystokinin), PV (parvalbumin), and CORT (cortistatin) expressing GABAergic neurons)(n=4 per group, GAD2-PV: ***p=0.0003, CCK-PV: ****p<0.0001, CCK-Cort *p=0.0363, PV-Cort: **p=0.0059). ( h ) mRNA levels of IFITM3 in human iPSC-derived neurons (n=4) and human primary astrocytes (n=3) were measured by qPCR (****p<0.0001). ( i-j ) Human iPSC-derived neurons (i) and human primary astrocytes (j) were stained for IFITM3 with MAP2 (neuronal marker) or S100β (astrocyte marker). DAPI was used for nucleus staining. Scale bar = 200 and 500 μm. ( k ) Induction of IFITM3 by IFN-α in primary neurons. E16 mouse primary neurons were treated with 100 ng/ml of IFN-α at DIV12 for 24 hours. The protein levels of γ-secretase and IFITM3 were analyzed by WB (n=4 per group). β-Tubulin III was used as a loading control. ( l ) Effect of IFITM3 induction on γ-secretase activity for Aβ40 (*p=0.0116) and Aβ42 (*p=0.0319) activity. Membranes from primary neurons were incubated with the recombinant APP substrate C100-ΔID-FLAG and γ-secretase activity (Aβ cleavage rate) was assayed by human Aβ three-plex MSD kits (n=12, 10). ( m ) JC8 whole cell photolabeling. Neuronal membranes were photolabeled with JC8 in the absence or presence of L458 and analyzed by anti-PS1-NTF antibody. Photolabeled PS1-NTF protein level was quantified by Odyssey imaging (n=3, *p=0.0210). (n) Spearman’s correlation between the expression level of IFITM3 and viruses. In the Brodmann Area 22 (BA-22 region) in the Mount Sinai Brain Bank (MSBB) cohort, the expression level of IFITM3 is positively correlated with the expression level of the human herpesvirus-6B (HHV-6B) (rho=0.248, p=0.044, n=66). In the Brodmann Area 36 (BA-36 region), the expression level of IFITM3 is positively correlated with the expression of hepatitis C virus genotype 4 (rho=0.255, p=0.033, n=70). All WB images and graphs are representative of two independent experiments (except; b/e: 1 replicate, h: data pooled from 2 experiments, k: 3 replicates, l: data pooled from 4 experiments). Bar graphs are mean ± SD. Violin plots represent median (middle line) and interquartile range (outer lines). ns, not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001, two-sided Student’s t-test (except; g: One-Way ANOVA followed by Tukey).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Expressing, Control, Membrane, Derivative Assay, Staining, Marker, Activity Assay, Incubation, Recombinant, Imaging, Virus

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Schematic model showing L458 binding to the subsites (S2-S3’) in the active site of γ-secretase. (b) Photolabeling of IFITM3 and PS1 by four inhibitors. (c) Photolabeling of IFITM3 and PS1 by L505 in human brains (control: n=4, LOAD-L: n=5, LOAD-H: n=5). (d) Pearson’s correlation between γ-secretase activity and L505 labeled IFITM3 (Fig. 5c) in LOAD samples (n=10). (e) Double cross-linking of γ-secretase and IFITM3 by the dual probe, L631. WB reveals multiple protein complex species containing IFITM3, PS1-NTF, PS1-CTF, IFITM3 homodimer, IFITM3-PS1 and PS1-NTF-CTF heterodimers. (f) Schematic representation of the interaction between IFITM3 and γ-secretase, IFITM3 is near the active site and can be crosslinked with PS1-NTF. (g) IFITM3 connects infections and innate immunity with Aβ production and AD risk. (A) Pathogenic challenge, or other inflammatory conditions, induce the release of proinflammatory cytokines from astrocytes and microglia. (B) Cytokines upregulate IFITM3 expression in neurons and astrocytes that potentiates γ-secretase, increasing Aβ production. (C) As part of an innate immune response, Aβ acts as an antimicrobial or antiviral peptide. In turn, Aβ accumulation also triggers AD pathology. All WB images and graphs are representative of three independent experiments (except; c: 2 replicates).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Binding Assay, Control, Activity Assay, Labeling, Expressing

Journal: Investigative and clinical urology

Article Title: IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

doi: 10.4111/icu.20230294

Figure Lengend Snippet: Fig. 1. Interferon-induced transmem- brane protein 3 (IFITM3) expression in the normal kidney and clear cell renal cell carcinoma (ccRCC). (A) In normal renal parenchyma, IFITM3 was immuno- reactive in glomerular parietal epithelial cells, capillaries (left), and damaged or atrophic tubules (right). IFITM3 ex- pression was inconspicuous in tubular epithelial cells (middle). (B) In ccRCC, IFITM3 was immunoreactive as a mem- branous pattern in low-grade tumors (left), whereas cytoplasmic patterns are observed in high-grade tumors (right) (magnification, ×400).

Article Snippet: For ectopic overexpression of IFITM3 proteins, a lentivirus containing the pCMV6-Entry vector cloned with IFITM3 ORF construct was purchased (RC201635, OriGene).

Techniques: Expressing

Journal: Investigative and clinical urology

Article Title: IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

doi: 10.4111/icu.20230294

Figure Lengend Snippet: Fig. 3. (A) The tumorigenicity of 786-O P/empty, 786-O suR/empty, 786-O P/IFITM3-overexpression, and 786-O suR/IFITM3-shRNA was evaluated in a xenograft model. Sunitinib was administered 31 days after transplantation. (B) Representative images 786-O P/IFITM3-overexpression (left upper), 786-O P/empty, 786-O suR/IFITM3-shRNA, and 786-O suR/empty were presented clockwise. Representative images of the high-power field were presented as inset. IFITM3, interferon-induced transmembrane protein 3 (magnification, ×40; ×400).

Article Snippet: For ectopic overexpression of IFITM3 proteins, a lentivirus containing the pCMV6-Entry vector cloned with IFITM3 ORF construct was purchased (RC201635, OriGene).

Techniques: Over Expression, shRNA, Transplantation Assay

Journal: Investigative and clinical urology

Article Title: IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

doi: 10.4111/icu.20230294

Figure Lengend Snippet: Fig. 5. The interferon-induced trans- membrane protein 3 (IFITM3)-induced acquired tyrosine kinase inhibitor (TKI) resistance mechanism represented in a schematic diagram. In patients resistant to sunitinib, overexpression of IFITM3 is observed. The binding of IFITM3 to TRAF6 leads to the activation of TRAF6 associated with IRAK1, which induces oncogenic signaling pathways, includ- ing MAPK/AP-1 pathways. As a result, clear cell renal cell carcinoma (ccRCC) can accelerate tumor growth, survival, and proliferation despite TKI treatment.

Article Snippet: For ectopic overexpression of IFITM3 proteins, a lentivirus containing the pCMV6-Entry vector cloned with IFITM3 ORF construct was purchased (RC201635, OriGene).

Techniques: Membrane, Over Expression, Binding Assay, Activation Assay, Protein-Protein interactions

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: a , Changes of Ifitm3 mRNA levels were monitored in murine splenic B cells upon BCR-engagement (mean ± s.e.; n=2). Pten fl/fl pre-B cells carrying 4-OHT-inducible Cre-ER T2 or ER T2 were treated with 4-OHT and studied by RNA-seq for Ifitm3 transcript expression ( b ) and Western blot for protein levels of Pten, phospho-Akt-S 473 , Akt and Ifitm3 ( c ; n=3). d , Phospho-STAT5-Y 694 , STAT5 and IFITM3 levels in patient-derived B-ALL cells (PDX2) measured by Western blotting upon Imatinib treatment (n=3). e , Scenario of the PI3K-pathway as positive regulator of IFITM3, which in turn amplifies BCR and oncogenic signaling. f , IFITM3 mRNA levels across human normal and malignant hematopoietic and B-lymphoid samples (source data and statistics ; two-tailed t-test). g , IFITM3 mRNA levels in pre-B cells from healthy donors and B-ALL patient samples were compared for two clinical cohorts (ECOG E2993 and St. Jude). In ECOG E2993, bone marrow samples were obtained at diagnosis before treatment from 83 adults with B-ALL with a confirmed purity of >90% leukemic blasts. For the St. Jude data sets, 15 pediatric B-ALL samples prior to treatment were compared to flow-sorted pre-B cells from bone marrow aspirates of healthy donors. (two-tailed Wilcoxon). h , Minimal residual disease (MRD) was determined in the COG P9906 cohort, IFITM3 mRNA levels were compared in MRD+ (n=67) and MRD- (n=124) patients (two-tailed Wilcoxon). i-l , Patients with leukemia and lymphoma from five clinical cohorts were segregated into two groups based on higher ( IFITM3 high ) or lower ( IFITM3 low ) than median IFITM3 mRNA levels. Overall survival was compared by two-tailed log-rank test. m , ChIP-seq enrichment of RNAPII and H3K4me3 at the Ifitm3 locus in pre-B cells (top) from Ikzf1 exon5fl/fl mice upon Cre-mediated deletion of Ikzf1 (GSE86897). Binding of IKZF1 to the promoter region of IFITM3 was also analyzed in ChIP-seq data from patient-derived B-ALL cells (bottom, LAX2, GSE58825). n , Human B-ALL cells (BV173) carrying IKZF1 -deletions were reconstituted with doxycycline-inducible IKZF1 or EV. Levels of IFITM3 were assessed by Western blotting upon doxycycline-induction (n=3). o , Multivariate analysis of established risk factors in pediatric B-ALL patients (COG P9906, n=207), including mutation or deletion of IKZF1 . Patients (n=207) were separated into IKZF +/+ or IKZF Del groups, then further segregated based on higher or lower than median expression levels of IFITM3 . The comparison of these four groups established IFITM3 mRNA levels as an independent risk factor regardless of IKZF1 -deletion status (two-sided log-rank test; P= 0.0045). c-d, n , For gel source data, see .

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: RNA Sequencing, Expressing, Western Blot, Derivative Assay, Two Tailed Test, Biomarker Discovery, ChIP-sequencing, Binding Assay, Mutagenesis, Comparison

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: a , CD19 surface expression (top), cell cycle progression (middle; percentages in S phase) and cell viability (bottom; percentages of Annexin V + 7-AAD + cells) were measured. b , Number of viable Ifitm3 +/+ and Ifitm3 −/− pre-B cells were counted at times indicated. c , Levels of phospho-Akt-S 473 , Akt, Myc, p53, p21 and Bcl2 were assessed in Ifitm3 +/+ and Ifitm3 −/− pre-B cells. d , Splenic B-cells from Ifitm3 +/+ and Ifitm3 −/− mice (n=5) were analyzed for CD21 high CD23 low/− marginal zone B-cells (MZB), peritoneal cavity B-cells for Mac-1 + IgM + B-1, and CD5 + IgM + B-1a cells. e , Splenic B-cells from Ifitm3 +/+ and Ifitm3 −/− mice were adoptively transferred to μMT recipient mice (n=10) followed by immunization with 0.5 mg of NP-KLH or Vehicle (Veh). Spleens were collected on day 12 after immunization and subjected to immunofluorescence staining of tissue sections with B220, CD3 and peanut agglutinin (PNA). f-g , Splenocytes harvested from μMT mice (n=10) were analyzed by flow cytometry 12 days post immunization for CD95, GL7 and NP to identify NP-specific GC-B cells. Relative fractions , absolute numbers , and representative flow cytometry plots ( h ) are shown. , Levels of serum immunoglobulin isotypes in μMT recipient mice transplanted with Ifitm3 +/+ or Ifitm3 −/− B-cells are shown before and after immunization (n=10; day 12). Serum levels of IgM, IgG1 and IgG2b were determined by ELISA. a-c, For gel source data, see , – , , Mean ± s.d. indicated, significance determined by two-tailed t -test.

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: Expressing, Immunofluorescence, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: a , Hardy fractions of B-cell subsets isolated from bone marrow of Ifitm3 +/+ and Ifitm3 ˉ / ˉ littermates analyzed by flow cytometry (n=3). b , Surface expression of IgM, CD20, CD19, IgD, CD2 and CD21 measured by flow cytometry in enriched bone marrow (Gr-1 − , Nk1.1 − and B220 + ) and splenic B-cells (CD3 − and B220 + ) from Ifitm3 +/+ or Ifitm3 ˉ / ˉ mice (n=7; mean±s.d.). Mean fluorescence intensities (MFI) values for individual measurement compared by two-tailed t -test. c , Ca 2+- mobilization from cytoplasmic stores in response to BCR (IgM)-engagement was measured in Ifitm3 +/+ and Ifitm3 ˉ / ˉ splenic B cells. Ca 2+ release was induced by addition of 10 μg ml −1 anti-mouse IgM 60 seconds after acquisition of background fluorescence. Ca 2+ release was measured over 300 seconds with cell permeant Rhod-2 dye; MFI compared between replicates (n=3). d , Ca 2+ mobilization in response to BCR-engagement measured upon CRISPR-Cas9-mediated deletion of IFITM3 in Jeko1 mantle cell lymphoma (MCL) cells. Ca 2+ -release upon addition of 10 μg ml −1 of polyclonal F(ab’) 2 anti-human IgM was measured for 300 seconds with cell permeant Fluo-4 dye; MFI compared between replicates (left; n=3). Surface expression of CD19 following deletion of IFITM3 in Jeko1 MCL cells, MFIs for CD19 indicated (right; n=3). e , Jeko1 MCL cells were electroporated with non-targeting RNP (Cas9-gRNA ribonucleoproteins, gNT) or IFITM3-targeting RNP complex (gIFITM3). Following electroporation, MCL cells were treated with vehicle (DMSO) or 25 nmol l −1 of Dasatinib for 3 hours. Cells were stimulated with 10 μg ml −1 of anti-human IgM F(ab’)2 for indicated timepoints and subjected to co-immunoprecipitation with an anti-CD19 antibody. Immunoblots were performed to measure levels of CD19-tyrosine phosphorylation, and binding of LYN to CD19. Levels of IFITM3, Src-pY 416 and Lyn were assessed in whole cell lysates (10% input) with β-actin as loading control. (n=3; gel source data ). f-h , Relative fractions (left) and absolute cell counts (right) of total B-1 ( f ), B-1a ( g ) cells in the peritoneal cavity and marginal zone B cells ( h ) in spleen of Ifitm3 +/+ and Ifitm3 −/− littermates (n=5) are shown (means ± s.d.; two-tailed t -test).

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: Isolation, Flow Cytometry, Expressing, Fluorescence, Two Tailed Test, CRISPR, Electroporation, Immunoprecipitation, Western Blot, Phospho-proteomics, Binding Assay, Control

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: a , CD19 surface expression (top), cell cycle progression (middle; percentages in S phase) and cell viability (bottom; percentages of Annexin V + 7-AAD + cells) were measured. b , Ifitm3 +/+ and Ifitm3 −/− BCR-ABL1 or NRAS G12D B-ALL cells were assayed for levels of phospho-Akt-S 473 , Akt, Myc, p53, p21 and Bcl2 (n=3) and c , plated in semi-solid methylcellulose. d , Kaplan-Meier analyses of NSG recipient mice injected with indicated numbers of Ifitm3 +/+ and Ifitm3 −/− BCR-ABL1 (left) or NRAS G12D (right) B-ALL cells (n=5) e , Frequencies of leukemia-initiating cells (LIC) estimated with extreme limiting dilution analysis (ELDA; 90% CI; likelihood ratio test). f , Jeko1 mantle cell lymphoma (MCL) cells expressing doxycycline (Dox)-inducible Cas9 were transduced with IFITM3 -targeting or non-targeting (NT) sgRNAs. Enrichment or depletion of targeted cells (Cas9 + gRNA + ) monitored by flow cytometry upon Dox-treatment (mean±s.d.), and IFITM3 levels measured by Western blot. , Pre-malignant LSL- Bcr BCR-ABL1 x Mb1-Cre pre-B cells expressing IFITM3, IFITM3 Y20E (Y20E) or EV were plated for colony forming assays (7 days; representative images shown at ×1 (top) and ×10 (bottom) magnification). h , Survival analyses ( P =0.0001, log-rank test) of congenic recipient mice transplanted with LSL- Bcr BCR-ABL1 x Mb1-Cre B-cells transduced with EV, IFITM3 or Y20E (n=7). i , Engraftment and expansion of luciferase-labeled leukemia cells were monitored by luciferase bioimaging at times indicated. j , Effects of IFITM3 or IFITM3 Y20E on oncogenic signaling in LSL- Bcr BCR-ABL1 x Mb1-Cre B-cell precursors measured by Western blot and compared to EV. Phosphorylation of Cd19-Y531, Src/Lyn-Y416 and Akt -S473 were examined. a-c,f-g,j, For gel source data, see , , Two-tailed t -test.

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: Expressing, Injection, Transduction, Flow Cytometry, Western Blot, Luciferase, Labeling, Phospho-proteomics, Two Tailed Test

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: a , Numbers of viable Ifitm3 +/+ and Ifitm3 −/− BCR-ABL1 or NRAS G12D B-ALL cells were counted by Trypan blue dye exclusion (n=3; mean±s.d.; two-tailed t -test). b , RNA-seq was performed for Ifitm3 +/+ and Ifitm3 −/− BCR-ABL1 and NRAS G12D B-ALL cells. Relative rlog normalized gene expression values for all strongly differentially expressed genes ( P < 1e-5 & L2FC > 1; Wald test with BH correction) in both BCR-ABL1 and NRAS G12D conditions plotted as a heatmap with row-scaling. B-cell signaling related genes are labeled in red, anergy-related genes are labeled in blue and PI3K signaling related genes are labeled in gray. c , Gene set enrichment analysis for genes ranked by ratio of Ifitm3 −/− to Ifitm3 +/+ as log2 fold change; red lines indicates running enrichment score (right axis), gray bars indicates fold change (left axis). Statistical significance was determined by two-tailed Kolmogorov-Smirnov test. d , Patient-derived B-ALL cells (PDX2) were transduced with N-terminally FLAG-tagged or C-terminally HA-tagged IFITM3 constructs. Combinations of intracellular and surface staining were performed to examine IFITM3 topology at the cell membrane. e , Patient-derived B-ALL cells (PDX2) were transduced with C-terminally HA-tagged IFITM3 or the IFITM3 Y20E phosphomimetic. Combinations of intracellular and surface staining, with or without Src-kinase inhibition by dasatinib, were performed to examine IFITM3 topology at the cell membrane and its regulation by Src-kinases. d-e , Representative plots from 3 independent experiments f , A scenario of the topology of IFITM3 regulated by Src (Lyn)- or oncogenic tyrosine kinases (BCR-ABL1) at the plasma membrane is shown. Phosphorylation of Y20 hinders the recognition of YEM endocytosis motif by the AP-2 complex, thereby antagonizes endocytosis and endosomal trafficking of IFITM3.

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: Two Tailed Test, RNA Sequencing, Gene Expression, Labeling, Derivative Assay, Transduction, Construct, Staining, Membrane, Inhibition, Clinical Proteomics, Phospho-proteomics

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: a , Phosphorylated protein levels in PDX2 B-ALL cells transduced with HA-tagged IFITM3 Y20E or empty vector (EV) control identified by mass spectrometry upon IFITM3-crosslinking with anti-HA antibodies. b , Feature set enrichment analysis (FSEA) for phosphorylated proteins in the BCR-(red), integrin (blue) and PI3K- (gray) pathways, ranked by log2-fold change (Kolmogorov-Smirnov). c, Levels of phospho-SRC-Y 416 , LYN, phospho-CD19-Y 531 , CD19, phospho-AKT-S 473 and AKT assessed in ICN12 cells expressing IFITM3-HA, IFITM3 Y20E -HA or EV-HA upon IFITM3-crosslinking. d-e, Interactomes of BirA-IFITM3 Y20E or EV control expressed in PDX2 B-ALL and Jeko1 MCL cells. f-g , Validation of IFITM3 interacting proteins by anti-FLAG co-immunoprecipitation in PDX2 cells transduced with FLAG-IFITM3 or FLAG-EV, followed by Western blotting for validation of interacting proteins and STAT5 and eIF4E as specificity controls ( g ). h-i, PLA in Jeko1 cells upon BCR-stimulation, assessed for proximity of CD79B to IFITM3. Representative images with PLA signal (red), nuclei (DAPI) and plasma membrane (WGA; green). ( h ). Quantitation of PLA signals per cell (two-tailed t -test). j , Structural model of IFITM3-mediated signal amplification between CD19 and LYN. In resting B-cells (left), IFITM3 is localized in endosomes. Upon antigen-encounter (right), LYN-mediated phosphorylation of IFITM3 induces membrane translocation, acting as a scaffold for CD19 and LYN in proximity with BCR-molecules. BCR-CD19-IFITM3 complexes form clusters for PI3K-activation and accumulation of PIP3 in lipid rafts. a-i, n=3, For gel source data, see , , , Row-scaled protein abundance ranked by fold change, key proteins of interest are highlighted.

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: Transduction, Plasmid Preparation, Control, Mass Spectrometry, Expressing, Biomarker Discovery, Immunoprecipitation, Western Blot, Clinical Proteomics, Membrane, Quantitation Assay, Two Tailed Test, Amplification, Phospho-proteomics, Translocation Assay, Activation Assay, Quantitative Proteomics

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: a , Volcano plot of differentially phosphorylated proteins in patient-derived B-ALL (PDX2) cells transduced with IFITM3 Y20E compared to empty vector (EV) control (n=3; Wald test with BH correction). b , F(ab) fragments of the anti-HA antibody or isotype control were purified and their identity confirmed by Western blot ( left ). 8 million patient-derived B-ALL (PDX2) cells carrying IFITM3-HA, IFITM3 Y20E -HA or EV control were resuspended into complete medium and treated with either 2.5 μg ml −1 of full antibodies or F(ab) fragments of anti-HA or isotype control for the indicated times. Levels of phospho-AKT-S 473 , AKT and HA tagged IFITM3 were assessed by Western blots using β-actin as loading control ( right ). Data from three independent experiments. For gel source data, see . c , Ca 2+ -mobilization in response to TCR-engagement using CD3ɛ-specific antibodies was measured upon CRISPR-Cas9-mediated deletion of IFITM3 in T-ALL cells (Jurkat; left ). Ca 2+ release from cytoplasmic stores was induced by adding 10 μg ml −1 of monoclonal (OKT3) anti-human CD3ɛ at 50 seconds after acquisition of background fluorescence. Surface expression of CD3 was measured following deletion of IFITM3 in Jurkat cells ( right ). MFIs for CD3 are indicated. Shown are representative plots from 3 independent biological experiments. MFI values for individual measurement were compared by two-tailed t -test. d , Surface proteins on Ifitm3 +/+ and Ifitm3 −/− B-ALL cells were labeled with biotin and enriched with streptavidin affinity pull-down followed by on-bead trypsin digestion, mass spectrometry and quantified with Lable-Free Quantification (LFQ). Differentially expressed cell surface proteins on Ifitm3 +/+ and Ifitm3 −/− B-ALL cells are shown with the mean difference of LFQ plotted against the P -value (Welch’s t-test). All experiments were performed in biological triplicates. e-g , Validation of differential expression of surface receptors between Ifitm3 +/+ , Ifitm3 −/− and Ifitm3 Y20E -overexpressing B-ALL cells. Flow cytometry analyses show surface expression of BCR-signaling components ( e ), integrins and adhesion receptors ( f ) and other surface receptors ( g ) in Ifitm3 +/+ , Ifitm3 −/− and B-ALL cells expressing Ifitm3 Y20E (n=3).

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: Derivative Assay, Transduction, Plasmid Preparation, Control, Purification, Western Blot, CRISPR, Fluorescence, Expressing, Two Tailed Test, Labeling, Mass Spectrometry, Biomarker Discovery, Quantitative Proteomics, Flow Cytometry

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: a , Surface expression of Cd19 was assessed by flow cytometry following forced expression of Cd19 for >1 week in Ifitm3 +/+ and Ifitm3 −/− B-ALL cells. b , Western blot analyses of phospho-Akt-S 473 , Akt, Myc and Bcl2 upon forced expression of Cd19 for >1 week in murine Ifitm3 +/+ and Ifitm3 −/− B-ALL cells. Colony forming ability ( c ) and cell cycle progression ( d ) of Ifitm3 +/+ and Ifitm3 −/− B-ALL cells upon forced expression of Cd19 for >1 week was examined. c , Colony numbers for individual measurement were compared by two-tailed t -test. d , Numbers indicate percentage of cells in S phase. e , Numbers of viable Ifitm3 +/+ and Ifitm3 −/− B-ALL cells following forced expression of Cd19 were counted by Trypan blue dye exclusion (left). Ifitm3 ˉ / ˉ B-ALL cells were transduced with GFP-tagged constructs for expression of Cd19 with an intact (Y531) or mutant (Y531F) PI3K-activation motif in its cytoplasmic tail. Relative changes of GFP + cells (transduced with Cd19-Y531 or Cd19-F531) were plotted over time (means ± s.d.). a-e, Data from three independent experiments. b, For gel source data, see . f , Murine Cd19 −/− B-ALL clones were generated by electroporation of murine B-ALL cells with Cd19-targeting RNP (Cas9-gRNA ribonucleoproteins, gCd19 ALL) and single-cell clones with biallelic deletion were selected. Cd19 −/− B-ALL cells were transduced with Cd19-ER T2 , a fusion of the ER-ligand binding domain to the C terminus of Cd19, or ER T2 as empty vector control. Reconstitution of Cd19 −/− B-ALL cells with Cd19-ER T2 resulted in stable expression of the fusion proteins that were retained in complex with cytoplasmic heatshock proteins. Addition of 4-OHT released Cd19-ER T2 from its cytoplasmic heatshock chaperone and enable cell surface expression within 30 minutes of 4-OHT addition. g , To test the effect of inducible Cd19 membrane translocation in Ifitm3 −/− B-ALL cells, Ifitm3 +/+ and Ifitm3 −/− B-ALL cells were transduced with Cd19-ER T2 or ER T2 empty vector control. 4-OHT-mediated translocation of Cd19 to the cell surface was assessed by flow cytometry for indicated times (0 to 3 hours). h , Ifitm3 +/+ and Ifitm3 −/− B-ALL cells were transduced with Cd19-ER T2 or ER T2 empty vector control. Cells were treated for 0, 1 and 3 hours with 4-OHT for surface-translocation of Cd19. Cell lysates from these populations were analyzed by Western blot for phospho-Cd19-Y 531 , Cd19, phospho-Src-Y416, Lyn, phospho-AKT-S 473 , AKT, Myc and Bcl2. While Cd19-ER T2 reconstitutes Cd19 protein levels in Ifitm3 −/− B-ALL cells and rapid translocation to the cell surface ( g ), this change alone was not sufficient to induce proper phosphorylation of Cd19, Src-kinases and PI3K-signaling via Akt f-h, Data from three independent experiments. h, For gel source data, see .

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: Expressing, Flow Cytometry, Western Blot, Two Tailed Test, Transduction, Construct, Mutagenesis, Activation Assay, Clone Assay, Generated, Electroporation, Ligand Binding Assay, Plasmid Preparation, Control, Membrane, Translocation Assay, Phospho-proteomics

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: a , Schematic of HA-tagged-IFITM3 Y20E BirA-fusion proteins used for TurboID interactome analyses. BirA (biotin ligase) was fused to N-terminal IFITM3 carrying the phosphomimetic Y20E mutation for membrane-localization. HA-IFITM3 Y20E -BirA or HA-BirA controls were expressed in PDX2 B-ALL ( b ) or Jeko1 MCL cells ( c and d ). c,d Cells were incubated with exogenous biotin for 10 min upon IFITM3- (anti-HA) or BCR- (anti-IgM) engagement. IFITM3 Y20E interactome analyses identified interacting proteins by mass spectrometry, plotted based on significance and log2-fold enrichment over EV control. Essential interactors as BCR component (red), integrin (blue) and PI3K-signaling (gray) are highlighted. Data from three independent biological replicates. e , Proximity ligation assays were performed with Jeko1 MCL cells upon engagement of BCR. Jeko1 MCL cells were stimulated by BCR-engagement for 0, 3 and 30 minutes, then fixed, permeabilized and assessed for the proximity of CD79B to IFITM3. Representative microscopic images with PLA signal (red dot) and nuclei stained with DAPI as blue are shown. LAMP1 was used as a marker for endosomes to distinguish plasma membrane-bound from endosomal localization of CD79B:IFITM3 complexes. Scale bars, 5 mm. Data from three independent replicates.

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: Mutagenesis, Membrane, Incubation, Mass Spectrometry, Control, Ligation, Staining, Marker, Clinical Proteomics

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: a , Homotypic aggregation was studied in Ifitm3 −/− B-ALL cells that were reconstituted with C-terminally HA-tagged Ifitm3, Ifitm3 Y20E or empty vector (EV) and incubated with anti-CD19, anti-HA antibodies or isotype control for 24 hours. Data from three independent experiments. b , Ifitm3 +/+ or Ifitm3 −/− B-ALL cells were transduced with GFP-tagged IK6 or GFP alone. IK6 levels in flow-sorted GFP + cells assessed by Western blot analysis using β-actin as loading control ( right ). 10,000 Ifitm3 +/+ or Ifitm3 −/− B-ALL cells carrying IK6 or EV were plated for colony forming assays. Colonies were imaged and counted after 7 days. Representative images are shown with colony numbers, Data from three independent experiments and assessed by two-tailed t -test (means ± s.d.). For gel source data, see . c , 100,000 Ifitm3 +/+ or Ifitm3 −/− B-ALL cells carrying IK6 or EV were cultured on OP9 stroma cells. Ratios of adherent cells to nonadherent cells were calculated. Data are analyzed from three independent biological experiments (right) and assessed by two-tailed t -test (means ± s.d.). d , Representative images of adherent B-ALL cells on OP9 stroma are shown. Round and light-refracting cells are adherent B-ALL cells attached to stroma cells. Dark and round cells are adherent B-ALL cells incorporated into stromal layer. Data from three independent biological replicates. e , Surface expression levels of integrins on adherent B-ALL cells were measured by flow cytometry. MFI values are indicated for individual measurements. Data from three independent biological replicates.

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: Plasmid Preparation, Incubation, Control, Transduction, Western Blot, Two Tailed Test, Cell Culture, Expressing, Flow Cytometry

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: a , Ganglioside GM1 and cholesterol (stained with filipin III) levels measured in Ifitm3 +/+ and Ifitm3 −/− pre-B (top) and B-ALL (bottom) cells. b-c , Ratios of PIP3 and PIP2 were measured in Ifitm3 +/+ and Ifitm3 −/− murine ( b , left), and human B-ALL cells (PDX2; b , right), and upon expression of EV, IFITM3 or IFITM3 Y20E ( ; Y20E). d , In vitro lipid-binding assays for GST-IFITM3 and 15 lipid classes. Recombinant GST-tag used as baseline. , Lipid-binding assays were performed for biotin-IFITM3 N-terminus (1–57), first intramembrane α-helix (IM-α; 58–70), conserved intracellular loop (CIL; 71–105) and transmembrane α-helix (TM-α; 89–105). f , Lipid-binding assays to study interactions of PIP3 with biotinylated IFITM3-CIL fragments carrying mutations of basic residues. g-i , PIP3 binding to K83/K104 and R85/R87/K88 basic residue patches ( g ), dashed lines indicate PIP3 contacts within 3.5 Å. , Interaction energies of PIP3 with basic residues K83 or R85 averaged over MD simulation trajectories (median levels in red dotted line, see ). Binding affinity for PIP3 as shown by heatmap of contact frequencies between PIP3 and each residue ( i ). j-l , Levels of phospho-AKT-S 473 , AKT, phospho-S6K-T 389/421 S 424 , S6K, phospho-CD19-Y 531 , CD19, phospho-SRC-Y 416 , LYN, phospho-PAK1/3-S 199/204 , phospho-PAK2-S 192/197 , PAK1, phospho-FAK-Y 397/576/577 , FAK, phospho-CXCR4-S 339 , CXCR4, and Integrin-β1 in PDX2 expressing EV, IFITM3-HA, IFITM3 Y20E -HA, and R85A, K83A, R85A/R87A/K88A and K83A/K104A mutants upon IFITM3-crosslinking with anti-HA antibodies. a-f,j-l, n=3, For gel source data, see – , , , two-tailed t -test.

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: Staining, Expressing, In Vitro, Binding Assay, Recombinant, Residue, Two Tailed Test

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: a , Single-cell size-normalized acoustic scattering (SNACS) was measured using a previously established microfluidic method as a metric for cell surface stiffness. Jeko1 mantle cell lymphoma cells were flown through a standing acoustic wave generated inside a vibrating suspended microchannel resonator. The cantilever vibration frequency was monitored, and its shift was used to quantify the acoustic scattering from the cells as well as the buoyant mass of the cells. The data displayed was obtained using a 350 μm long cantilever with 15 × 20 μm sized channel inside of the cantilever and a ~200 ms transit time through the cantilever. All the regulators, valves and data acquisition were controlled by custom software coded in LabVIEW 2017 (National Instruments). A parallel volume measurement using Coulter Counter was carried out to quantify average cell volume, which was used together with the single-cell buoyant mass measurements to calculate SNACS for each cell. Jeko1 mantle cell lymphoma cells without ( IFITM3 +/+ ) and with ( IFITM3 −/− ) CRISPR/Cas9-mediated deletion of IFITM3 were kept under cell culture conditions and treated with an anti-IgM antibody. Fixation with paraformaldehyde (PFA) was used as s positive control. Shown are representative plots from 3 independent experiments (median levels in red dotted line). Statistical significance was determined by two-tailed t-test. Numbers indicated cells studied for SNACS measurement. b , Ifitm3 +/+ and Ifitm3 −/− B-ALL cells were incubated with 30 μM of the PIP3 carrier histone H1 or PIP3-histone H1 complex for 30 minutes. Levels of phospho-CD19-Y 531 , CD19, phospho-Src-Y 416 , Lyn, phospho-Akt-S 473 , Akt and Myc were measured by Western blot using β-actin as loading control. Data from three independent replicates. For gel source data, see . c , Colony formation assays were performed for Ifitm3 +/+ and Ifitm3 −/− B-ALL cells that were treated with 30 μM of PIP3-histone H1 or the shuttle protein histone H1 (H1) alone. Photomicrographs and colony numbers per 10,000 plated cells are shown. Data are presented as means ± standard derivation (s.d.) from three independent experiments. Statistical significance was determined by two-tailed t-test. d , Flow cytometry analyses of surface expression of CD19, CD44, CD25 and CD44 in Ifitm3 +/+ and Ifitm3 −/− B-ALL cells treated with 30 μM of PIP3-histone H1 or the shuttle protein histone H1 (H1) alone for 72 hours. Data from three independent replicates.

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: Generated, Software, CRISPR, Cell Culture, Positive Control, Two Tailed Test, Incubation, Western Blot, Control, Flow Cytometry, Expressing

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: The hierarchical scheme of MD simulations ( a-c ) to delineate the structural and dynamical basis of PIP2 and PIP3 binding to IFITM3 (yellow). a , Coarse grained simulations of IFITM3 in composite cell membrane. The side views of the lipid bilayer are shown. The lipid raft markers are shown in color with GM1 (pink), cholesterol (green) and sphingomyelin (blue). The gray surface represents all the other lipids. b , The simulation cell extracted from the coarse grained simulation as the starting structure for all-atom MD simulations. The IFITM3 protein is shown in yellow and the PIP2 and PIP3 are shown in green and red stick representations. c , Close up view of one of the predicted binding poses of PIP2 and PIP3 in the most populated conformation of IFITM3. The dashed lines shown are the PIP2 or PIP3 contacts with the basic residues in IFITM3 ( top ). The amino acid sequence of IFITM3 in the stretch of residues between TM1 and TM2 from 57 to 128 modeled in this work is shown. The conserved intracellular loop (CIL) region is boxed and basic amino acids are highlighted with blue. The average interaction energy of PIP2 or PIP3 with the two basic batches measured in all-atom MD simulations is indicated (see ). d, e , The average interaction energy of PIP2 (green) or PIP3 (red) with the IFITM3 (residues 57–128) was measured in all-atom MD simulations. The 1-residue contact indicates the conformation of PIP2 or PIP3 binding with only one basic residue in the basic amino patch KSRDRK of IFITM3. The 2-residue contact refer to the binding conformation of PIP2 or PIP3 that show contacts with two basic residues. Shown are representative plots from at least 4 independent experiments (mean ± s.d.). P -values were determined by two-tailed t -test ( d ). Populations showing one or two-residue contacts of PIP2 (green) or PIP3 (red) to IFITM3 were quantitated from all-atom MD simulations. The population density was assessed by the normalization of number of events with the total number of frames. Shown are representative plots from 3 independent experiments (mean ± s.d.). Statistical significance was determined by two-tailed t-test ( e ).

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: Binding Assay, Membrane, Sequencing, Residue, Two Tailed Test

Journal: Nature

Article Title: IFITM3 functions as PIP3-scaffold to amplify PI3K signaling in B-cells

doi: 10.1038/s41586-020-2884-6

Figure Lengend Snippet: a-c , Levels of differentially phosphorylated proteins in patient-derived B-ALL (PDX2) cells transduced with IFITM3 Y20E , K83A, R85A or empty vector (EV) control were identified by mass spectrometry (n=3). Relative abundance values are plotted for all sites ranked by fold-change as indicated, phosphosites of interest are highlighted. b , Feature set enrichment analysis (FSEA) ranked by log2 fold change are shown for phosphosites in PI3K signaling (gray), BCR-signaling (red) and integrin and adhesion receptor elements (blue). Statistical significance was determined by two-tailed Kolmogorov-Smirnov test. d, Cumulative distribution frequencies for log2-fold changes in phosphosite abundance between IFITM3 transduced and EV conditions were calculated for all sites and globally increased in IFITM3 Y20E over EV. The analysis was repeated for the K83A- (red) and R85A- (green) mutants of IFITM3 Y20E . The light gray line indicates background variance observed between EV replicates. Shifts that were caused by the K83A and R85A mutants are indicated by arrows. Overall changes in distribution (shifts) between IFITM3 Y20E and K83A and R85A mutants were measured by two-tailed Kolmogorov-Smirnov test. e , Schematic of BirA (engineered biotin ligase) was fused at N-terminal ends of HA-tagged IFITM3 Y20E or its K83A mutant and expressed in PDX2 B-ALL or Jeko1 MCL cells. e-g , Interactomes of IFITM3 Y20E and its K83A mutant were compared in PDX2 B-ALL cells upon IFITM3-engagement (anti-HA; e ) or in Jeko1 MCL cells upon IFITM3- (anti-HA; f ) or BCR- (anti-IgM; g ) engagement. Phosphosites of interest, including BCR-signaling, PI3K-signaling and integrins and adhesion receptor elements were highlighted.

Article Snippet: Recombinant human IFITM3 fragments listed in were synthesized at LifeTein, South Plainfield, NJ.

Techniques: Derivative Assay, Transduction, Plasmid Preparation, Control, Mass Spectrometry, Two Tailed Test, Phospho-proteomics, Mutagenesis

Journal: Frontiers in Oncology

Article Title: IONPs combined with cytarabine downregulated IFITM3 expression to inhibit acute myeloid leukemia

doi: 10.3389/fonc.2025.1515956

Figure Lengend Snippet: Analyzing the knockdown of IFITM3 efficiency in LV-shIFITM3 KG-1a cells. (A) The efficiency of lentivirus infection of the KG-1a cells was observed under a conventional fluorescent microscope. (B) The IFITM3 mRNA expression in the KG-1a cells was detected by qRT-PCR. (C) The expression of the IFITM3 protein in the KG-1a cells was detected by Western blot. (D) The results from the semi-quantitative analysis of protein expression levels. ****P<0.00001. ns, no statistical significance.

Article Snippet: The membrane was then incubated with the rabbit anti-human GAPDH monoclonal antibody and the rabbit anti-human IFITM3 polyclonal antibody (Wuhan Sanying Biotechnology Co. LTD), respectively, for overnight at 4°C.

Techniques: Knockdown, Infection, Microscopy, Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Oncology

Article Title: IONPs combined with cytarabine downregulated IFITM3 expression to inhibit acute myeloid leukemia

doi: 10.3389/fonc.2025.1515956

Figure Lengend Snippet: Down-regulation of IFITM3 changed the biological properties of the KG-1a cells. (A) The effect of down-regulated IFITM3 on the proliferation ability of KG-1a cells as detected by CCK8 assay. (B) The colony formations of various KG-1a cells detected by soft agar cloning assay (50×); (C) Comparisons of the colony formations; (D) The FCM analysis results of the KG-1a cell cycle changes; (E) Comparisons of the cell cycles; (F) The effects of Ara-C (0.4μM) on the apoptosis of various KG-1a cells analyzed by FCM (24h). (G) The FCM results; (H) The mRNA expression level of caspase 3 in various KG-1a cells as detected by qRT-PCR. *P < 0.05, ***P < 0.001. ns, no statistical significance.

Article Snippet: The membrane was then incubated with the rabbit anti-human GAPDH monoclonal antibody and the rabbit anti-human IFITM3 polyclonal antibody (Wuhan Sanying Biotechnology Co. LTD), respectively, for overnight at 4°C.

Techniques: CCK-8 Assay, Cloning, Expressing, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: IONPs combined with cytarabine downregulated IFITM3 expression to inhibit acute myeloid leukemia

doi: 10.3389/fonc.2025.1515956

Figure Lengend Snippet: The IFITM3 expression and the proliferation of the KG-1a cells. (A) The IFITM3 expression as detected by Western blot after the KG-1a cells were treated with PBS, PBNPs and IONPs, respectively, for 72h; (B) The semi-quantitative analysis of the Western blot results; (C) The IFITM3 expression in the KG-1a cells as detected by Western blot after the cells were treated with PBS, PBNPs and IONPs, respectively, for 72h; (D) The proliferation of the KG-1a cells as detected by the CCK8 assay in the KG-1a cells that were treated with different drugs for 96h; (E) The qRT-PCR-detected expression level of c-myc in the KG-1a cells treated with different drugs for 24h. *P < 0.05, **P < 0.01, ***P < 0.001. ns, no statistical significance.

Article Snippet: The membrane was then incubated with the rabbit anti-human GAPDH monoclonal antibody and the rabbit anti-human IFITM3 polyclonal antibody (Wuhan Sanying Biotechnology Co. LTD), respectively, for overnight at 4°C.

Techniques: Expressing, Western Blot, CCK-8 Assay, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: IONPs combined with cytarabine downregulated IFITM3 expression to inhibit acute myeloid leukemia

doi: 10.3389/fonc.2025.1515956

Figure Lengend Snippet: Impact of IFITM3 knockdown on disease progression in AML model mice. (A) Leukemia cells in peripheral blood and bone marrow; (B) With Reichsen-Giemsa staining; (C) Leukemia cells of bone marrow (left: under a light microscope; right: under a fluorescence microscope) in the AML-bearing mice injected with the Scrambled or the Lv-shIFITM3-KG1a cells; (D) The expression levels of CD33, CD123 and CD11b detected by FCM. (E) Statistical results of the CD33, CD123 and CD11b expression levels. The experiment was repeated twice. *P < 0.05, ***P < 0.001.

Article Snippet: The membrane was then incubated with the rabbit anti-human GAPDH monoclonal antibody and the rabbit anti-human IFITM3 polyclonal antibody (Wuhan Sanying Biotechnology Co. LTD), respectively, for overnight at 4°C.

Techniques: Knockdown, Biomarker Discovery, Staining, Light Microscopy, Fluorescence, Microscopy, Injection, Expressing