Journal: Nature Immunology

Article Title: Human T H 17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation

doi: 10.1038/s41590-022-01386-w

Figure Lengend Snippet: a , b , Imaging flow cytometry with T H 17 cells on day 5 after stimulation with plate-bound anti-CD3 and anti-CD28 monoclonal antibodies and macrophages after 24 h of stimulation with LPS and ATP for the last 30 min. a , Representative experiment. BF, bright-field. b , Cumulative data with n = 3 biological samples, presented as mean ± s.e.m. Left, P values calculated using one-way ANOVA with Tukey’s multiple-comparison test. Right, P values calculated using two-tailed, paired Student’s t -test. c , RT–qPCR analysis of T H 17 cells stimulated as in a and restimulated with PMA and ionomycin for 3 h. Data represent three independent experiments with n = 9 biological replicates (two-tailed, paired Student’s t -test). d , ELISA of cell culture supernatants after stimulation of T H 17 cells for 5 d with anti-CD3 and anti-CD28 monoclonal antibodies. Data represent three experiments with n = 7 biological replicates (two-tailed, paired Student’s t -test). e , Immunoblot analysis of cell lysates from T H 17 cells after 5 d of stimulation with anti-CD3 and anti-CD28 monoclonal antibodies and of monocyte lysates after stimulation with LPS for 24 h and nigericin (Nig.) for the last 30 min. The conditions from the same blot after removal of irrelevant conditions or replicates are shown. f , ELISA of cell culture supernatants from anti-CD3- and anti-CD28-activated T H 17 cells (5 d) and LPS (24 h)- and nigericin (30 min)-stimulated monocytes ( n = 3 biological samples presented as mean ± s.e.m.; one-way ANOVA with Tukey’s multiple-comparison test). g , ELISA of cell culture supernatants from anti-CD3- and anti-CD28-activated T H 17 cells after depletion of CASP1 by CRISPR–Cas9 gene editing. Data represent five independent experiments. P values were calculated using two-tailed, paired Student’s t -test. Each circle indicates an independent blood donor.

Article Snippet: The transcripts were quantified by RT–qPCR with predesigned TaqMan Gene Expression Assays ( IL1A , catalog no. HS00174092-m1; IL1B , catalog no. Hs01555410_m1; NLRP3 , catalog no. Hs00918082_m1; CASP1 , catalog no. Hs00354836_m1, CAPN2 , catalog no. Hs00965097_m1; GSDMD , catalog no. Hs00986739_g1; DFNA5 , catalog no. Hs00903185_m1; and 18 S, catalog no. Hs03928990_g1) and reagents (Applied Biosystems).

Techniques: Imaging, Flow Cytometry, Bioprocessing, Comparison, Two Tailed Test, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, CRISPR

Journal: Nature Immunology

Article Title: Human T H 17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation

doi: 10.1038/s41590-022-01386-w

Figure Lengend Snippet: a , Th17 cells were isolated ex vivo by FACS-sorting and stimulated for 5 days with anti-CD3 and anti-CD28 mAbs (48 h plate-bound) in the presence or absence of the indicated cytokines before flow cytometric analysis of intracellular IL-1α and active caspase-1 after PMA and ionomycin restimulation. The results of one representative experiment are shown. b , Cumulative data for active caspase-1 as shown in (a). One-way ANOVA. n = 3 biologically independent samples examined over 3 independent experiments. Data are presented as mean ± SEM. c , Intracellular staining and flow cytometry of monocytes following 24 h stimulation with LPS and 30 min with nigericin (Nig.). The results shown are from one representative experiment. d, e , cumulative data of (d), Two-sided paired t-test. Each circle indicates an individual healthy blood donor (n = 3-4 biologically independent samples examined over 3 independent experiment). Data are presented as mean ± SEM. f , ELISA of Th17 cells, which were stimulated as in (a) and of monocytes were stimulated with LPS (24 h) and ATP (30 min). n = 3 biologically independent samples examined over 2 independent experiments. Data are presented as mean ± SEM. g , Flow cytometric analysis of cells as in (g). h , Intracellular cytokine staining and flow cytometric analysis of T-cell clones generated from the Th17-cell subset. Each circle indicates an individual T cell clone (n = 64). Data are presented as mean ± SEM. i , scRNA-seq and UMAP showing IL-1β expression in human Th17 cells stimulated for 5 days with anti-CD3 and anti-CD28 mAbs.

Article Snippet: The transcripts were quantified by RT–qPCR with predesigned TaqMan Gene Expression Assays ( IL1A , catalog no. HS00174092-m1; IL1B , catalog no. Hs01555410_m1; NLRP3 , catalog no. Hs00918082_m1; CASP1 , catalog no. Hs00354836_m1, CAPN2 , catalog no. Hs00965097_m1; GSDMD , catalog no. Hs00986739_g1; DFNA5 , catalog no. Hs00903185_m1; and 18 S, catalog no. Hs03928990_g1) and reagents (Applied Biosystems).

Techniques: Isolation, Ex Vivo, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Clone Assay, Generated, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: The Regulation of Trophoblastic p53 Homeostasis by the p38-Wip1 Feedback Loop is Disturbed in Placentas from Pregnancies Complicated by Preeclampsia.

doi: 10.33594/000000023

Figure Lengend Snippet: Fig. 2. Wip1 activates p38 and dephosphorylates p53 in trophoblasts under conditions of hypoxia. A: Lentiviral transfection efficiency was examined by FCM and IF. Scale bar 400 µm; B: Wip1 protein expression was confirmed by western blotting, n=4, *P<0.05 vs. scrambled shRNA, one-way ANOVA, Dunnett-t multiple comparison test; C-D. Representative Western blots of p-p38Thr180/Tyr182, p38, cl-cas9, Mdm2, p-p53Ser15, and p53 in HTR8/SVneo cells after 24 h of HII (A) or SIB (B) in the presence of 20 µM GSK2830371 (GSK), DMSO (0.1%), shWip1, or scrambled shRNA (MOI=50). n=4, (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one- way ANOVA, Sidak’s multiple comparisons test. Experiments were performed in triplicate.

Article Snippet: KG Tan et al.: p38-Wip1 Loop Regulates p53 in Trophoblast p38 (1:1000), and cleaved caspase-9 (cl-cas9, 1:1000) were purchased from Cell Signaling Technology (Danvers, USA); p53 (1:2000) and β-actin (1:1000) were purchased from Proteintech (Rosemont, USA); Wip1 (1:1000) and Mdm2 (1:1000) were purchased from Abcam (Cambridge, UK) and Invitrogen (Waltham, USA), respectively.

Techniques: Transfection, Expressing, Western Blot, shRNA, Comparison

Journal: Cell reports

Article Title: Lidocaine induces apoptosis in head and neck squamous cell carcinoma through activation of bitter taste receptor T2R14

doi: 10.1016/j.celrep.2023.113437

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-caspase-7 (D2Q3L) , Cell Signaling , Cat# 9492; RRID: AB_2228313.

Techniques: Virus, Recombinant, Reverse Transcription, DC Protein Assay, Western Blot, CRISPR, Control, Expressing, TaqMan Assay, shRNA, Software

Journal: The Journal of Clinical Investigation

Article Title: Renal immune surveillance and dipeptidase-1 contribute to contrast-induced acute kidney injury

doi: 10.1172/JCI96640

Figure Lengend Snippet: (A) Immunoblot probing for caspase-1 (Casp1) (pro- and cleaved, p11 CARD domain) and cleaved IL-1β (p17) in extracts (ext) and supernatants (sup) of THP-1 cells untreated (NT) or treated with ioversol (1:1 to 1:8 dilution) at 6 hours. Nigericin (NGC) was used as positive control. Image is representative of 3 independent experiments. (B) THP-1 cells were treated with DTA or nigericin (NGC). IL-1β production was measured using ELISA at 6 hours (n = 3/group). (C) Confocal microscopy for ASC or activated caspase-1 in ioversol-treated THP-1 cells at 6 hours. Arrowheads indicate ASC- and caspase-1–positive specks. Image is representative of 3 independent experiments. Scale bars: 10 μm. (D) IL-1β ELISA in Nlrp3+/+ and CRISPR-deleted Nlrp3–/– THP-1 cells treated with ioversol or the positive control ATP (Nlrp3+/+ vs. Nlrp3–/–, ATP: **P = 0.01, ioversol: **P = 0.01, n = 3/group, ANOVA). (E) IL-1β ELISA from Nlrp3+/+ and Nlrp3–/– mouse BMDMs treated with ioversol at 6 hours (Nlrp3+/+ vs. Nlrp3–/–, IVRS: **P = 0.01, n = 3/group, 2-tailed Student’s t test). (F) IL-1β ELISA in kidneys of Nlrp3+/+ and Nlrp3–/– mice treated with ioversol over 3 days (Nlrp3+/+ vs. Nlrp3–/–, 6 hours: **P = 0.006, n = 3–5/group, ANOVA). (G) Nlrp3+/+ and Nlrp3–/– mice were depleted using irradiation and clodronate-liposomes before bone marrow (BM) transplant (Nlrp3+/+ BM to Nlrp3–/–, Nlrp3–/– BM to Nlrp3+/+). BM chimeras were then volume depleted and treated with ioversol for 6 hours, and IL-1β ELISA was performed from kidney lysates (*P = 0.03, n = 4/group, 2-tailed Student’s t test). (H) DAMPs were generated from HPTCs using ioversol and used with or without ioversol to activate the inflammasome in THP-1 cells as measured by IL-1β ELISA. NT vs. ioversol+HPTC DAMPs, ***P = 0.0001, n = 3/group; HPTC DAMPs vs. ioversol+HPTC DAMPs, ***P = 0.0008, n = 3/group, ANOVA.

Article Snippet: Antibodies used were as follows: caspase-8 (clone 1C12, Cell Signaling Technology), caspase-3 (Cell Signaling Technology, catalog 9662), PARP (Cell Signaling Technology, catalog 9542), MLKL (clone D6H3V, Cell Signaling Technology), RIPK3 (clone E1Z1D, Cell Signaling Technology), caspase-1 (Santa Cruz Biotechnology Inc., catalog SC-622 targeting N-terminal CARD domain), Nlrp3 (clone CRYO-2, Adipogen), ASC (Adipogen, catalog AL177), IL-1β (pro-IL1β: clone 3A6; cleaved-IL-1β: clone D3A3Z, Cell Signaling Technology), GAPDH (Abcam, catalog ab9485), and β-tubulin (Sigma-Aldrich, catalog T0198).

Techniques: Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, CRISPR, Irradiation, Generated

Journal: The Journal of Clinical Investigation

Article Title: Renal immune surveillance and dipeptidase-1 contribute to contrast-induced acute kidney injury

doi: 10.1172/JCI96640

Figure Lengend Snippet: LysM(gfp/gfp) and Nlrp3–/– LysM(gfp/gfp) mice were treated with vehicle or ioversol and assessed at various time points. (A) LysM-GFP+CD11b+ leukocytes were isolated from vehicle- or ioversol-treated LysM(gfp/gfp) mice at 6 hours and analyzed for inflammasome activation (active caspase-1) in infiltrating monocytes and resident macrophages by flow cytometry using CX3CR1, Ly6C, F4/80, and CCR2. Images are representative of 3 independent experiments. (B) LysM-GFP+CD11b+CX3CR1+Ly6C+ monocytes/macrophages were isolated from LysM(gfp/gfp) and Nlrp3–/– LysM(gfp/gfp) mice treated with vehicle or ioversol and analyzed at 6 hours or 3 days using CCR2 and F4/80. Images are representative of 3 independent experiments.

Article Snippet: Antibodies used were as follows: caspase-8 (clone 1C12, Cell Signaling Technology), caspase-3 (Cell Signaling Technology, catalog 9662), PARP (Cell Signaling Technology, catalog 9542), MLKL (clone D6H3V, Cell Signaling Technology), RIPK3 (clone E1Z1D, Cell Signaling Technology), caspase-1 (Santa Cruz Biotechnology Inc., catalog SC-622 targeting N-terminal CARD domain), Nlrp3 (clone CRYO-2, Adipogen), ASC (Adipogen, catalog AL177), IL-1β (pro-IL1β: clone 3A6; cleaved-IL-1β: clone D3A3Z, Cell Signaling Technology), GAPDH (Abcam, catalog ab9485), and β-tubulin (Sigma-Aldrich, catalog T0198).

Techniques: Isolation, Activation Assay, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Renal immune surveillance and dipeptidase-1 contribute to contrast-induced acute kidney injury

doi: 10.1172/JCI96640

Figure Lengend Snippet: (A) Urinary IL-18 and (B) KIM-1 before and less than 24 hours after coronary angiogram as determined by Meso Scale Discovery assay. Data points normalized to urine creatinine are shown (IL-18 before vs. after, P = 0.005, n = 42/group; KIM-1 before vs. after, P = 0.03, n = 42/group, Wilcoxon’s signed-rank test). (C) Caspase-1 immunoblotting from urine pellets in 2 healthy control individuals and 8 patients before and less than 24 hours after coronary angiogram. Nigericin-treated THP-1 macrophages were used as a positive control. Arrow indicates cleaved capase-1 N-terminal CARD domain. (D) Caspase-1 immunoblotting from urine pellets in 9 patients before and less than 24 hours after coronary angiogram, with corresponding urine leukocyte score. (E) Quantification of caspase-1 expression versus leukocyte score in 17 patient urine samples before and less than 24 hours after coronary angiogram (caspase-1 densitometry normalized to urine creatinine per urine leukocyte score, 0 vs. 1: *P = 0.011, 0 vs. 2: ***P = 0.0001, n = 21, 7, and 6/group, ANOVA).

Article Snippet: Antibodies used were as follows: caspase-8 (clone 1C12, Cell Signaling Technology), caspase-3 (Cell Signaling Technology, catalog 9662), PARP (Cell Signaling Technology, catalog 9542), MLKL (clone D6H3V, Cell Signaling Technology), RIPK3 (clone E1Z1D, Cell Signaling Technology), caspase-1 (Santa Cruz Biotechnology Inc., catalog SC-622 targeting N-terminal CARD domain), Nlrp3 (clone CRYO-2, Adipogen), ASC (Adipogen, catalog AL177), IL-1β (pro-IL1β: clone 3A6; cleaved-IL-1β: clone D3A3Z, Cell Signaling Technology), GAPDH (Abcam, catalog ab9485), and β-tubulin (Sigma-Aldrich, catalog T0198).

Techniques: Western Blot, Positive Control, Expressing

Journal: The Journal of Cell Biology

Article Title: The microRNA-200 family coordinately regulates cell adhesion and proliferation in hair morphogenesis

doi: 10.1083/jcb.201708173

Figure Lengend Snippet: miR-200 overexpression represses proliferation and migration and affects FA and AJ formation. (A) miR-200 targets involved with cell cycle. (B and C) Colony formation assay of Tg keratinocytes untreated (Unt) or treated with doxycycline (Dox). n = 6. (D) miR-200 targets involved with FA and actin cytoskeleton. (E) Scratch assay on Tg keratinocytes untreated or treated with doxycycline 0 or 8 h after scratching. (F) Number of cells migrated in E. n = 3. (G and H) Immunofluorescence and quantification of vinculin and phalloidin stain for actin on Tg keratinocytes untreated or treated with doxycycline. n = 76 untreated; n = 77 doxycycline treated. (I) Immunofluorescence of E-cadherin (Ecad) and phalloidin staining for actin on Tg keratinocytes untreated or treated with doxycycline and induced with calcium for 3 or 7 h. Arrowheads indicate actin localization to E-cadherin. Red-green-blue (RGB) line scans over 90 pixels across each cell junction. Representative images from n > 20 cell junctions imaged for each treatment. (J) Percentage of peaks per junction shared between E-cadherin and actin or exclusive to E-cadherin or actin treated as in I. 3-h untreated, n = 21; 3-h doxycycline, n = 23; 7-h untreated, n = 19; 7-h doxycycline, n = 26. *, P < 0.05; ***, P < 0.001; Student’s two-tailed t test; error bars are SD. Bars: (E) 100 µm; (G and I) 20 µm.

Article Snippet: After being filtered through a 40-μm filter, cells were spun down for 10 min at 300 g and resuspended in 1× PBS with 3% chelexed FBS on ice for 1 h with the following antibodies: p-cadherin (1:100; FAB761P; R&D Systems) and α6-integrin (1:75; clone NKI-GoH3; MCA699A647; AbD Serotec).

Techniques: Over Expression, Migration, Colony Assay, Wound Healing Assay, Immunofluorescence, Staining, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: The microRNA-200 family coordinately regulates cell adhesion and proliferation in hair morphogenesis

doi: 10.1083/jcb.201708173

Figure Lengend Snippet: Multiple miR-200 targets are involved with FA and AJ formation. (A) Immunofluorescence of vinculin and phalloidin stain for actin on WT keratinocytes infected with shScr , shCfl2 , shEgfr , shRock2 , shFak , or a combination of all shRNAs. (B) Quantification of FAs per cell in A. n = 34 Scr, n = 19 shCfl2, n = 26 shEgfr, n = 35 shFak, n = 20 shRock2, and n = 24 combined. (C) Immunofluorescence of E-cadherin and phalloidin stain for actin on WT keratinocytes infected with shScr , shCfl2 , shEgfr , shRock2 , shFak , or a combination of all shRNAs and induced with calcium for 3 or 7 h. *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s two-tailed t test. Bars, 20 µm.

Article Snippet: After being filtered through a 40-μm filter, cells were spun down for 10 min at 300 g and resuspended in 1× PBS with 3% chelexed FBS on ice for 1 h with the following antibodies: p-cadherin (1:100; FAB761P; R&D Systems) and α6-integrin (1:75; clone NKI-GoH3; MCA699A647; AbD Serotec).

Techniques: Immunofluorescence, Staining, Infection, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: The microRNA-200 family coordinately regulates cell adhesion and proliferation in hair morphogenesis

doi: 10.1083/jcb.201708173

Figure Lengend Snippet: Ablation of all five miR-200 members enhances proliferation, cell migration, and cell adhesion and also affects FA and AJ formation. (A) Schematic of sequence targeted and guide RNAs used to delete miR-200b cluster. Arrows indicate predicted CRISPR cleavage sites. These sites were subsequently validated in Fig. S4 D. (B) Quantitative RT-PCR from whole epidermis of control and dKO animals at P0.5. n = 3. (C) Quantitative RT-PCR for miR-200 targets in control and dKO keratinocytes. n = 3. (D) Colony formation assay on control and dKO keratinocytes. (E and F) Number (E) and size (F) of colonies formed by control and dKO keratinocytes. n = 3. (G) Immunofluorescence for EdU and Krt5 in back skin of control and dKO animals at P0.5. (H) Percentage of EdU + /K5 + cells in control and dKO epidermis. n = 3. (I) Crystal violet absorbance of control and dKO keratinocytes plated in cell adhesion assay. n = 3. (J) Scratch assays on control and dKO keratinocytes 0 or 8 h after scratching. (K) Number of migrated cells from J. n = 3. (L) Immunofluorescence of vinculin and phalloidin stain for actin on control and dKO keratinocytes. Arrowheads indicate examples of FAs. (M) Number of FAs per cell in L. n = 43 control cells; n = 40 dKO cells. (N) Immunofluorescence of E-cadherin and phalloidin staining for actin on control and dKO keratinocytes induced with calcium for 3 or 7 h. Arrowheads indicate E-cadherin localization to cell cortex. RGB line scans over 90 pixels across each cell junction. Representative images from n > 20 cell junctions imaged for each treatment. Bars: (G) 100 µm; (L and N) 20 µm. (O) Mean fluorescence intensity of E-cadherin across cell junction. Distance of 0 and dashed line indicate center of junction. 3-h K14cre, n = 22; 3-h dKO, n = 22; 7-h K14cre, n = 21; 7-h dKO, n = 29; normalized to maximum intensity for each condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s two-tailed t test; error bars are SD.

Article Snippet: After being filtered through a 40-μm filter, cells were spun down for 10 min at 300 g and resuspended in 1× PBS with 3% chelexed FBS on ice for 1 h with the following antibodies: p-cadherin (1:100; FAB761P; R&D Systems) and α6-integrin (1:75; clone NKI-GoH3; MCA699A647; AbD Serotec).

Techniques: Migration, Sequencing, CRISPR, Quantitative RT-PCR, Control, Colony Assay, Immunofluorescence, Cell Adhesion Assay, Staining, Fluorescence, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: The microRNA-200 family coordinately regulates cell adhesion and proliferation in hair morphogenesis

doi: 10.1083/jcb.201708173

Figure Lengend Snippet: Simultaneous KD of multiple miR-200 targets in dKO cells rescues FA and cell junction phenotypes. (A) Immunofluorescence of vinculin and phalloidin stain for actin on dKO keratinocytes infected with Scr, Rock2 , or a combination of Rock2 , Cfl2 , and Fak shRNAs. (B) Number of FAs per cell in A. n = 42 Scr; n = 25 shRock2; n = 42 combined. (C) Immunofluorescence of E-cadherin and phalloidin staining for actin on dKO keratinocytes infected with Scr, shRock2, or a combination of Cfl2 , Rock2 , and Fak shRNAs and induced with calcium for 3 or 7 h; line scans over 90 pixels across each cell junction; representative images from n > 20 cell junctions imaged for each treatment. (D) Mean fluorescence intensity of E-cadherin across cell junction. Distance of 0 and dashed line indicate center of junction. 3-h Scr, n = 10; 3-h combined, n = 28; 7-h Scr, n = 18; 7-h combined, n = 38; normalized to maximum intensity for each condition. *, P < 0.05; Student’s two-tailed t test. Bars, 20 µm.

Article Snippet: After being filtered through a 40-μm filter, cells were spun down for 10 min at 300 g and resuspended in 1× PBS with 3% chelexed FBS on ice for 1 h with the following antibodies: p-cadherin (1:100; FAB761P; R&D Systems) and α6-integrin (1:75; clone NKI-GoH3; MCA699A647; AbD Serotec).

Techniques: Immunofluorescence, Staining, Infection, Fluorescence, Two Tailed Test

Journal: eLife

Article Title: Diverse viral proteases activate the NLRP1 inflammasome

doi: 10.7554/eLife.60609

Figure Lengend Snippet: ( A ) Immunoblot depicting human NLRP1 activation (maturation of IL-1β) by CVB3 3C pro and TEV protease. HEK293T cells were co-transfected using 100 ng of the indicated protease, 50 ng V5-IL-1β, 100 ng CASP1, 5 ng ASC, and 4 ng of the indicated Myc-tagged NLRP1, and immunoblotted with the indicated antibodies. Appearance of the mature p17 band of IL-1β indicates successful assembly of the NLRP1 inflammasome and activation of CASP1. ( B ) Bioactive IL-1β in the culture supernatant was measured using HEK-Blue IL-1β reporter cells, which express secreted embryonic alkaline phosphatase (SEAP) in response to extracellular IL-1β. Supernatant from cells transfected as in ( A ) was added to HEK-Blue IL-1β reporter cells and SEAP levels in the culture supernatant from HEK-Blue IL-1β reporter cells were quantified by the QUANTI-Blue colorimetric substrate. Transfections were performed in triplicate and compared to the standard curve generated from concurrent treatment of HEK-Blue IL-1β reporter cells with purified human IL-1β . Data were analyzed using two-way ANOVA with Sidak’s post-test. **** = p<0.0001. ( C ) CVB3 infection of inflammasome-reconstituted HEK293T cells results in IL-1β release when NLRP1 can be cleaved by 3C pro . Cells were transfected with the indicated NLRP1 construct and other NLRP1 inflammasome components as in ( B ). Sixteen hours post-transfection, cells were mock infected or infected with 250,000 PFU (MOI = ~1) CVB3. Eight hours post-infection, culture supernatant was collected and bioactive IL-1β was measured as in ( B ). ( D ) CVB3 infection of an immortalized human keratinocyte cell line, HaCaT, activates the NLRP1 inflammasome. WT or knockout HaCaT cell lines were mock infected or infected with 100,000 PFU (MOI = ~0.4) CVB3. Forty-eight hours post-infection, culture supernatant was collected and bioactive IL-1β was measured as in ( B ). Figure 3—source data 1. Individual data values for .

Article Snippet: To establish NLRP1 and CASP1 knockouts in human immortalized keratinocyte HaCaT cells, lentivirus-like particles were made by transfecting HEK293T cells with the plasmids psPAX2 (gift from Didier Trono, Addgene plasmid # 12260) and pMD2.G (gift from Didier Trono, Addgene plasmid # 12259) and either the pLB-Cas9 (gift from Feng Zhang, Addgene plasmid # 52962) ( ) or a plentiGuide-Puro, which was adapted for ligation-independent cloning (kindly gifted by Moritz Gaidt) ( ).

Techniques: Western Blot, Activation Assay, Transfection, Generated, Purification, Infection, Construct, Knock-Out

Journal: eLife

Article Title: Diverse viral proteases activate the NLRP1 inflammasome

doi: 10.7554/eLife.60609

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with either WT NLRP1 or a FIIND auto-processed defective mutant (S1213A) along with other components of the NLRP1 inflammasome (CASP1, ASC, and IL-1β) as in . Only cells transfected with WT NLRP1 can produce mature IL-1β upon co-transfection with CVB3 3C pro or TEV protease as indicated by the appearance of the p17 band. ( B ) HEK293T cells were transfected as in ( A ), and then treated with the indicated inhibitors of proteasomal-mediated degradation (0.5 µM MG132) or the N-glycine degron pathway (1.0 µM MLN4924) for 6 hr prior to harvest. Addition of 10 µM VbP, an inhibitor of the NLRP1 inhibitors DPP8/9 , was used as a control for protease-independent activation of the inflammasome and is thus unaffected by MLN4924.

Article Snippet: To establish NLRP1 and CASP1 knockouts in human immortalized keratinocyte HaCaT cells, lentivirus-like particles were made by transfecting HEK293T cells with the plasmids psPAX2 (gift from Didier Trono, Addgene plasmid # 12260) and pMD2.G (gift from Didier Trono, Addgene plasmid # 12259) and either the pLB-Cas9 (gift from Feng Zhang, Addgene plasmid # 52962) ( ) or a plentiGuide-Puro, which was adapted for ligation-independent cloning (kindly gifted by Moritz Gaidt) ( ).

Techniques: Transfection, Mutagenesis, Cotransfection, Control, Activation Assay

Journal: eLife

Article Title: Diverse viral proteases activate the NLRP1 inflammasome

doi: 10.7554/eLife.60609

Figure Lengend Snippet: ( A ) The WT NLRP1 (exon 5 or exon 2) sequence is shown with CRISPR-targeting sgRNA and PAM sequences indicated. Below each WT sequence is shown the Sanger sequencing chromatogram and associated mutant sequence for each indicated clone. ( B ) Same as panel A, except indicating WT CASP1 intron 1/exon two sequence and sequencing data from two independently isolated knockout clones.

Article Snippet: To establish NLRP1 and CASP1 knockouts in human immortalized keratinocyte HaCaT cells, lentivirus-like particles were made by transfecting HEK293T cells with the plasmids psPAX2 (gift from Didier Trono, Addgene plasmid # 12260) and pMD2.G (gift from Didier Trono, Addgene plasmid # 12259) and either the pLB-Cas9 (gift from Feng Zhang, Addgene plasmid # 52962) ( ) or a plentiGuide-Puro, which was adapted for ligation-independent cloning (kindly gifted by Moritz Gaidt) ( ).

Techniques: Sequencing, CRISPR, Mutagenesis, Isolation, Knock-Out, Clone Assay

Journal: eLife

Article Title: Diverse viral proteases activate the NLRP1 inflammasome

doi: 10.7554/eLife.60609

Figure Lengend Snippet: ( A ) Immunoblot depicting CVB3 3C pro cleavage susceptibility of two alleles (129 and B6) of mouse NLRP1B. Assays were performed as in . ( B ) Immunoblot depicting mouse NLRP1B activation (maturation of IL-1β) by enterovirus 3C pro and TEV protease. HEK293T cells were co-transfected using 100 ng of the indicated protease, 50 ng mouse IL-1β, 50 ng mouse CASP1, and either 4 ng of 129 NLRP1B or 2.5 ng of B6 NLRP1B constructs, and immunoblotted with the indicated antibodies. Appearance of the mature p17 band of IL-1β indicates successful assembly of the NLRP1B inflammasome and activation of CASP1.

Article Snippet: To establish NLRP1 and CASP1 knockouts in human immortalized keratinocyte HaCaT cells, lentivirus-like particles were made by transfecting HEK293T cells with the plasmids psPAX2 (gift from Didier Trono, Addgene plasmid # 12260) and pMD2.G (gift from Didier Trono, Addgene plasmid # 12259) and either the pLB-Cas9 (gift from Feng Zhang, Addgene plasmid # 52962) ( ) or a plentiGuide-Puro, which was adapted for ligation-independent cloning (kindly gifted by Moritz Gaidt) ( ).

Techniques: Western Blot, Activation Assay, Transfection, Construct

Journal: eLife

Article Title: Diverse viral proteases activate the NLRP1 inflammasome

doi: 10.7554/eLife.60609

Figure Lengend Snippet:

Article Snippet: To establish NLRP1 and CASP1 knockouts in human immortalized keratinocyte HaCaT cells, lentivirus-like particles were made by transfecting HEK293T cells with the plasmids psPAX2 (gift from Didier Trono, Addgene plasmid # 12260) and pMD2.G (gift from Didier Trono, Addgene plasmid # 12259) and either the pLB-Cas9 (gift from Feng Zhang, Addgene plasmid # 52962) ( ) or a plentiGuide-Puro, which was adapted for ligation-independent cloning (kindly gifted by Moritz Gaidt) ( ).

Techniques: Cell Culture, Recombinant, Plasmid Preparation, Sequencing, Software

Journal: bioRxiv

Article Title: Direct cleavage of human NLRP1 by enteroviral 3C protease triggers inflammasome activation in airway epithelium

doi: 10.1101/2020.10.14.325076

Figure Lengend Snippet: A. Domain structures of human NLRP1 and rodent Nlrp1 homologues. B. Percentage of 293T-ASC-GFP cells with ASC-GFP specks after over-expression of vector control or NLRP1 with various viral proteases. Cells were fixed 24 hours after co-transfection of the indicated plasmids and >100 cells were scored for ASC-GFP speck formation. n=3 biological replicates. ****: p<0.0001 (two-way ANOVA). N.s: p>0.5 (two-way ANOVA). C. Representative images of ASC-GFP speck formation in NLRP1-expressing 293T-ASC-GFP cells transfected with the indicated viral proteases. D. NLRP1 oligomerization assayed by Blue-Native PAGE. 293T cells were transfected with NLRP1-HA and 3Cpros. Cells were lysed 48 hours after transfection. 20 μg of total lysate was used for Blue-Native PAGE followed by Western blot. E. HRV14-3Cpro induces mature IL-1B secretion and ASC oligomerization in immortalized human keratinocytes. N/TERT keratinocytes and conditioned media were harvested 48 hours after transfection. Conditioned media were concentrated 10 times before SDS-PAGE and IL-1B Western blotting. Endogenous ASC oligomers were extracted by 1% SDS after covalent crosslinking of the detergent (1% NP40)-insoluble pellets with 1% DSS. F. Live-cell imaging of ASC-GFP speck formation and lytic cell death in 3Cpro-transfected immortalized N/TERT keratinocytes. Yellow arrows, ASC-GFP specks. G. CRISPR/Cas9-deletion of NLRP1, ASC and CASP1 abrogates 3Cpro-induced IL-1B secretion in immortalized human keratinocytes. Cas9-control, NLRP1, ASC and CASP1 knockout keratinocytes were transfected with the indicated 3Cpro plasmids in parallel. Conditioned media were harvested 48 hours post transfection and analyzed by ELISA. **: p<0.005 (Two-way ANOVA), n=3 biological replicates.

Article Snippet: The following antibodies were used in this study: c-Myc (Santa Cruz Biotechnology #sc-40), HA tag (Santa Cruz Biotechnology, #sc-805), GAPDH (Santa Cruz Biotechnology, #sc-47724), ASC (Apipogen, #AL-177), CASP1 (Santa Cruz Biotechnology, #sc-622), IL1B (R&D systems, #AF-201), FLAG (SigmaAldrich, #F3165), GFP (Abcam, #ab290), NLRP1 (R&D systems, #AF6788), IL18 (R&D systems, MAB9124) and VP2 (QED Bioscience, #18758).

Techniques: Over Expression, Plasmid Preparation, Control, Cotransfection, Expressing, Transfection, Blue Native PAGE, Western Blot, SDS Page, Live Cell Imaging, CRISPR, Knock-Out, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Direct cleavage of human NLRP1 by enteroviral 3C protease triggers inflammasome activation in airway epithelium

doi: 10.1101/2020.10.14.325076

Figure Lengend Snippet: A. Polio- and EV71-3Cpros do not significantly induce ASC-GFP speck formation in 293T-ASC-GFP cells expressing NLRP1. B. Expression levels of viral proteases in 293T-ASC-GFP cells. All indicated viral proteases were tagged with Myc at the N-terminus. Cell pellets were harvested 48 hours post plasmid transfection. C. Validation of pooled CRISPR/Cas9 PYCARD/ASC knockout in immortalized keratinocytes. The cell lines that were used for downstream analyses are highlighted in red. D. Validation of pooled CRISPR/Cas9 CASP1 knockout in immortalized keratinocytes. E. NLRP1 Q130A is resistant to cleavage by multiple 3Cpros. 293T-ASC-GFP cells were transfected as in . Cell pellets were harvested 48 hours post transfection. Asterisk, endogenous c-myc in 293T cells (unrelated to myc-3Cpros). F. NLRP1 WT -HA and NLRP1 Q130A -HA were expressed to similar levels in NLRP1 KO immortalized keratinocytes.

Article Snippet: The following antibodies were used in this study: c-Myc (Santa Cruz Biotechnology #sc-40), HA tag (Santa Cruz Biotechnology, #sc-805), GAPDH (Santa Cruz Biotechnology, #sc-47724), ASC (Apipogen, #AL-177), CASP1 (Santa Cruz Biotechnology, #sc-622), IL1B (R&D systems, #AF-201), FLAG (SigmaAldrich, #F3165), GFP (Abcam, #ab290), NLRP1 (R&D systems, #AF6788), IL18 (R&D systems, MAB9124) and VP2 (QED Bioscience, #18758).

Techniques: Expressing, Plasmid Preparation, Transfection, Biomarker Discovery, CRISPR, Knock-Out

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Schematic of integrating computational rankings of adhesion proteins into a four-way Venn diagram, based on mass spectrometry proteomic abundance in primary breast tumors (N=122) and TNBC cell lines, altered protein expression in TNBC tumor vs. normal tissue voxels, and breast cancer cell-secreted EVs; to identify novel candidates associated with patient outcomes. B ) Representative IHC images of human PB2 + TNBC tumor and PB2 - normal breast tissue (adjacent to tumor) from a patient with TNBC. C ) KM plot for overall survival of all patients with breast cancer, divided by best cut-off of high vs. low PB2 protein expression in primary tumors, N=108. D ) KM plot for distant metastasis-free survival (DMFS) of patients with ER - breast cancer, divided by median cut-off of high vs. low PB2 mRNA expression, N=218. E ) Schematic depicting the patient blood sample analysis workflow for CTC analysis on CellSearch. F ) Representative CellSearch images of a homotypic PB2 + CTC-CTC (CD45 - CK + DAPI + ) cluster, a heterotypic PB2 + CTC-WBC (CD45 + CK - DAPI + ) cluster, and a single PB2 - CTC, stained after anti-EpCAM bead enrichment. Scale bar = 5 µm. G ) Portion of CellSearch-analyzed PB2 + CTCs (%) in single CTCs, homotypic CTC clusters, and heterotypic CTC-WBC clusters, as shown in (F), N=10. Data are presented as mean values +/- SD, p-values reported are from two-sided unpaired t-tests.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Mass Spectrometry, Expressing, Staining

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Average spectral counts-based abundance ranking of 398 identified adhesion/surface proteins across 122 treatment-naïve primary breast patient samples (N=122) ( https://doi.org/10.1016/j.cell.2020.10.036 ), highlighting key proteins previously identified to be significant in CTCs (CD44, ICAM1, CD81 in green) and a novel candidate PB2 at a higher rank (red). B ) Average peptide spectrum match (PSM)-based abundance ranking of 252 identified adhesion/surface proteins in MDA-MB-231 triple negative breast cancer (TNBC) cell lines (N=3) ( https://doi.org/10.1021/acs.jproteome.1c00293 ). C ) Categories of MS-identified extracellular vesicle proteins (total 1603 peptides) secreted by normal and cancer cells. D ) Heat map of MS-identified differentially expressed proteins between EVs derived from breast cancer cell lines vs. EVs from immortalized normal epithelial cells (unpaired t test P<0.05). PB2 is indicated by a red box. E ) Table showing the top 11 proteins significantly up-regulated in breast tumor tissue vs. normal tissue and whether or not they significantly correlate to poor prognosis in breast cancer. F ) Average intensity expression (in millions) of PB2 in EVs of normal non-tumor epithelial cells compared to that of metastatic breast cancer cells. Data are reported as mean +/- SD, p-value is from a two-sided unpaired t-test.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) KM plot for distant metastasis-free survival (DMFS) of grade 3 breast cancer with high vs. low PB2 mRNA expression, N=836 (data from KMPlotter). B ) Tissue microarray of advanced breast cancer patient samples subjected to IHC staining for PB2 (N=86). The first 11 cases from the B06 cohort of patients (STU00203283), as highlighted by the green box on the TMA (see Suppl. Excel 3), had simultaneous analyses for the number of CTCs in the blood. C & D) Annotation of PB2 status of TMA patients stratified by specific molecular subtype of breast cancer (C) and tabular annotation of the cases (D). E ) PB2 status of first 11 patients from the B06 cohort in correlation with CTC status in blood.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Expressing, Microarray, Immunohistochemistry

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Schematic showing workflow for flow cytometry analysis of blood cells from patients with breast cancer. B ) Representative flow cytometry gating of single and clustered blood cells, by PB2 +/- and CD45 +/- status, from the B06 cohort of advanced breast cancer patients. C ) Average percentage of cells expressing PB2 in CD45 - single and clustered CTCs from flow analysis, including all EpCAM +/- CTCs, N=17 patients, data reported as mean values +/- SD, p-value from a two-sided unpaired t-test. D ) Percentage of PB2 + CD45 - single and CD45 - clustered CTCs from advanced breast cancer patients at the time of blood draw, N=17 patients, data stratified by individual patient. E ) Table showing total number and proportion of CTC single cells or CTC clusters that are PB2 + from breast cancer patients as reported by flow analysis, N=23.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A) Flow panel showing the gating of PB2 + and PB2 - cells sorted from dissociated primary TN3 L2G + PDX cells for clustering in (B-C). B & C) Representative images at 0 and 6 h of clustering (B) and cluster count curves of sorted PB2 high and PB2 low TN3 PDX cells (C), measured by IncuCyte imaging system; N=3 experiments with at least 5 technical replicates each. D & E) Representative images (D) and cluster area growth curves (E) of MDA-MB-231 cells treated with control (siCon), PB2 KD via SmartPool siRNA (siPB2), and single target siRNA (siPB2-10), N=3 experiments with at least 5 technical replicates each. F & G) Representative images (F) and cluster area growth curves (G) of MDA-MB-468 cells treated with siCon, PB2 KD via siPB2 SmartPool (siPB2), and siPB2-10. H ) Schematic of PB2 domains: Extracellular (Ecto) domains include Sema = Semaphorin domain; IPT = Ig-like fold domain; and PSI = Plexin-semaphorin-integrin domain. Intracellular domains include RBD = Rho-binding domain; GAP = GTPase activating protein domain; and VTDL = PDZ-domain binding site (Rho-GEF binding). I ) Immunoblots of overexpressed PB2 mutants with either full-length (fl) or truncated (tr) depletions: mutated RBD (mRBD) tr, depleted Ecto domain (dECTO) fl and depleted VTDL (dVTDL) tr in MDA-MB-231 PB2 KO cells. J & K) Representative images (J) and cluster area growth curves (K) of MDA-MB-231 PB2 KO clusters with overexpression of PB2 full-length or mutants (mRBD, dECTO, or dVTDL) over 6 h as measured via IncuCyte; Con vs. KO+PB2 p=0.67, Con vs. KO1 p=0.0002, Con vs. KO2 p=1.43e-6, KO+PB2 vs. KO+mRBD p=0.04, KO+PB2 vs. KO+dECTO p=1.69e-6, KO+PB2 vs. KO+dVTDL p=0.005, KO+PB2 vs. KO1 p=0.0002. L-O) IVIS bioluminescence images (L, N) of NSG mice on days 0 and 4, and quantified signals of metastasis (M, O) of dissected lungs ex vivo on day 4 after tail vein injection of MDA-MB- 231 cells transfected with siCon, siPB2 (L), or siPB-11 (N), N=4 (L-M) and 3 (N-O) biological replicates. Data are presented as mean values +/- SD, p-values reported are from two-sided unpaired t-tests.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Imaging, Control, Binding Assay, Western Blot, Over Expression, Ex Vivo, Injection, Transfection

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Left panel: Western blot showing MDA-MB-231 KD efficiency of PB2 using SmartPool (siPB2) siRNA as well as single siRNAs (si09, si10, si11), N=3 experiments; Right panel: Proliferation of MDA-MB-231 cells measured as percent confluence using Incucyte. Breast cancer cells plated after double transfection with either SmartPool siRNA (siPB2) or single siRNA KD of PB2 (siPB2-11), N=3 biological replicates with at least 3 technical replicates each, data are presented as mean +/- SD, p-value at 84 h reported as two-sided unpaired t-test. B ) Flow analysis showing MDA-MB-468 KD of PB2 using SmartPool (siPB2) siRNA as well as single siRNA (siPB2-10), N=3 experiments. C ) Flow panel showing KD efficiency of SKBR3 breast cancer cells after double transfection with PB2 SmartPool siRNA. D ) Representative images of SKBR3 cell clusters after 4 h as taken by the Incucyte Live Cell Imager. E ) Quantification of clusters of SKBR3 cells with PB2 KD after 4 h, average cluster area measured by IncuCyte, with N=4 experiments. Data are presented as mean values +/- SD, p- value is from a two-sided unpaired t-test. F ) Flow panel showing KD efficiency of 4T1 mouse breast cancer cells after double transfection with PB2 SmartPool siRNA. G ) Representative images of 4T1 cell clusters after 6 h as taken by the Incucyte Live Cell Imager. H ) Quantification of clustering assay of 4T1 cells with PB2 KD after 6 h, average cluster area measured by Incucyte, with N=3 experiments. Data are presented as mean values +/- SD, p- value is from a two-sided unpaired t-test. I ) Flow panel showing KD efficiency of HS578T breast cancer cells after double transfection with PB2 SmartPool siRNA and siPB2-10 single PB2 siRNA. J ) Representative images of HS578T cell clusters after 4 h as taken by the Incucyte Live Cell Imager. K ) Quantification of clustering assay of HS578T cells with PB2 KD after 4 h, average cluster area measured by Incucyte, with N=3 experiments. Data are presented as mean values +/- SD, p-value is from a two-sided unpaired t-test.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Western Blot, Transfection

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Immunoblots of full-length (FL) and truncated (Tr) PB2 in MDA-MB-231 cells with two pooled clones of CRISPR/Cas9-mediated KO, KO1 and KO2, with PB2-specific bands at 250 kDa and 75 kDa, N=3 experiments; B-D) Flow cytometry analysis of MDA-MB-231 Cas9 control cells for PB2, KO1, and KO2. E) Proliferation assay of MDA-MB-231 control and KO cells over 60+ h measured as percent confluence by IncuCyte, N=3 experiments, data reported as mean +/- SD, p-value is from a two-sided unpaired t-test. F-G) Representative images (F) and cluster size quantification (G) of MDA-MB-231 PB2 control, KO1, and KO2 cell clusters after 6 h as taken by the IncuCyte Live Cell Imager; N=3 experiments with at least 3 technical replicates each, data are presented as mean values +/- SD, p-value is from a two-sided unpaired t-test. H ) Flow cytometry analysis of PB2 overexpression in MDA-MB-231 PB2 KO cells. I & J) Representative images (I) of average mammosphere counts of 10 cells or more (J) of MDA-MB-231 WT cells with scramble (siCon), PB2 SmartPool (siPB2), and PB2 single (siPB2-10) siRNA KD after 7 days; N=3 experiments with at least 5 technical replicates each, data are presented as mean values +/- SD, p-values are from a two-sided unpaired t-test. K & L) Mammosphere images (K) and counts (L) of MDA-MB-231 KO2 cells with overexpression of PB2 full-length (PB2 OE) and a PB2 mutant with depleted extracellular domain (dECTO) for 10 days, N=4 replicates (bottom). Mammospheres larger than 100 microns across were counted. Data are presented as mean +/-SD, p-value is from a two-sided unpaired t-test.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Western Blot, Clone Assay, CRISPR, Flow Cytometry, Control, Proliferation Assay, Over Expression, Mutagenesis

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Abundance ranking of PB2 and Sema4 family members based on average spectral counts among 398 identified adhesion/surface proteins across 122 treatment-naïve primary breast patient samples ( https://doi.org/10.1016/j.cell.2020.10.036 ); the canonical binding ligands of PB2, Sema4C, Sema4D, and Sema4A, are shown by yellow dots. B ) KM plot of distant metastasis-free survival (DMFS) of breast cancer patients based on mRNA expression of Sema4C, p=0.0051. C ) KM plot of overall survival (OS) of breast cancer patients based on mRNA expression of Sema4C, p=0.049.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Binding Assay, Expressing

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Schematic of PB2-Sema4C intercellular interactions in homotypic tumor cell clustering between two cancer cells. B ) Immunoblots of PB2 and Sema4C expression in different breast cancer cell lines and PDX models using antibodies that are specific to detect both human and mouse isoforms. C ) Immunoblots of Sema4C, CDC42, and PB2 in the protein complex immunoprecipitated by anti-PB2 antibody compared to IgG. D ) Immunoblot image with reduced Sema4C after Sema4C KD in MDA-MB-231 tumor cells. E & F) Representative images (E) and average cluster growth area curves (F) of MDA-MB-231 control and PB2 KO cells in co-culture with Con or Sema4C KD cells, measured by IncuCyte, N=3 experiments with at least 5 technical replicates each, PB2 + Sema4C + vs. PB2 + Sema4 - p=1.86e-9, PB2 + Sema4C + vs. PB2 - Sema4C - p=6.81e-10, PB2 - Sema4C + vs. PB2 - Sema4C - p=0.22, PB2 + Sema4C - vs. PB2 - Sema4C - p=0.47. G ) Principal component analysis clusters of MDA-MB-231 single cells, siCon clusters, and siPB2 clusters analyzed from global mass spectrometry with N=3 replicates per group. H ) Heat map of differentially expressed proteins from global mass spectrometry analysis of siCon single cells (blue) N=3, siCon clusters (green) N=3, and siPB2 clusters (red) N=3 I ) Gene ontology biological processes analysis of significantly up-regulated and down-regulated proteins in PB2 control vs. PB2 KD clusters in breast cancer cells analyzed by mass spectrometry. Data are presented as mean values +/- SD, p-values reported are from two-sided unpaired t-tests.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Western Blot, Expressing, Immunoprecipitation, Control, Co-Culture Assay, Mass Spectrometry

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Table showing fold change in the top hits from GO analysis of biological processes for proteins that were significantly up-regulated in PB2 KD compared to control cells by global mass spectrometry analysis of clustered cells. B ) GO biological processes analysis of altered proteins in Groups C and F in , with clustering-specific up-regulation and down-regulation, respectively, and reversed in a siPB2- dependent manner. C ) Western blot validation of top hits from Group B (see ) of global mass spectrometry analysis of siPB2 single cells vs. clusters. D & E) Representative images (D) and average cluster area (E) of MDA-MB-231 control vs. MCM7 KD cells as taken by the IncuCyte. N=3 experiments with at least 3 technical replicates each, data are presented as mean values +/- SD, p-value is from a two-sided unpaired t-test.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Control, Mass Spectrometry, Western Blot, Biomarker Discovery

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Schematic of the interactions between breast cancer cell PB2 and monocyte Sema4A for heterotypic cluster formation. B ) Representative images at 4 h co-culture of ex vivo heterotypic clusters of L2G + MDA-MB- 231 control or PB2 KO cells with WBCs from breast cancer patients (Pt). Top row: fluorescent images with WBCs from Pt 396 (minimal cytotox red-stained dead cells). Bottom row: brightfield images with WBCs from Pt 431. C ) Area curves of heterotypic tumor cell clusters co-cultured with WBCs from patients (N=4) as shown in (B), measured by Incucyte imaging. Data are reported as mean +/-SD, with p-values from two-sided unpaired t-tests. D ) Abundance ranking of 96 identified adhesion/surface proteins in human monocytes (N=3), based on mass spectrometry (MS) quantitative analysis ( https://doi.org/10.1038/s41598-020-61356-w ). E ) Sema4A expression on granulocytes, monocytes, and lymphocytes from patients with advanced breast cancer (N=4). F ) Percent of heterotypic CD45 + EpCAM + CTC-WBC clusters vs. CD45 + EpCAM - WBC clusters that express both PB2 and Sema4A, as analyzed by flow analysis, N=4 patients. G ) PB2 immunoprecipitation of MDA-MB-231 tumor-monocyte (THP1) clusters or THP1 monocytes only, blotted for Sema4A and CDC42. H & I) Representative images at 4 h (H) and cluster area curves (I) of heterotypic clustering with MDA-MB-231 tumor cells (green) and THP1 monocytes (red) mixed at a 1:4 ratio, reported as mean +/-SD, N=3 with at least 3 technical replicates each. J & K) Representative images at 6 h (J) and cluster size curves (K) of heterotypic clustering with L2G + TN1 PDX tumor cells (green, sorted by PB2 high and PB2 low ) and THP1 monocytes (red), N=2 experiments with at least 3 technical replicates each. L & M) Representative images at 6 h (L) and cluster size curves (M) of MDA-MB-231 WT (Con) or PB2 KO cells cocultured with THP1 monocytes (sorted by Sema4A+/- expression), N=3 experiments with at least 5 technical replicates each. Data are presented as mean values +/- SD, p-values reported are from two-sided unpaired t-tests.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Co-Culture Assay, Ex Vivo, Control, Staining, Cell Culture, Imaging, Mass Spectrometry, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Quantification of normalized cytotoxic red dye-labeled dead cell events as measured by IncuCyte at 12 h during the MDA-MB-231 breast cancer cell co-culture with WBCs isolated from breast cancer patients, N=4. Data reported as mean +/-SD, p-value is from a two-sided unpaired t-test. B ) Sema4A expression in human PBMCs, analyzed via single-cell sequencing of the Human Protein Atlas. C ) Representative flow plots of Sema4A expression in monocytes, granulocytes, and lymphocytes derived from the blood of patients with breast cancer. D ) Representative gating strategy of flow cytometry analysis of advanced stage breast cancer patient WBCs stained for EpCAM, CD45, PB2, and Sema4A to identify PB2 + Sema4A + heterotypic CTC clusters. E ) Quantification of double positive PB2 + Sema4A + clusters (CD45 +/- ) in advanced stage breast cancer PBMCs, N=4 patients, data are presented as mean +/-SD, p-value is from a two-sided unpaired t-test. F ) Flow cytometry analysis of THP1 monocyte cell line for PB2 and Sema4A expression, N=3. G ) Average Cytotox Red counts in heterotypic THP1 co-culture system as counted by IncuCyte, N=3, data are presented as mean +/-SD, p-value is from an unpaired two-sided t-test.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Labeling, Co-Culture Assay, Isolation, Expressing, Sequencing, Derivative Assay, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A) Schematic showing the experimental workflow of orthotopic implantation of tumor cells into mouse mammary fat pads and subsequent analyses of lung metastases and blood CTCs. B-C) Representative images (B) and weight quantification of PB2 Con and KO tumors (C) upon harvest from mice at 10 weeks (N=8). D-E) Bioluminescence images of mouse lungs ex vivo (D) and normalized lung metastasis burden (E) at 10 weeks, with bioluminescence counts normalized to cumulative tumor weight, N=5 biological replicates. F-G) Representative images (F) and metastatic burden (G) in control and PB2 KO metastatic cells of mouse lungs with H&E staining at 10 weeks after orthotopic implantation, scale bar = 250 µm; experiments were repeated with the PB KO2 clone. H ) L2G + CTC counts (single and clusters) detected in control and PB2 KO mouse blood via flow cytometry at 10 weeks of spontaneous metastasis, N=4. I ) IVIS bioluminescence images of NSG mice after tail vein injection of pre-clustered MDA- MB-231 cells alone (PB2 Con or KO), and with THP1 cells (PB2 Con/THP1, KO/THP1) on day 0 (D0), 12 h, day 4 (D4). N=3 biological replicates. J-K) Mean photon counts/s of ROI (region of interest) of lungs ex vivo after harvesting lungs at 12 h (J) and day 4 (K), N=3 biological replicates per time point. Data are presented as mean values +/-SD, with p-values reported from two-sided unpaired t-tests.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Ex Vivo, Control, Staining, Flow Cytometry, Injection