Journal: Viral immunology

Article Title: Strain-dependent requirement for IFN-γ for respiratory control and immunotherapy in murine gammaherpesvirus infection.

doi: 10.1089/vim.2011.0004

Figure Lengend Snippet: FIG. 3. Absence of IFN-g does not affect recruitment of other cell populations or other CD8 T-cell effector functions. (A) Total cell numbers of various cell populations in the lungs at day 10 post-infection. (B) Representative FACS plots showing the distribution of cells of various phenotypes in the lungs at day 10 post-infection. Numbers indicate percentages of the relevant populations in the gates shown. (A and B) Data are representative of three experiments. (C) Frequencies of granzyme B-producing CD8 T cells. Granzyme B ELISPOT was performed at day 11 post-infection. Numbers indicate the frequency of granzyme B-producing cells among the CD8 T-cell population. Each dot represents data from an individual animal and horizontal bars indicate the mean. Data are representative of two experiments.

Article Snippet: The Granzyme B ELISPOT kit was purchased from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Infection, Enzyme-linked Immunospot

Journal: Cancers

Article Title: ROR1 as an Immunotherapeutic Target for Inducing Antitumor Helper T Cell Responses Against Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cancers17142326

Figure Lengend Snippet: Induction of ROR1-specific T cells by a novel epitope. ( a ) Flow cytometric analysis of ROR1 expression in the HNSCC cell lines HPC92Y, Sa3, and HSC3. ( b ) Evaluation of the peptide dose–responses of ROR1-reactive helper T lymphocytes (HTLs) designated as R1 and R2. These HTLs were co-cultured with autologous peripheral blood mononuclear cells (PBMCs) used as antigen-presenting cells (APCs) and pulsed with increasing concentrations of the ROR1 403–417 peptide. ( c ) Human leukocyte antigen (HLA) restriction analysis of ROR1-reactive HTLs. These HTLs were cocultured with autologous PBMCs pulsed with 3 μg/mL ROR1 403–417 peptide with or without anti-HLA-DR or anti-HLA class I antibodies. IFN-γ levels secreted from the co-cultured HTLs were measured using ELISA. ( d ) HLA-DR allele restriction of ROR1-reactive HTLs. HTL responses to the ROR1 403–417 peptide were evaluated using peptide-pulsed (3 μg/mL) L-cells expressing individual HLA-DR alleles. IFN-γ secretion was quantified via ELISA after 48 h of co-culture. Experiments were performed in triplicate. Data are presented as mean ± standard deviation. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To investigate the anti-tumor activity of ROR1-reactive HTLs against HNSCC, HTLs were co-cultured with ROR1+ tumor cell lines (3 × 10 4 ) for 48 h. Supernatants were collected and analyzed using Human IFN-γ and Granzyme B ELISA kits (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Standard Deviation

Journal: Cancers

Article Title: ROR1 as an Immunotherapeutic Target for Inducing Antitumor Helper T Cell Responses Against Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cancers17142326

Figure Lengend Snippet: Anti-tumor activity of ROR1-reactive HTLs against ROR1-expressing HNSCC cell lines. ( a ) Direct recognition of tumor cells by ROR1-reactive HTLs was assessed by coculturing HLA-DR-matched or -mismatched HNSCC cell lines with or without anti-HLA-DR antibody. IFN-γ secretion in culture supernatants was measured using ELISA after 48 h of coculture. ( b ) Granzyme B production by ROR1-reactive HTLs under the same conditions was evaluated. ( c ) Cytotoxicity assays of ROR1-reactive HTLs in HLA-DR-matched and -mismatched tumor cell lines. HTLs were co-cultured with tumor cell lines with several effector-to-target (E:T) ratios. ( d ) Representative flow cytometry plots showing tumor cell killing at an E:T ratio of 20:1. Experiments were performed in triplicate ( a , b ) or duplicate ( c , d ). Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To investigate the anti-tumor activity of ROR1-reactive HTLs against HNSCC, HTLs were co-cultured with ROR1+ tumor cell lines (3 × 10 4 ) for 48 h. Supernatants were collected and analyzed using Human IFN-γ and Granzyme B ELISA kits (R&D Systems, Minneapolis, MN, USA).

Techniques: Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Standard Deviation

Journal: Cancers

Article Title: ROR1 as an Immunotherapeutic Target for Inducing Antitumor Helper T Cell Responses Against Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cancers17142326

Figure Lengend Snippet: Enhanced antitumor activity of ROR1-reactive HTLs by blockade of PD-L1/PD-1 and PD-L2/PD-1 axes. ( a , b ) Expression levels of PD-L1 ( a ) and PD-L2 ( b ) on the HNSCC cell line, HPC92Y, were assessed using flow cytometry. ROR1-reactive HTLs were co-cultured with HPC92Y cells in the presence of immune checkpoint inhibitors targeting PD-1, PD-L1, PD-L2, or a combination of PD-L1 and PD-L2. ( c , d ) Levels of IFN-γ ( c ) and granzyme B ( d ) secreted into the supernatants were measured via ELISA after 48 h of coculture. Experiments were performed in triplicate. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To investigate the anti-tumor activity of ROR1-reactive HTLs against HNSCC, HTLs were co-cultured with ROR1+ tumor cell lines (3 × 10 4 ) for 48 h. Supernatants were collected and analyzed using Human IFN-γ and Granzyme B ELISA kits (R&D Systems, Minneapolis, MN, USA).

Techniques: Activity Assay, Expressing, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Nature Communications

Article Title: Molecular basis promoting centriole triplet microtubule assembly

doi: 10.1038/s41467-024-46454-x

Figure Lengend Snippet: a Heatmap of Chronos scores of the indicated genes for 1031 human cell lines. b Scatter plots of Chronos scores of CEP295 and CEP44 . c Schematic of the two-step screening. d Heatmap of correlation coefficients between Chronos scores of the indicated genes and those of 17,393 human genes. The genes were sorted in order of mean values. e Hierarchical clustering analysis of the top 100 hits using their Chronos scores. The cluster enclosed by orange rectangle included CEP295 , CEP44 , CEP120 , C2CD3 , and HYLS1 . f STED microscopy images of HeLa mCherry-HYLS1 knock-in cells. Scale bar: 100 nm. MC: mother centriole, PC: procentriole. Ace-tub: acetylated tubulin. g Confocal microscopy images of RPE-1 cells transfected with pCMV-FLAG-HYLS1 and fixed with the indicated methods. Scale bars: 10 μm and 1 μm. Large arrowheads: magnified areas, small arrowheads: superstructures. h Time-lapse images of RPE-1 cells stably expressing TUBB-mNeonGreen transfected with pCMV-mScarlet-i-HYLS1. Scale bars: 10 μm and 1 μm. Arrowheads: first confirmed accumulations. i STED microscopy images of the tubulin-based superstructures in RPE-1 cells transfected with pCMV-HA-HYLS1 and centrioles in RPE-1 cells. Scale bars: 1 μm. Ace-tub: acetylated tubulin. j Quantification of diameter of the tubulin-based superstructures and the centrioles in ( i ). n = 50 structures. k IF images of RPE-1 cells transfected with pCMV-FLAG-HYLS1 and then incubated on ice for 1 h before fixation. Scale bar: 10 μm. Red arrowheads: superstructures, blue arrowheads: centrioles. l Quantification of frequency of interphase cells with the tubulin-based superstructures in ( k ). n = 3 independent experiments, 50 cells each. m Schematic showing whether each structure remains at low temperature. Data are represented as mean ± s.d. P value was calculated by two-tailed unpaired Student’s t -test ( l ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibodies against RFP/mCherry [MBL, PM005, Lot 046, immunofluorescence (IF) 1:500, immunoblotting (IB) 1:1000], CEP97 [Novus Biologicals, NBP1-83591, Lot A107064, IF 1:500], α-tubulin [MBL, PM054, Lot 007, IF 1:500, IB 1:1000], β-tubulin [Thermo Fisher Scientific, PA5-16863, Lot XG3654589, IF 1:500], acetylated tubulin [Abcam, ab179484, IF 1:500], FLAG [Sigma, F7425, Lot 086M4803V, IF 1:500], HA [Abcam, ab9110, IF 1:1000], PCNT [Abcam, ab4448, IF 1:1000], HYLS1 [Novus Biologicals, NBP1-56899, Lot QC25211-90821, IB 1:1000], CEP152 [Bethyl Laboratories, A302-480A, IF 1:1000], CEP192 [Bethyl Laboratories, A302-324A, IF 1:1000], CP110 [Proteintech, 12780-1-AP, IF 1:500], ANKRD26 [GeneTex, GTX128255, Lot 41458, IF 1:1000], CEP44 [Proteintech, 24457-1-AP, IF 1:500], CEP57 [GeneTex, GTX115931, Lot 40289, IF 1:500], CEP63 [Proteintech, 16268-1-AP, IF 1:500], CEP135 [Abcam, ab196809, Lot GR199116-6, IF 1:500], CEP295 [Sigma, HPA038596, Lot R33456, IF 1:500], CPAP [Proteintech, 11517-1-AP, IF 1:500], PLK1 [Bethyl Laboratories, A300-251A, IF 1:500], and ARL13B [Proteintech, 17711-1-AP, IF 1:500]; goat polyclonal antibody against PCNT [Santa Cruz Biotechnology, sc-28145, Lot C1716, IF 1:500]; mouse monoclonal antibodies against Centrin [Merck Millipore, 04-1624, Clone 20H5, IF 1:1000], γ-tubulin [Sigma, T5326, Clone GTU88, IF 1:1000], α-tubulin [Sigma, T5168, Clone B-5-1-2, IF 1:1000], acetylated tubulin [Sigma, T7451, Clone 6-11B-1, IF 1:500], polyglutamylation modification [AdipoGen, AG-20B-0020-C100, Clone GT335, IF 1:500], monoglycylated tubulin [Merck Millipore, MABS277, Clone TAP952, IF 1:500], polyglycylated tubulin [Merck Millipore, MABS276, Clone AXO49, IF 1:500], FLAG [Sigma, F1804, Clone M2, IF 1:1000, IB 1:1000], HA [Biolegend, 901501, Clone 16B12, IF 1:500], SAS6 [Santa Cruz Biotechnology, sc-81431, IF 1:500], HSP90 [BD Biosciences, 610419, IB 1:2000], EB1 [BD Biosciences, 610534, Clone 5/EB1, IF 1:500]; rat monoclonal antibodies against HA [Merck, 11867423001, Clone 3F10, IF 1:500], Centrin 2 [BioLegend, 698602, Clone W16110A, IF 1:500].

Techniques: Microscopy, Knock-In, Confocal Microscopy, Transfection, Stable Transfection, Expressing, Incubation, Two Tailed Test

Journal: Nature Communications

Article Title: Molecular basis promoting centriole triplet microtubule assembly

doi: 10.1038/s41467-024-46454-x

Figure Lengend Snippet: a TEM images of vertically oriented superstructures in RPE-1 cells transfected with pCMV-mScarlet-i-HYLS1. Scale bars: 1 μm, 100 nm, and 10 nm. Large arrowheads (red & black): superstructures, small arrowheads (orange, blue, purple, and cyan): magnified areas in the wall of the superstructure highlighted in red. b TEM images of horizontal superstructures in RPE-1 cells transfected with pCMV-mScarlet-i-HYLS1 and singlet microtubules in RPE-1 cells. Scale bars: 100 nm and 10 nm. Large arrowheads: both sides of the lateral surface of the horizontal superstructure (red and yellow) or magnified areas (red and blue), small arrowheads (white): measured intervals. c Quantification of widths of the superstructure interlayer distances and those of the singlet microtubules in ( b ). n = 55 structures. d TEM images of younger centrioles in G1 phase RPE-1 cells transfected with siControl or siHYLS1. Scale bar: 100 nm. n = 7 (siControl) or 12 (siHYLS1) cells. e Representative IF images of RPE-1 cells subjected to centriole stability assay with STLC. Before the STLC treatment, the cells were transfected with siControl or siHYLS1. Scale bar: 5 μm. Arrowheads: Centrin foci (centrioles). f Quantification of frequency of mitotic cells with <4 Centrin foci in e . Only groups treated with STLC for 18 h are shown. n = 3 independent experiments, 50 cells each. g IF images of RPE-1 C-Nap1 KO cells subjected to microtubule regrowth assay (30°C, 5 seconds). The cells were transfected with siControl or siHYLS1 before cold-induced microtubule depolymerization. Scale bars: 10 μm and 1 μm. Arrowheads: magnified areas with older (white) or younger (orange) centrosomes. h Quantification of average EB1 intensity around younger centrosomes in ( g ). A.U.: arbitrary unit. n = 150 cells pooled from 3 independent experiments, 50 cells each. i IF images of mitotic RPE-1 cells treated with siControl or siHYLS1 for 72 h. Scale bar: 10 μm. Arrowheads: Centrin foci (centrioles). j Quantification of frequency of mitotic cells with abnormal mitotic spindles, such as monopolar spindles, asymmetric bipolar spindles with non-centrosomal poles, and bipolar spindles with precociously disengaged centrioles, in ( i ). n = 3 independent experiments, 31, 32, and 33 cells (siControl) or 31, 31, and 32 cells (siHYLS1). k IF images of RPE-1 cells transfected with siControl or siHYLS1 and then serum starved for 48 h. Scale bar: 5 μm. l Quantification of frequency of interphase cells with primary cilia in ( k ). n = 3 independent experiments, 50 cells each. m Quantification of length of primary cilia in ( k ). n = 50 (siControl) or 39 (siHYLS1) cells pooled from 3 independent experiments. n Schematic showing the phenotypes resulting from HYLS1 depletion in centrioles, centrosomes, and primary cilia. MT: microtubule. Data are represented as mean ± s.d. P values were calculated by Mann–Whitney U test ( c , h , m ), one-way ANOVA with Tukey’s multiple comparisons test ( f ), or two-tailed unpaired Student’s t -test ( j , l ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibodies against RFP/mCherry [MBL, PM005, Lot 046, immunofluorescence (IF) 1:500, immunoblotting (IB) 1:1000], CEP97 [Novus Biologicals, NBP1-83591, Lot A107064, IF 1:500], α-tubulin [MBL, PM054, Lot 007, IF 1:500, IB 1:1000], β-tubulin [Thermo Fisher Scientific, PA5-16863, Lot XG3654589, IF 1:500], acetylated tubulin [Abcam, ab179484, IF 1:500], FLAG [Sigma, F7425, Lot 086M4803V, IF 1:500], HA [Abcam, ab9110, IF 1:1000], PCNT [Abcam, ab4448, IF 1:1000], HYLS1 [Novus Biologicals, NBP1-56899, Lot QC25211-90821, IB 1:1000], CEP152 [Bethyl Laboratories, A302-480A, IF 1:1000], CEP192 [Bethyl Laboratories, A302-324A, IF 1:1000], CP110 [Proteintech, 12780-1-AP, IF 1:500], ANKRD26 [GeneTex, GTX128255, Lot 41458, IF 1:1000], CEP44 [Proteintech, 24457-1-AP, IF 1:500], CEP57 [GeneTex, GTX115931, Lot 40289, IF 1:500], CEP63 [Proteintech, 16268-1-AP, IF 1:500], CEP135 [Abcam, ab196809, Lot GR199116-6, IF 1:500], CEP295 [Sigma, HPA038596, Lot R33456, IF 1:500], CPAP [Proteintech, 11517-1-AP, IF 1:500], PLK1 [Bethyl Laboratories, A300-251A, IF 1:500], and ARL13B [Proteintech, 17711-1-AP, IF 1:500]; goat polyclonal antibody against PCNT [Santa Cruz Biotechnology, sc-28145, Lot C1716, IF 1:500]; mouse monoclonal antibodies against Centrin [Merck Millipore, 04-1624, Clone 20H5, IF 1:1000], γ-tubulin [Sigma, T5326, Clone GTU88, IF 1:1000], α-tubulin [Sigma, T5168, Clone B-5-1-2, IF 1:1000], acetylated tubulin [Sigma, T7451, Clone 6-11B-1, IF 1:500], polyglutamylation modification [AdipoGen, AG-20B-0020-C100, Clone GT335, IF 1:500], monoglycylated tubulin [Merck Millipore, MABS277, Clone TAP952, IF 1:500], polyglycylated tubulin [Merck Millipore, MABS276, Clone AXO49, IF 1:500], FLAG [Sigma, F1804, Clone M2, IF 1:1000, IB 1:1000], HA [Biolegend, 901501, Clone 16B12, IF 1:500], SAS6 [Santa Cruz Biotechnology, sc-81431, IF 1:500], HSP90 [BD Biosciences, 610419, IB 1:2000], EB1 [BD Biosciences, 610534, Clone 5/EB1, IF 1:500]; rat monoclonal antibodies against HA [Merck, 11867423001, Clone 3F10, IF 1:500], Centrin 2 [BioLegend, 698602, Clone W16110A, IF 1:500].

Techniques: Transfection, Stability Assay, Regrowth Assay, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Molecular basis promoting centriole triplet microtubule assembly

doi: 10.1038/s41467-024-46454-x

Figure Lengend Snippet: a Representative IF images of RPE-1 cells transfected with the indicated constructs. Scale bar: 10 μm. b Quantification of frequency of interphase cells with incomplete MT superstructures in ( a ). n = 3 independent experiments, 30 cells each. c Schematic showing a specific region of HYLS1 that is required for the assembly of incomplete MT superstructures (superstructure domain). d Schematic showing amino acid sequences of HYLS-1 Box in the indicated species. e IF images of RPE-1 cells transfected with pCMV-FLAG-HYLS1 WT or pCMV-FLAG-HYLS1 D211G. Scale bar: 10 μm. Arrowheads: incomplete MT superstructures. f Quantification of frequency of interphase cells with incomplete MT superstructures in ( e ). n = 3 independent experiments, 30 cells each. g Representative IF images of RPE-1 TetOn-mNeonGreen-X cells (X = HYLS1 WT or D211G) subjected to centriole stability assay with STLC. Before the STLC treatment, the cells were transfected with siControl or siHYLS1 and were treated with doxycycline. Scale bar: 5 μm. Arrowheads: Centrin foci (centrioles). h Quantification of frequency of mitotic cells with <4 Centrin foci in ( g ). Only groups treated with STLC for 18 h are shown. n = 3 independent experiments, 50 cells each. Data are represented as mean ± s.d. P values were calculated by one-way ANOVA with Tukey’s multiple comparisons test ( h ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibodies against RFP/mCherry [MBL, PM005, Lot 046, immunofluorescence (IF) 1:500, immunoblotting (IB) 1:1000], CEP97 [Novus Biologicals, NBP1-83591, Lot A107064, IF 1:500], α-tubulin [MBL, PM054, Lot 007, IF 1:500, IB 1:1000], β-tubulin [Thermo Fisher Scientific, PA5-16863, Lot XG3654589, IF 1:500], acetylated tubulin [Abcam, ab179484, IF 1:500], FLAG [Sigma, F7425, Lot 086M4803V, IF 1:500], HA [Abcam, ab9110, IF 1:1000], PCNT [Abcam, ab4448, IF 1:1000], HYLS1 [Novus Biologicals, NBP1-56899, Lot QC25211-90821, IB 1:1000], CEP152 [Bethyl Laboratories, A302-480A, IF 1:1000], CEP192 [Bethyl Laboratories, A302-324A, IF 1:1000], CP110 [Proteintech, 12780-1-AP, IF 1:500], ANKRD26 [GeneTex, GTX128255, Lot 41458, IF 1:1000], CEP44 [Proteintech, 24457-1-AP, IF 1:500], CEP57 [GeneTex, GTX115931, Lot 40289, IF 1:500], CEP63 [Proteintech, 16268-1-AP, IF 1:500], CEP135 [Abcam, ab196809, Lot GR199116-6, IF 1:500], CEP295 [Sigma, HPA038596, Lot R33456, IF 1:500], CPAP [Proteintech, 11517-1-AP, IF 1:500], PLK1 [Bethyl Laboratories, A300-251A, IF 1:500], and ARL13B [Proteintech, 17711-1-AP, IF 1:500]; goat polyclonal antibody against PCNT [Santa Cruz Biotechnology, sc-28145, Lot C1716, IF 1:500]; mouse monoclonal antibodies against Centrin [Merck Millipore, 04-1624, Clone 20H5, IF 1:1000], γ-tubulin [Sigma, T5326, Clone GTU88, IF 1:1000], α-tubulin [Sigma, T5168, Clone B-5-1-2, IF 1:1000], acetylated tubulin [Sigma, T7451, Clone 6-11B-1, IF 1:500], polyglutamylation modification [AdipoGen, AG-20B-0020-C100, Clone GT335, IF 1:500], monoglycylated tubulin [Merck Millipore, MABS277, Clone TAP952, IF 1:500], polyglycylated tubulin [Merck Millipore, MABS276, Clone AXO49, IF 1:500], FLAG [Sigma, F1804, Clone M2, IF 1:1000, IB 1:1000], HA [Biolegend, 901501, Clone 16B12, IF 1:500], SAS6 [Santa Cruz Biotechnology, sc-81431, IF 1:500], HSP90 [BD Biosciences, 610419, IB 1:2000], EB1 [BD Biosciences, 610534, Clone 5/EB1, IF 1:500]; rat monoclonal antibodies against HA [Merck, 11867423001, Clone 3F10, IF 1:500], Centrin 2 [BioLegend, 698602, Clone W16110A, IF 1:500].

Techniques: Transfection, Construct, Stability Assay

Journal: Nature Communications

Article Title: Molecular basis promoting centriole triplet microtubule assembly

doi: 10.1038/s41467-024-46454-x

Figure Lengend Snippet: a Schematic of AlphaFold-based screening. PAE: predicted aligned error. b , c Structural model and PAE plot of the protein complex of HYLS1 and each α-tubulin ( b ) or β-tubulin ( c ) isotype generated by AlphaFold-Multimer. d Immunoblotting images of immunoprecipitated lysates from HEK293 cells transfected with the indicated constructs. Immunoprecipitation was performed using an antibody against FLAG-tag. IP: immunoprecipitation, IB: immunoblotting. e Representative IF images of RPE-1 cells transfected with pCMV-FLAG-HYLS1 and the indicated siRNAs. For each group, two distinct siRNAs were mixed and used, except for siTUBA3D. Scale bar: 10 μm. Arrowheads: incomplete MT superstructures. f Quantification of frequency of interphase cells with incomplete MT superstructures in ( e ). Cont.: Control. n = 3 independent experiments, 30 cells each. Data are represented as mean ± s.d. P value was calculated by one-way ANOVA with Dunnett’s multiple comparisons test ( f ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibodies against RFP/mCherry [MBL, PM005, Lot 046, immunofluorescence (IF) 1:500, immunoblotting (IB) 1:1000], CEP97 [Novus Biologicals, NBP1-83591, Lot A107064, IF 1:500], α-tubulin [MBL, PM054, Lot 007, IF 1:500, IB 1:1000], β-tubulin [Thermo Fisher Scientific, PA5-16863, Lot XG3654589, IF 1:500], acetylated tubulin [Abcam, ab179484, IF 1:500], FLAG [Sigma, F7425, Lot 086M4803V, IF 1:500], HA [Abcam, ab9110, IF 1:1000], PCNT [Abcam, ab4448, IF 1:1000], HYLS1 [Novus Biologicals, NBP1-56899, Lot QC25211-90821, IB 1:1000], CEP152 [Bethyl Laboratories, A302-480A, IF 1:1000], CEP192 [Bethyl Laboratories, A302-324A, IF 1:1000], CP110 [Proteintech, 12780-1-AP, IF 1:500], ANKRD26 [GeneTex, GTX128255, Lot 41458, IF 1:1000], CEP44 [Proteintech, 24457-1-AP, IF 1:500], CEP57 [GeneTex, GTX115931, Lot 40289, IF 1:500], CEP63 [Proteintech, 16268-1-AP, IF 1:500], CEP135 [Abcam, ab196809, Lot GR199116-6, IF 1:500], CEP295 [Sigma, HPA038596, Lot R33456, IF 1:500], CPAP [Proteintech, 11517-1-AP, IF 1:500], PLK1 [Bethyl Laboratories, A300-251A, IF 1:500], and ARL13B [Proteintech, 17711-1-AP, IF 1:500]; goat polyclonal antibody against PCNT [Santa Cruz Biotechnology, sc-28145, Lot C1716, IF 1:500]; mouse monoclonal antibodies against Centrin [Merck Millipore, 04-1624, Clone 20H5, IF 1:1000], γ-tubulin [Sigma, T5326, Clone GTU88, IF 1:1000], α-tubulin [Sigma, T5168, Clone B-5-1-2, IF 1:1000], acetylated tubulin [Sigma, T7451, Clone 6-11B-1, IF 1:500], polyglutamylation modification [AdipoGen, AG-20B-0020-C100, Clone GT335, IF 1:500], monoglycylated tubulin [Merck Millipore, MABS277, Clone TAP952, IF 1:500], polyglycylated tubulin [Merck Millipore, MABS276, Clone AXO49, IF 1:500], FLAG [Sigma, F1804, Clone M2, IF 1:1000, IB 1:1000], HA [Biolegend, 901501, Clone 16B12, IF 1:500], SAS6 [Santa Cruz Biotechnology, sc-81431, IF 1:500], HSP90 [BD Biosciences, 610419, IB 1:2000], EB1 [BD Biosciences, 610534, Clone 5/EB1, IF 1:500]; rat monoclonal antibodies against HA [Merck, 11867423001, Clone 3F10, IF 1:500], Centrin 2 [BioLegend, 698602, Clone W16110A, IF 1:500].

Techniques: Generated, Western Blot, Immunoprecipitation, Transfection, Construct, FLAG-tag, Control

Journal: Nature Communications

Article Title: Molecular basis promoting centriole triplet microtubule assembly

doi: 10.1038/s41467-024-46454-x

Figure Lengend Snippet: a Structural model of HYLS1-TUBB complex generated by AlphaFold-Multimer. C-terminal tail (CTT) of TUBB is magnified. Orange: HYLS1, red: HYLS1 D211, cyan: TUBB, blue: TUBB G437. b Schematic of the CTT amino acid sequences of β-tubulin isotypes. Orange: aromatic, light green: uncharged, yellow: negatively charged. c Structural models of TUBB either in HYLS1-TUBB complex or TUBB-TUBA1B complex generated by AlphaFold-Multimer. For each complex, five models were overlaid. d Structural model of TUBB lacking the CTT generated by AlphaFold-Multimer and IF images of RPE-1 cells transfected with pCMV-TUBB with or without the CTT. Scale bars: 10 μm and 1 μm. Arrowheads: magnified area. e Quantification of frequency of interphase cells with tubulin-based superstructures similar to incomplete MT superstructures in ( d ). n = 3 independent experiments, 30 cells each. f Representative IF images of RPE-1 TetOn-X-FLAG cells (X = TUBB WT or G437E) transfected with pCMV-HA-HYLS1 and siControl or siTUBB, and treated with doxycycline. Scale bar: 10 μm. Arrowheads: incomplete MT superstructures. g Quantification of frequency of interphase cells with incomplete MT superstructures in ( f ). n = 3 independent experiments, 30 cells each. h Representative IF images of RPE-1 TetOn-X-FLAG cells (X = TUBB WT or G437E) subjected to centriole stability assay with STLC. Before the STLC treatment, the cells were transfected with siControl or siTUBB and were treated with doxycycline. Scale bar: 5 μm. Arrowheads: Centrin foci (centrioles). i Quantification of frequency of mitotic cells with <4 Centrin foci in ( h ). Only groups treated with STLC for 18 h are shown. n = 3 independent experiments, 50 cells each. j Schematic showing a speculative model of centriole triplet microtubule assembly promoted by HYLS1. Data are represented as mean ± s.d. P values were calculated by one-way ANOVA with Tukey’s multiple comparisons test ( g , i ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibodies against RFP/mCherry [MBL, PM005, Lot 046, immunofluorescence (IF) 1:500, immunoblotting (IB) 1:1000], CEP97 [Novus Biologicals, NBP1-83591, Lot A107064, IF 1:500], α-tubulin [MBL, PM054, Lot 007, IF 1:500, IB 1:1000], β-tubulin [Thermo Fisher Scientific, PA5-16863, Lot XG3654589, IF 1:500], acetylated tubulin [Abcam, ab179484, IF 1:500], FLAG [Sigma, F7425, Lot 086M4803V, IF 1:500], HA [Abcam, ab9110, IF 1:1000], PCNT [Abcam, ab4448, IF 1:1000], HYLS1 [Novus Biologicals, NBP1-56899, Lot QC25211-90821, IB 1:1000], CEP152 [Bethyl Laboratories, A302-480A, IF 1:1000], CEP192 [Bethyl Laboratories, A302-324A, IF 1:1000], CP110 [Proteintech, 12780-1-AP, IF 1:500], ANKRD26 [GeneTex, GTX128255, Lot 41458, IF 1:1000], CEP44 [Proteintech, 24457-1-AP, IF 1:500], CEP57 [GeneTex, GTX115931, Lot 40289, IF 1:500], CEP63 [Proteintech, 16268-1-AP, IF 1:500], CEP135 [Abcam, ab196809, Lot GR199116-6, IF 1:500], CEP295 [Sigma, HPA038596, Lot R33456, IF 1:500], CPAP [Proteintech, 11517-1-AP, IF 1:500], PLK1 [Bethyl Laboratories, A300-251A, IF 1:500], and ARL13B [Proteintech, 17711-1-AP, IF 1:500]; goat polyclonal antibody against PCNT [Santa Cruz Biotechnology, sc-28145, Lot C1716, IF 1:500]; mouse monoclonal antibodies against Centrin [Merck Millipore, 04-1624, Clone 20H5, IF 1:1000], γ-tubulin [Sigma, T5326, Clone GTU88, IF 1:1000], α-tubulin [Sigma, T5168, Clone B-5-1-2, IF 1:1000], acetylated tubulin [Sigma, T7451, Clone 6-11B-1, IF 1:500], polyglutamylation modification [AdipoGen, AG-20B-0020-C100, Clone GT335, IF 1:500], monoglycylated tubulin [Merck Millipore, MABS277, Clone TAP952, IF 1:500], polyglycylated tubulin [Merck Millipore, MABS276, Clone AXO49, IF 1:500], FLAG [Sigma, F1804, Clone M2, IF 1:1000, IB 1:1000], HA [Biolegend, 901501, Clone 16B12, IF 1:500], SAS6 [Santa Cruz Biotechnology, sc-81431, IF 1:500], HSP90 [BD Biosciences, 610419, IB 1:2000], EB1 [BD Biosciences, 610534, Clone 5/EB1, IF 1:500]; rat monoclonal antibodies against HA [Merck, 11867423001, Clone 3F10, IF 1:500], Centrin 2 [BioLegend, 698602, Clone W16110A, IF 1:500].

Techniques: Generated, Transfection, Stability Assay