Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Expression and functional analysis of TRPV1 in BMMs and Osteoclasts. (a) BMMs stained for the macrophage marker CD11b (red) and TRPV1 (green) depict the latter’s expression in these cells, both in the absence (upper panel) and presence (lower panel) of RANKL. (b) Expression of TRPV1 (red) in phalloidin-stained osteoclasts (green, upper panel) is confirmed by a peptide segment against anti-TRPV1 antibody (lower panel) that reduces the specific fluorescence signal intensity of the TRPV1 channel.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: Expressing, Functional Assay, Staining, Marker, Fluorescence

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Functional analysis of TRPV1 in BMMs. (a) BMMs were assessed for intracellular Ca 2+ levels upon TRPV1 modulation. Representative intensity profiles of Fluo4-AM intensity at different frames are indicated. (b) Time series graphs of intracellular Fluo4-AM intensities across 200 frames of live imaging. The arrow at the x-axis signifies the time of addition of the respective drugs (20th frame). Gray traces are of individual cells, and the black trace represents the average of 50 cells. (c) Compiled average of different treatments of BMMs, individual cell traces omitted.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: Functional Assay, Imaging

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Functional analysis of TRPV1 in BMMs grown on the CMT:HEMA hydrogel. (a) BMMs grown on hydrogels to check for the endogenous levels of Ca 2+ using Fluo4-AM Ca 2+ -sensitive dye. TRPV1 activation elevates the intracellular Ca 2+ levels, as is quantified in (b); n = 100 cells; one-way ANOVA; ns: non-significant, **** p < 0.0001. (c) Correlation representation of the area of cells and per unit area intensity of Fluo4-AM depicts strong positive correlations under basal and TRPV1-activated conditions but not upon inhibition of the channel.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: Functional Assay, Activation Assay, Inhibition

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Morphological analysis of BMMs grown on the hydrogel. (a) Representative images of BMMs grown on glass or hydrogel in the presence of RANKL and TRPV1 modulators. Right panels denote marked inset of respective images. Phalloidin intensity (b) and morphometric analyses of BMM’s area (c), perimeter (d), length (e), width (f), and LWR (g). n = 18–51 cells per group; one-way ANOVA; ns: non-significant, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques:

Journal: ACS Omega

Article Title: Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

doi: 10.1021/acsomega.1c06915

Figure Lengend Snippet: Differentiation propensities of BMMs into osteoclasts grown on hydrogel. (a) Representative TRAP assay of BMMs grown on the hydrogel in the presence of the TRPV1 activator (RTX) and inhibitor (5′-IRTX) under differentiating conditions (MCSF + RANKL). (b,c) Quantitation of TRAP-positive cells and multinucleated cells in the presence of capsaicin (b) and RTX (c) shows elevated osteoclastogenesis as compared to MCSF and CMT:HEMA control groups. n = 5–10; one-way ANOVA; ** p < 0.01, *** p < 0.005, **** p < 0.001.

Article Snippet: For confirming the specificity of the antibody, TRPV1 epitope-specific peptide (Alomone Labs) was used.

Techniques: TRAP Assay, Quantitation Assay

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Immunostaining of original tumor, low passage, and high passage KCI-MENG1 cells. The original patient-derived tumor ( top row ) showed moderate and patchy immunoreactivity for epithelial membrane antigen (EMA); strong and diffuse immunostaining for progesterone receptor (PR); and a Ki-67 proliferative index of 2–3%. There was also strong immunostaining for N-cadherin and vimentin. KCI-MENG1-LP cells ( middle row ) and KCI-MENG1-HP cells ( bottom row ) maintained expression of EMA, N-cadherin, and vimentin but had significantly reduced PR expression compared to the original tumor. Whereas Ki-67 labeling was found in only a small number of cells in the original tumor and low passage cells, it was positive in virtually all P84 cells. Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunostaining, Derivative Assay, Membrane, Expressing, Labeling

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Immunostaining of original patient tumor, low and high passage KCI-MENG1 cells, and subcutaneous xenograft tumor. The original patient-derived tumor showed moderate immunoreactivity for E-cadherin which was maintained in all in vitro and in vivo models. Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunostaining, Derivative Assay, In Vitro, In Vivo

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Meningioma cell lines reported in the literature

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Southern Blot, Injection, Expressing, TRAP Assay, Activity Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Human meningioma mouse xenograft model KCI-MENG1-LPSX generated with the spontaneously immortal cell line KCI-MENG1-LP. Tumors from immunocompromised SCID mice were dissected ( a ) and the derivative cell line KCI-MENG1-LPSX CL was generated. The H&E staining of the mouse tumor revealed a pattern of moderately cellular meningothelial cells similar to the original patient tumor ( b ). The KCI-MENG1-LPSX CL cells were composed of the round-shaped cells similar to the high passage parent cell line KCI-MENG1-HP ( c ). The EMA, PR, and N-cadherin IHC of the mouse tumor highly resembled the original patient-derived tumor ( d top row ). The vimentin- and Ki-67-stained cells in the mouse tumor tissue were markedly more abundant and more intensely stained than in the original tumor ( d top row ). KCI-MENG1-LPSX CL cells displayed the same patterns of immunostaining as the high passage parent cell line KCI-MENG1-HP, including the loss of PR staining ( d bottom row ). Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Generated, Staining, Derivative Assay, Immunostaining

Journal: Frontiers in endocrinology

Article Title: A novel link between chronic inflammation and humanin regulation in children.

doi: 10.3389/fendo.2023.1142310

Figure Lengend Snippet: FIGURE 4 Local effects of TNF on humanin, PCNA, SOX9 and TRAF2 expressions in growth plates tissue specimens (n=3) in patient 1 and patient 2 analyzed separately. Quantitative analysis of (A, E) humanin staining, (B, F) PCNA staining, (C, G) SOX9 staining, and (D, H) TRAF2 staining. Error bars indicate mean ± SE. Students t-test was used to analyze differences between groups.

Article Snippet: Next, slides were incubated with anti-humanin antibody (NB100-56877; Novus Biologicals, Littleton, Colorado, USA), anti-proliferating cell nuclear antigen (PCNA) antibody (ab-18197; Abcam, Cambridge, United Kingdom), anti-SOX9 antibody (ab-5355; Sigma-Aldrich, Burlington, MA, USA), and anti-TRAF2 antibody (NB100-56173SS; Novus Biologicals, Littleton, Colorado, USA) overnight at 4°C, 1:300 diluted for all antibodies.

Techniques: Staining

Journal: Frontiers in endocrinology

Article Title: A novel link between chronic inflammation and humanin regulation in children.

doi: 10.3389/fendo.2023.1142310

Figure Lengend Snippet: FIGURE 5 TNF suppressed SOX9, PCNA and TRAF2 expressions in human growth plate tissue specimens (n=6) obtained from 2 children or human chondrocytes. (A, B) Quantitative analysis of SOX9 staining (yellow arrows), calculated as number of positive cells per mm². (C) Relative expression of SOX9 assessed by qPCR in HCS-2/8 cells treated for 72 hours with TNF at 10, 30, 100, 300 ng/ml concentrations (n=3). (D, E) Quantitative analysis of PCNA staining (yellow arrows), calculated as number of positive cells per mm². (F) Relative expression of PCNA assessed by qPCR in HCS-2/8 cells treated for 72 hours with TNF at 10, 30, 100, 300 ng/ml concentrations (n=3). (G) Western blot analysis of SOX9 and PCNA expressions in HCS- 2/8 cells treated with TNF (100 ng/ml). (H, I) Quantification of SOX9 and PCNA expressions by three independent Western blot experiments. (J, K) Quantitative analysis of TRAF2 staining (yellow arrows), calculated as number of positive cells per mm². Error bars indicate mean ± SE. Students t- test was used to analyze differences between groups.

Article Snippet: Next, slides were incubated with anti-humanin antibody (NB100-56877; Novus Biologicals, Littleton, Colorado, USA), anti-proliferating cell nuclear antigen (PCNA) antibody (ab-18197; Abcam, Cambridge, United Kingdom), anti-SOX9 antibody (ab-5355; Sigma-Aldrich, Burlington, MA, USA), and anti-TRAF2 antibody (NB100-56173SS; Novus Biologicals, Littleton, Colorado, USA) overnight at 4°C, 1:300 diluted for all antibodies.

Techniques: Staining, Expressing, Western Blot

Journal: The EMBO Journal

Article Title: Cell‐to‐cell transmission of C9orf72 poly‐(Gly‐Ala) triggers key features of ALS / FTD

doi: 10.15252/embj.2019102811

Figure Lengend Snippet: A–C Primary hippocampal neurons were transduced with GFP or GA 175 ‐GFP (DIV4 + 4) and co‐cultured with naïve primary neurons for 4 days. Endogenous TDP‐43 and poly‐GA aggregates in donor and receiver coverslips were analyzed by immunofluorescence. (A) Schematic representation of co‐culture experiments. (B) Cytoplasmic TDP‐43 immunostaining is elevated not only in poly‐GA‐transduced neurons, but also in the non‐transduced receiver cells. White and red arrows indicate cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. (C) Automated quantification of cells with cytoplasmic TDP‐43 in GFP‐ or GA 175 ‐GFP‐transduced (donor) and non‐transduced (receiver) neurons. Cells with and without GFP signal were counted separately (indicated by +/−). Two groups (GFP‐negative donor and GFP‐positive receiver) were excluded due to very high GFP transduction rate and very low GFP transmission rate. n = 4 biological replicates. In total, 283 donor GFP, 273 donor GA 175 ‐GFP, 284 receiver GFP, and 266 receiver GA 175 ‐GFP cells were analyzed. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. *** P < 0.001. D–G Co‐culture model in HeLa cells transfected with iRFP or GA 175 ‐iRFP in the donor compartment and TDP‐43 ΔNLS ‐GFP in donor and receiver compartments. (D) Immunofluorescence staining and (E) automatic quantification of TDP‐43 ΔNLS aggregate number per cell, (F) in addition to filter trap assay of SDS‐insoluble TDP‐43 ΔNLS ‐GFP aggregates compared in iRFP‐ or GA 175 ‐iRFP‐transfected cells. In (E) n = 3 biological replicates with 368 donor iRFP, 251 donor GA 175 ‐iRFP, 430 receiver iRFP, and 328 GA 175 ‐iRFP cells were analyzed. Cells with and without GFP signal were analyzed separately (indicated by +/−). White and red arrows indicate cells with cytoplasmic TDP‐43 in GFP‐positive and GFP‐negative cells, respectively. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. (G) GFP mRNA expression levels were measured by qPCR. RNA levels were normalized to GAPDH, β‐actin, and β2‐microglobulin mRNA. Bar graphs of mean ± SD. Unpaired two‐tailed t ‐test with Welch's correction. * P < 0.05, and ** P < 0.01. Data information: Scale bars denote 20 μm. Additional larger fields of view for Fig B and D are shown in Appendix Fig S2A and E . Source data are available online for this figure.

Article Snippet: The following primers were used: EGFP (Mr04097229_mr, Thermo Fisher Scientific), ACTB (Hs01060665_g1, Thermo Fisher Scientific), B2M (4326319E, Thermo Fisher Scientific), GAPDH (Hs02758991_g1, Thermo Fisher Scientific), and tagRFP (PrimerQuest Tool and Supply, Integrated DNA Technologies).

Techniques: Transduction, Cell Culture, Immunofluorescence, Co-Culture Assay, Immunostaining, Transmission Assay, Transfection, Staining, TRAP Assay, Expressing, Two Tailed Test

Journal: The EMBO Journal

Article Title: Cell‐to‐cell transmission of C9orf72 poly‐(Gly‐Ala) triggers key features of ALS / FTD

doi: 10.15252/embj.2019102811

Figure Lengend Snippet: A, B Double immunofluorescence of the proteasome subunit PSMC4 and poly‐GA inclusions in spinal cord of GA 149 ‐CFP transgenic mouse and cortex of a C9orf72 patient compared with controls. Scale bar denotes 20 μm. C, D Co‐culture model of HeLa cells transfected with iRFP or GA 175 ‐iRFP in the donor compartment and an Ub G76V ‐GFP proteostasis reporter in donor and receiver compartments (48 h). (C) Separate analysis of both compartments by immunoblot and (D) immunoblot quantification. For quantitative analysis of immunoblots, Ub G76V ‐GFP was normalized to calnexin. n = 3 biological replicates. Scatter plot with mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. Red dashed line indicates the control's expression level. * P < 0.05, *** P < 0.001. E GFP mRNA expression levels were measured by qPCR. RNA levels were normalized to GAPDH, β‐actin, and β2‐microglobulin mRNA. Bar graphs of mean ± SD. n = 3 biological replicates. Unpaired two‐tailed t ‐test with Welch's correction. F, G Flow cytometry analysis of a Ub G76V ‐GFP reporter cell line incubated 48 h with conditioned media from RFP or GA 175 ‐RFP‐transfected cells upon immunodepletion of poly‐GA or control depletion using unspecific IgG. n = 3 biological replicates. Scatter plot with bar graphs of mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. * P < 0.05. (G) Comparisons of the corresponding histograms for compensated RFP and Ub G76V ‐GFP fluorescence from one representative experiment that shows specific transmission of GA 175 ‐RFP associated with accumulation of Ub G76V ‐GFP in cells incubated with GA 175 ‐RFP conditioned media. For flow cytometry analysis of co‐culture experiments, see Fig E–H. Source data are available online for this figure.

Article Snippet: The following primers were used: EGFP (Mr04097229_mr, Thermo Fisher Scientific), ACTB (Hs01060665_g1, Thermo Fisher Scientific), B2M (4326319E, Thermo Fisher Scientific), GAPDH (Hs02758991_g1, Thermo Fisher Scientific), and tagRFP (PrimerQuest Tool and Supply, Integrated DNA Technologies).

Techniques: Immunofluorescence, Transgenic Assay, Co-Culture Assay, Transfection, Western Blot, Control, Expressing, Two Tailed Test, Flow Cytometry, Incubation, Immunodepletion, Fluorescence, Transmission Assay

Journal: Nature Communications

Article Title: Selenoprotein W ensures physiological bone remodeling by preventing hyperactivity of osteoclasts

doi: 10.1038/s41467-021-22565-7

Figure Lengend Snippet: a Downregulation of SELENOW during osteoclastogenesis. Osteoclast precursors were cultured with RANKL and M-CSF, and SELENOW gene expression was analysed by RT-PCR, northern blotting (NB), and immunoblotting (IB). b , c RANKL/RANK/TRAF6 axis-dependent downregulation of SELENOW . Osteoclast precursors were pretreated with interferon-γ (IFN-γ; 150 U/ml), which degrades TRAF6, 30 min prior to RANKL stimulation. Osteoclast precursors treated with IFN-γ ( b ) and TRAF6-deficient osteoclast precursors ( c ) failed to induce RANKL-mediated SELENOW downregulation. d Up- and downregulation of SELENOW via ERK and p38 activation, respectively. Osteoclast precursors were pretreated with inhibitors of ERK (PD98059), JNK (SP600125), p38 (SB203580), NF-κB (SN50), and NFATc1 (cyclosporin A, CsA) for 30 min in the presence of M-CSF and then stimulated with RANKL for 2 days. The expression levels of SELENOW were analysed using RT-PCR. e , f Decreased and increased osteoclast formation following SELENOW knockdown ( e ) and overexpression ( f ), respectively. Osteoclast precursors infected with shRNA-mediated SELENOW gene-silencing lentivirus and SELENOW -overexpressing retrovirus were differentiated into osteoclasts and TRAP-positive multi-nucleated cells (TRAP + MNCs) with more than 3 nuclei were assessed ( n = 3). Scale bars, 100 μm. Images are representative of three independent experiments. Data represent the mean ± SD of triplicate samples. Statistical significance was determined by Student’s two-tailed t -test ( f ). One-way ANOVA was performed followed by Turkey’s test ( e ). Source data are provided as a Source Data file.

Article Snippet: For qPCR analysis, total RNA was reverse transcribed into cDNA with the Superscript First-Strand Synthesis System (Invitrogen) and the reaction was carried out on a 7500 Detection System (Applied Biosystems, Foster City, CA, USA) using the Real-time TaqMan PCR assay kit that included primer sets for NFATc1 (Mm00479445_m1), Acp5 (Mm00475698_m1), and OSCAR (Mm00558665_m1) (Thermo Fisher Scientific).

Techniques: Cell Culture, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Western Blot, Activation Assay, Expressing, Knockdown, Over Expression, Infection, shRNA, Two Tailed Test

Journal: Nature Communications

Article Title: Selenoprotein W ensures physiological bone remodeling by preventing hyperactivity of osteoclasts

doi: 10.1038/s41467-021-22565-7

Figure Lengend Snippet: a μCT analysis of proximal tibiae from wild-type (WT) male littermates and age/sex-matched SELENOW −/− mice at 10 weeks. BV/TV, trabecular bone volume per tissue volume; Tb.N, trabecular bone number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; BMD, bone mineral density. Scale bar, 0.5 mm. b , c Reduced osteoclast formation on the trabecular bone surface of SELENOW −/− mice. H&E- and TRAP-stained sections of tibiae were used to detect osteoblasts ( b ) and osteoclasts ( c ), respectively. NOb/BS, number of osteoblasts per bone surface; NOc/BS, BV/TV, trabecular bone volume per tissue volume; number of osteoclasts per bone surface. In addition, osteoclast size and eroded bone surface were analysed from the TRAP-stained sections. Scale bar, 100 μm. d Increased bone mass phenotype in μCT analysis of proximal tibiae from WT male littermates ( SELENOW tm1c/tm1c ; SeW fl/fl ) and age/sex-matched osteoclast-specific SELENOW knockout mice ( SELENOW tm/c/tm1c :LysM-Cre; SeW fl/fl ;LysM-Cre) at 10 weeks. Scale bar, 0.5 mm. e Analysis of NOb/BS, number of osteoblasts per bone surface, and BV/TV in H&E-stained sections. Scale bar, 100 μm. f Analysis of NOc/BS, number of osteoclasts per bone surface, osteoclast size and eroded bone surface from TRAP-stained sections. Scale bar, 100 μm. g Histomorphometric analysis of the tibia. BFR, bone formation rate. Scale bar, 10 μm. Data represent mean ± SD ( n = 7 mice per group in a – c ; n = 8 mice per group in d – g ). Statistical significance was determined by Student’s two-tailed t -test. Source data are provided as a Source Data file.

Article Snippet: For qPCR analysis, total RNA was reverse transcribed into cDNA with the Superscript First-Strand Synthesis System (Invitrogen) and the reaction was carried out on a 7500 Detection System (Applied Biosystems, Foster City, CA, USA) using the Real-time TaqMan PCR assay kit that included primer sets for NFATc1 (Mm00479445_m1), Acp5 (Mm00475698_m1), and OSCAR (Mm00558665_m1) (Thermo Fisher Scientific).

Techniques: Staining, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: Selenoprotein W ensures physiological bone remodeling by preventing hyperactivity of osteoclasts

doi: 10.1038/s41467-021-22565-7

Figure Lengend Snippet: a μCT analysis of proximal tibiae from wild-type (WT) male littermates and age/sex-matched transgenic (TG) mice at 10 weeks. Scale bar, 0.5 mm. b μCT images of calvaria and analysis of bone parameters [trabecular bone volume per tissue volume (BV/TV) and BMD]. scale bar, 3 mm. c , d Increased osteoclast formation on the trabecular bone surface of TG mice. Analysis of NOb/BS, number of osteoblasts per bone surface, from H&E-stained sections ( c ). Analysis of NOc/BS, number of osteoclasts per bone surface, osteoclast size and eroded bone surface from TRAP-stained sections ( d ). Scale bar, 100 μm. e – g Whole calvaria ( e ) and cross-sections ( f ) were stained with TRAP. The number of TRAP + osteoclasts and the calvarial marrow cavity area ( g ), which reflects the degree of osteoporosis, were measured in whole sections. Scale bar, 1 mm. h The level of urinary DPD, a marker of osteoporosis, was measured by enzyme immunoassay. Data represent mean ± SD ( n = 12 mice per group in a , c , d and f , and n = 7 mice per group in b and e – h ). Statistical significance was determined by Student’s two-tailed t -test. Source data are provided as a Source Data file.

Article Snippet: For qPCR analysis, total RNA was reverse transcribed into cDNA with the Superscript First-Strand Synthesis System (Invitrogen) and the reaction was carried out on a 7500 Detection System (Applied Biosystems, Foster City, CA, USA) using the Real-time TaqMan PCR assay kit that included primer sets for NFATc1 (Mm00479445_m1), Acp5 (Mm00475698_m1), and OSCAR (Mm00558665_m1) (Thermo Fisher Scientific).

Techniques: Transgenic Assay, Staining, Marker, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Selenoprotein W ensures physiological bone remodeling by preventing hyperactivity of osteoclasts

doi: 10.1038/s41467-021-22565-7

Figure Lengend Snippet: a Osteoclastogenic transcription factors and SELENOW co-translocate into the nucleus. Osteoclast precursors infected with SELENOW -harbouring retrovirus were cultured with M-CSF and RANKL for 2 days. After cells were exposing to RANKL-free condition for 3 h and treated without or with an inhibitor of NFATc1 (cyclosporin A, CsA), cells were stimulated with RANKL for 20 min. Cytosolic and nuclear proteins were fractionated and NF-κB, NFATc1, and SELENOW levels were determined by immunoblotting. b Luciferase reporter assay. RAW264.7 cells were transfected with AP-1-, NF-κB-, and NFATc1-luciferase reporter or pcDNA3.1-His-tagged SELENOW (SeCys-13) vector. Cells were stimulated with RANKL for 24 h and luciferase activity was measured ( n = 3). c , d SELENOW interacts with NF-κB and NFATc1. Cytosolic extracts from HEK 293 T cells expressing a His-tagged SELENOW (SeCys-13) were pulled down with an anti-His-Tag antibody ( c ). Also, cytosolic extracts from HEK 293T cells with a His-tagged wild-type SELENOW (SeCys-13) and His-tagged SELENOW mutants in which SeCys-13 was replaced by cysteine (SeCys13C) or serine (SeCys13S) were immunoprecipitated (IP) with anti-His-Tag antibody and then immunoblotted (IB) with the indicated antibodies ( d ). e ChIP assay. Osteoclast precursors were cultured with M-CSF alone (d0) or with M-CSF and RANKL for 3 days (d3; left panels). Also, osteoclast precursors from wild-type (WT) and SELENOW -overexpressing transgenic (TG) mice were cultured with M-CSF and RANKL for 3 days (right panels). Following immunoprecipitation (IP) of chromatin with anti-SELENOW antibody, ChIP assay was performed to detect the promoter for NF-κB- or NFATc1-binding sites. f , g 14-3-3γ mediates nuclear translocation of NFATc1, NF-κB, and SELENOW, and osteoclast differentiation. After osteoclast precursors from WT and TG mice were cultured with M-CSF and RANKL for 2 days to induce pre-osteoclasts, the cells were exposed to M-CSF- and RANKL-free condition for 3 h and were stimulated with RANKL for indicated times ( f ; left panel). In addition, this was performed in TG mice-derived pre-osteoclasts transduced with control lentivirus (pLKO) or 14-3-3γ-targeted shRNA-harbouring lentivirus ( f ; right panel). Nuclear proteins were fractionated and subjected to immunoblotting. Osteoclast precursors from TG mice were infected with shRNA-mediated 14-3-3γ gene-silencing lentivirus and differentiated into osteoclasts ( n = 3). TRAP + MNCs with more than 3 or 10 nuclei were assessed ( g ). Scale bar, 100 μm. Data represent the mean ± SD of triplicate samples. Statistical significance was determined by Student’s two-tailed t -test ( g ). One-way ANOVA was performed followed by Turkey’s test ( b ). Images are representative of three independent experiments. Source data are provided as a Source Data file.

Article Snippet: For qPCR analysis, total RNA was reverse transcribed into cDNA with the Superscript First-Strand Synthesis System (Invitrogen) and the reaction was carried out on a 7500 Detection System (Applied Biosystems, Foster City, CA, USA) using the Real-time TaqMan PCR assay kit that included primer sets for NFATc1 (Mm00479445_m1), Acp5 (Mm00475698_m1), and OSCAR (Mm00558665_m1) (Thermo Fisher Scientific).

Techniques: Infection, Cell Culture, Western Blot, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Activity Assay, Expressing, Immunoprecipitation, Transgenic Assay, Binding Assay, Translocation Assay, Derivative Assay, Transduction, Control, shRNA, Two Tailed Test

Journal: Nature Communications

Article Title: Selenoprotein W ensures physiological bone remodeling by preventing hyperactivity of osteoclasts

doi: 10.1038/s41467-021-22565-7

Figure Lengend Snippet: a , b Induction of pre-osteoclast fusion and osteoclastic bone resorption by SELENOW. Fusion assay in pre-osteoclasts from wild-type and SELENOW -overexpressing transgenic mice ( a ) or SELENOW −/− mice ( b ). Osteoclast precursors were treated with M-CSF and RANKL for 2 days to form pre-osteoclasts following fusion assay. Osteoclast fusion rate was determined by counting TRAP + MNCs with a diameter ≥100 μm ( n = 3). c , d Pit formation. Osteoclast precursors prepared from wild-type and SELENOW -overexpressing transgenic mice ( c ) or SELENOW −/− mice ( d ) were differentiated into osteoclasts for 4 days. After mature osteoclasts were detached from the culture dish and seeded on dentine slice, cells were further cultured with M-CSF and RANKL for 2 days to allow bone resorption. Pit formation by osteoclasts is expressed as a percentage of the resorbed area on the bone slice surface ( n = 3). e Anti-apoptotic effect of SELENOW. Mature osteoclasts were transduced with SELENOW -overexpressing retrovirus and cell survival was assessed 2 days later by staining with TRAP (upper panels) or FITC-labelled phalloidin (lower panels) to detect TRAP + osteoclasts with a full actin ring ( n = 3). f Caspase activity was assessed at indicated times after mature osteoclasts were cultured as in ( e , n = 3). g Increase in the cellular redox status by SELENOW. Osteoclasts were transduced with SELENOW -overexpressing retrovirus and total thiol content was assessed at indicated times ( n = 3). h Increase in the cellular redox status by NAC. After treatment with 4 mM NAC for 24 h or no treatment, cytosolic extracts of mature osteoclasts were prepared and assayed for free thiol level ( n = 3). i Increased mature osteoclast survival by NAC ( n = 3). Osteoclasts were treated as described in h and then stained as in e . Scale bars, 100 μm. Data represent mean ± SD of triplicate samples. Statistical significance was determined by Student’s two-tailed t -test ( a – e , i ). One-way ANOVA was performed followed by Turkey’s test ( f – h ). Source data are provided as a Source Data file.

Article Snippet: For qPCR analysis, total RNA was reverse transcribed into cDNA with the Superscript First-Strand Synthesis System (Invitrogen) and the reaction was carried out on a 7500 Detection System (Applied Biosystems, Foster City, CA, USA) using the Real-time TaqMan PCR assay kit that included primer sets for NFATc1 (Mm00479445_m1), Acp5 (Mm00475698_m1), and OSCAR (Mm00558665_m1) (Thermo Fisher Scientific).

Techniques: Single Vesicle Fusion Assay, Transgenic Assay, Cell Culture, Transduction, Staining, Activity Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: SNX10 gene mutation leading to osteopetrosis with dysfunctional osteoclasts

doi: 10.1038/s41598-017-02533-2

Figure Lengend Snippet: Osteoclast differentiation of CD14 + monocytes from peripheral blood. Photographs of TRAP staining ( A ) and quantification of surface area ( B ) of CD14 + cells cultured in M-CSF (M) or M-CSF and RANKL (M/RL) for four days (C1, C2, Pt2, Pt5) or five days (C1, lower photos, and Pt1). Surfaces are presented as mean ± SEM. ( C ) Actin ring staining of cells cultured in M/RL for four days. Scale bars: 100 μm. *** P ≤ 0.001.

Article Snippet: We analyzed ACP5 (Hs00356261_m1), CTSK (Hs01080388_m1), NFATc1 (Hs00542678_m1), TCIRG1 ( ATP6i , HS00990751_m1), CLC7 (Hs01126462_m1), ITGAV (Hs00233808_m1), ITGB3 (Hs01001469_m1), DC-STAMP (Hs00229255_m1), OC-STAMP (Hs00875776_m1), ERVW-1 ( Syncytin-1 , Hs02341206_g1), SLC1A5 ( Asct2 , Hs00194540_m1), SNX10 exon 2–3 (Hs01007224_m1) and SNX10 exon 4–5 (HS01007226_m1).

Techniques: Staining, Cell Culture

Journal: Scientific Reports

Article Title: SNX10 gene mutation leading to osteopetrosis with dysfunctional osteoclasts

doi: 10.1038/s41598-017-02533-2

Figure Lengend Snippet: Patient-derived osteoclasts form actin rings but are unable to resorb bone. ( A ) TRAP staining of CD14 + cells cultured in M/RL on bone for eight days. Scale bar 40× panel: 500 μm, scale bar 100× light microscope and reflective light panel: 200 μm, scale bar 100× toluidine blue panel: 100 μm. ( B , C ) Osteoclasts were analyzed by confocal microscopy; ( B ) stained with TRITC-phalloidin (red), DAPI (blue) and the bone surface using a fluorescent bisphosphonate (AF-ALN; green). Resorption pits stain brightly with AF-ALN and can be seen in the control only. Scale bar = 100 μm; ( C ) stained with TRITC-phalloidin (red), DAPI (blue) AF-ALN (magenta) and the cell membranes with wheat germ agglutinin (green). AF-ALN omitted from upper panel of Pt2 for clarity. Black patches in C2 xy image indicates resorption pits. Bottom panel of Pt2 is a zoom of the zx section illustrating lack of resorption. Scale bar = 20 μm; ( D ) CTX-1 release in media during day 6 to 8 of culture in M/RL. C1, C6 = controls, Pt2, Pt5 = patients (n = 4–5).

Article Snippet: We analyzed ACP5 (Hs00356261_m1), CTSK (Hs01080388_m1), NFATc1 (Hs00542678_m1), TCIRG1 ( ATP6i , HS00990751_m1), CLC7 (Hs01126462_m1), ITGAV (Hs00233808_m1), ITGB3 (Hs01001469_m1), DC-STAMP (Hs00229255_m1), OC-STAMP (Hs00875776_m1), ERVW-1 ( Syncytin-1 , Hs02341206_g1), SLC1A5 ( Asct2 , Hs00194540_m1), SNX10 exon 2–3 (Hs01007224_m1) and SNX10 exon 4–5 (HS01007226_m1).

Techniques: Derivative Assay, Staining, Cell Culture, Light Microscopy, Confocal Microscopy, Control

Journal: Frontiers in Immunology

Article Title: Multi-omic analysis of the tumor microenvironment shows clinical correlations in Ph1 study of atezolizumab +/- SoC in MM

doi: 10.3389/fimmu.2023.1085893

Figure Lengend Snippet: Unsupervised integrative clustering analysis at baseline schema (A) using (B) CyTOF, (C) Olink, (D) microenvironment-specific (CD138-negative), (E) tumor-specific (CD138-positive), and (F) SNF integrated data. Cohort A: Atezo monotherapy; B1: Atezo+Len; C: Atezo+Len; D1/D2/D3: Atezo+Dara; E1: Atezo+Dara+Len; F1/F2: Atezo+Dara+Pom; F3: Dara+Pom+Dex SNF, similarity network fusion; T, tumor; TME, tumor microenvironment. The heatmaps show clustered similarity matrices, with higher values depicted with darker shades indicating greater similarity between patients. In addition, the number of patients per cohort are color coded in the tables beneath each heatmap.

Article Snippet: CD8+ T-cell activation and proliferation were analyzed using %CD8+HLA-DR+Ki-67+.23 Formalin-fixed and decalcified paraffin-embedded tissue sections (4 mm thickness) taken at baseline were used for dual-plex immunohistochemistry (IHC) assay (CD138/tartrate-resistant acid phosphatase [TRAP]) at Histogenics Corp (MA, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Multi-omic analysis of the tumor microenvironment shows clinical correlations in Ph1 study of atezolizumab +/- SoC in MM

doi: 10.3389/fimmu.2023.1085893

Figure Lengend Snippet: Integrative analysis of on-treatment samples schema (A) using (B) CyTOF, (C) microenvironment-specific (CD138-negative), (D) tumor-specific (CD138-positive), and (E) SNF data. Cohort A: Atezo monotherapy; B1: Atezo+Len; C: Atezo+Len; D1/D2/D3: Atezo+Dara; E1: Atezo+Dara+Len; F1/F2: Atezo+Dara+Pom; F3: Dara+Pom+Dex. Atezo, atezolizumab; C, cycle; Combo, combination; D, day; Dara, daratumumab; Mono, monotherapy; SNF, similarity network fusion. The heatmaps show clustered similarity matrices, with higher values depicted with darker shades indicating greater similarity between patients.

Article Snippet: CD8+ T-cell activation and proliferation were analyzed using %CD8+HLA-DR+Ki-67+.23 Formalin-fixed and decalcified paraffin-embedded tissue sections (4 mm thickness) taken at baseline were used for dual-plex immunohistochemistry (IHC) assay (CD138/tartrate-resistant acid phosphatase [TRAP]) at Histogenics Corp (MA, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Multi-omic analysis of the tumor microenvironment shows clinical correlations in Ph1 study of atezolizumab +/- SoC in MM

doi: 10.3389/fimmu.2023.1085893

Figure Lengend Snippet: Pairwise longitudinal analysis of (A) CyTOF, (B) microenvironment-specific (CD138-negative), and (C) tumor-specific (CD138-positive) data, and comparison of cell frequency in responders and non-responders of (D) CD8 TEMRA cells, (E) CD4 TEMRA cells, (F) DN T cells and (G) Tregs, and their distribution in each cluster. Mann-Whitney and Tukey’s multiple comparisons tests, *P<0.05; **P<0.01; *** P<0.001. Atezo, atezolizumab; CL, cluster; Dara, daratumumab; DN, double-negative; Mono, monotherapy; NK, natural killer; NR, non-responder; PD, programmed death; PDL, programmed death ligand; R, responder; Refract., refractory; TEMRA, terminally differentiated effector memory T cell; Treg, regulatory T cell.

Article Snippet: CD8+ T-cell activation and proliferation were analyzed using %CD8+HLA-DR+Ki-67+.23 Formalin-fixed and decalcified paraffin-embedded tissue sections (4 mm thickness) taken at baseline were used for dual-plex immunohistochemistry (IHC) assay (CD138/tartrate-resistant acid phosphatase [TRAP]) at Histogenics Corp (MA, USA).

Techniques: MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Multi-omic analysis of the tumor microenvironment shows clinical correlations in Ph1 study of atezolizumab +/- SoC in MM

doi: 10.3389/fimmu.2023.1085893

Figure Lengend Snippet: Post-treatment one vs all gene set analysis using (A) microenvironment-specific (CD138-negative), and (B) tumor-specific (CD138-positive) data. mDC, myeloid dendritic cells; NK, natural killer.

Article Snippet: CD8+ T-cell activation and proliferation were analyzed using %CD8+HLA-DR+Ki-67+.23 Formalin-fixed and decalcified paraffin-embedded tissue sections (4 mm thickness) taken at baseline were used for dual-plex immunohistochemistry (IHC) assay (CD138/tartrate-resistant acid phosphatase [TRAP]) at Histogenics Corp (MA, USA).

Techniques:

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: A bi-allelic splicing donor site variant in LRRC23 was identified from asthenozoospermia patients. ( A ) A consanguineous pedigree with two infertile males (IV-1 and IV-2). IV-1 was subjected for WES (arrow). Genotypes of the variant (blue) in all attended family members (III-1, III-2, IV-1, IV-2, IV-3, and IV-4) are confirmed by Sanger sequencing. +, wild-type allele. An infertile female sibling (IV-4) is marked in black circle. ( B ) Papanicolaou-stained sperm from the infertile male (IV-2). ( C ) Mapping of the LRRC23 variant. Mutation of G to A at the splicing donor site in the 5 th intron is predicted to prevent LRRC23 mRNA from splicing. ( D ) Sequencing chromatograms presenting the LRRC23 variant in the infertile male (IV-1) and his father (III-2). The variant is underlined and normal splicing donor site (GT) is boxed. ( E-F ) Minigene assay for testing altered splicing of LRRC23 by the variant. ( E ) Minigene constructs expressing LRRC23 ORF containing the 5 th intron (sashed) with wild-type (WT) or mutant (Mut, red) splicing donor site were generated. The constructs are tagged with FLAG and HA at N- and C-termini, respectively. ( F ) RT-PCR of the 293T cells transfected with the minigene constructs reveals the 5 th intron is not spliced out and retained by the variant. Intron-spanning primers, F1 and R1, are used. Three times biological replicated.

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Variant Assay, Sequencing, Staining, Mutagenesis, Mini Gene Assay, Construct, Expressing, Generated, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: Lrrc23 mutant mice mimicking human splice variant phenocopy male infertility and reduced sperm motility. ( A-B ) Immunoblotting of LRRC23 in testis ( A ) and epididymal sperm ( B ) from mutant male mice. Truncated LRRC23 (arrowheads) is detected from testis microsome fraction (filled), but not in mature sperm (empty), of heterozygous (+/Δ) and homozygous (Δ/Δ) males. Acetylated tubulin (AcTub) is a loading control. ( C ) Confocal images of immunostained LRRC23 in Lrrc23 +/Δ and Lrrc23 Δ/Δ epididymal sperm. Samples from WT were used for positive or negative control of normal or truncated LRRC23 ( A, B , and C ). ( D ) Epididymal sperm counts. n.s., not significant. ( E ) Pregnancy rate of Lrrc23 +/Δ and Lrrc23 Δ/Δ males. ( F ) Number of litters from fertile females mated with Lrrc23 +/Δ and Lrrc23 Δ/Δ males. ( G ) Swimming trajectory of Lrrc23 +/Δ and Lrrc23 Δ/Δ sperm in viscous media (0.3% methylcellulose). Swimming trajectory for 2 seconds is overlaid. ( H ) Flagellar waveforms of Lrrc23 +/Δ and Lrrc23 Δ/Δ sperm before (0 minute) and after (90 minutes) inducing capacitation. Flagellar movements for two beat cycles are overlaid and color coded in time. Circles indicate sperm counts from individual males ( D ) and pup numbers from each litter ( F ). Data represented as mean ± SEM ( D and F ). Statistical comparison was perfomed by Mann-whiteny U test (D) or Student’s t-test (F). Experiments were repeated three times with biological replications ( A, B, C, G , and H ).

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Mutagenesis, Variant Assay, Western Blot, Control, Negative Control, Comparison

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: C-terminal truncation of human LRRC23 by the splicing site mutation prevents its interaction with radial spoke (RS) head. ( A ) Sub-tomogram averaging images of RSs from Chlamydomonas reinhardtii ( left ), Trypanosoma brucei ( middle ), and mouse sperm ( right ). Original data from Electron Microscopy Data Bank was rendered. ( B ) Structure of RS in C. reinhardtii . A schematic cartoon shows the RS1 and 2. The structure of RS2 stalk is shown in inset (PDB Id: 7JRJ). ( C - D ) Purification of normal (hLRRC23 WT ) and the mutant human LRRC23 (hLRRC23 Mut ) by the splicing site mutation (c.621+1G>A) in this study. ( C ) Diagrams for the purified recombinant normal and mutant proteins tagged with tagged with GST and HA at N- and C-termini, respectively. ( D ) Purified proteins by Coomassie blue staining ( left ) and immunoblotting with a-HA ( middle ) and a-LRRC23 ( right ). Proteins matched to the predicted size were marked with asterisks. ( E ) A cartoon of the RSPH-trap approach to test LRRC23 interaction with RS proteins. Individual human RS proteins tagged with FLAG (RSPH-FLAG) are expressed in 293T cells and enriched by α-FLAG resin from cell lysates. The recombinant RSPH proteins were incubated with the purified hLRRC23 WT or hLRRC23 Mut and subjected to immunoblotting. ( F ) Interaction of hLRRC23 to a RS head component, RSPH9. The purified hLRRC23 were incubated with the RSPH-Trap (RS head, RSPH6A and RSPH9; stalk, RSPH3 and RSPH22) and subjected to immunoblotting. 5% amount of the hLRRC23s used for the trap assay were loaded as inputs. White lines in individual α-HA blot images indicate marker information (75 kDa, left ; 50 kDa, right ). Experiments were repeated four times. Purified GST was used for negative control ( SI Appendix , Fig. S4 B ). ( G ) A phylogenetic tree constructed by Maximum-likelihood analysis of the protein sequences of the C. reinhardtii RSP15 and the orthologs of LRRC23 and LRRC34. LRR37, the first LRRC23 ortholog identified in Ciona intestinalis is marked in bold. ( H ) Comparison of the reported RSP15 from C. reinhardtii and the predicted structure of LRRC23 and LRRC34 from human. Atomic structure of the C. reinhardtii RS2 containing RSP15 are represented by ribbon (RS2) and surface (RSP15) diagram ( left , PDB Id: 7JU4). Ribbon diagrams of C. reinhardtii RSP15 and AlphaFold-predicted human LRRC23 ( middle ) and LRRC34 ( right ) are shown for structural comparison. Secondary structures are color-coded. Different from C. reinhardtii RSP15 and LRRC34, LRRC23 does not display repeated α-helix (magenta) between β-sheets (gold).

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Mutagenesis, Electron Microscopy, Purification, Recombinant, Staining, Western Blot, Incubation, TRAP Assay, Marker, Negative Control, Construct, Comparison

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: Head of the third radial spoke is absent in Lrrc23 Δ/Δ sperm flagella. ( A - B ) Sub-tomogram averaging (STA) to analyze structural defects at radial spoke (RS) of WT ( A ) and Lrrc23 Δ/Δ sperm ( B ). Shown are STA images resulted from 96-nm doublet repeats from WT and Lrrc23 Δ/Δ sperm. RS2 and 3 are magnified and density to represent RS3 head and the bridge between RS2 and RS3 (red circle) is missed in Lrrc23 Δ/Δ sperm specifically. ( C ) Overwrapped STA images from 96 nm-doublet repeats from WT (gray) and Lrrc23 Δ/Δ (gold) sperm, and Chlamydomonas reinhardtii (cyan). ( D ) A proposed model of impaired sperm motility and male infertility by the LRRC23 loss of function.

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques:

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: LRRC23 mutation disrupts the third radial spoke (RS) in sperm flagellum. ( A ) Immunostaining of flagellar proteins in different compartments. Shown are midpiece (TOM20), annulus (SEPT4 and SEPT12), fibrous sheath (AKAP4), outer dense fiber (ODF2), and axoneme (acetylated tubulin, AcTub) in Lrrc23 +/Δ ( top ) and Lrrc23 Δ/Δ ( bottom ) sperm. Magnified insets are represented for annulus proteins (scale bars in insets = 2μm). Fluorescence and corresponding DIC images are merged. Sperm heads were counter stained with Hoechst. Experiments were performed with three biological replications. (B) Transmission electron microscopy images of Lrrc23 +/Δ ( left ) and Lrrc23 Δ/Δ ( right ) sperm. Shown are longitudinal section of sperm flagella. M, mitochondria; ODF, outer dense fiber; AX, axoneme; CP, central pair; MT, microtubule; FS, fibrous sheath. ( C ) Cryo-electron tomography (cryo-ET) of WT and Lrrc23 Δ/Δ sperm flagella. Shown are representative tomographic slices from WT ( left ) and Lrrc23 Δ/Δ sperm ( right ). The 9+2 axonemal structure are shown in both WT and Lrrc23 Δ/Δ in cross-sectional view ( left ). Axonemal structures are shown with proximal side of the flagellum on the left in longitudinal view ( right ). Magnified insets ( bottom ) reveal that RS1, 2, and 3 are shown in WT sperm ( left , filled arrowheads) but RS3, especially head part, is not clearly visible ( right , red arrowheads) in Lrrc23 Δ/Δ sperm. Lrrc23 +/Δ ( A and B ) or WT ( C ) sperm were used for positive control.

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Mutagenesis, Immunostaining, Fluorescence, Staining, Transmission Assay, Electron Microscopy, Tomography, Positive Control