micromonospora nigra dsm 43818t x92609 micromonospora pallida dsm 43817t  (ATCC)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Multimodal regulation of the osteoclastogenesis process by secreted group IIA phospholipase A 2

doi: 10.3389/fcell.2022.966950

Figure Lengend Snippet: Modulation of PLA 2 mRNA levels during murine osteoclastogenesis. RAW264.7 cells were treated without (w/o) or with 15–30 ng/mL RANKL for 72 h, while M-CSF–expanded OCP from wild-type (wt) and Pla2g2a -ko mice were treated with 20 ng/mL M-CSF alone (M-CSF) or with 2.5 ng/mL RANKL (M-CSF+RANKL) for 3–6 days, before RNA extraction.

Article Snippet: After 24 and 48 h, cells were double transfected with 125 pmol/well of small-interfering (si)RNAs against sPLA 2 -IIA (si–sPLA 2 -IIA) (murine group II sPLA 2 siRNA, sc-43818, from Santa Cruz Biotecnology, San Diego, CA, United States), or with non-targeting siRNAs (si–NT) (siGENOME non-targeting siRNA pool #2, cat. No. D-001206–14 from Dharmacon, Chicago, IL, United States), using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, United States), according to the manufacturer instructions.

Techniques: RNA Extraction

Journal: Frontiers in Cell and Developmental Biology

Article Title: Multimodal regulation of the osteoclastogenesis process by secreted group IIA phospholipase A 2

doi: 10.3389/fcell.2022.966950

Figure Lengend Snippet: Secreted PLA 2 activity measured in cell lysates. RAW264.7 cells were treated without (w/o) or with 15 ng/mL RANKL for 72 h, in the absence or presence of 10 nM BPB. Phospholipase A 2 activity was analyzed in cell lysates (250 μg) in absence (‒) or presence of the indicated PLA 2 inhibitors.

Article Snippet: After 24 and 48 h, cells were double transfected with 125 pmol/well of small-interfering (si)RNAs against sPLA 2 -IIA (si–sPLA 2 -IIA) (murine group II sPLA 2 siRNA, sc-43818, from Santa Cruz Biotecnology, San Diego, CA, United States), or with non-targeting siRNAs (si–NT) (siGENOME non-targeting siRNA pool #2, cat. No. D-001206–14 from Dharmacon, Chicago, IL, United States), using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, United States), according to the manufacturer instructions.

Techniques: Activity Assay, Inhibition

Journal: Frontiers in Cell and Developmental Biology

Article Title: Multimodal regulation of the osteoclastogenesis process by secreted group IIA phospholipase A 2

doi: 10.3389/fcell.2022.966950

Figure Lengend Snippet: Secreted PLA 2 -IIA interference in RAW264.7 precursor cells inhibits RANKL-induced osteoclast differentiation. RAW264.7 cells were interfered for 72 h with no-targeting (si–NT) or sPLA 2 -IIA specific (si–sPLA 2 -IIA) siRNAs, and subsequently treated in the absence (w/o) or presence of 15–30 ng/mL RANKL for the following 48 h. (A) Pla2g2a mRNA levels of 72-h interfered RAW264.7 cells were quantified by real-time qPCR, and normalized using β2-microglobulin expression, as the housekeeping gene. Data are means ± SEM from six independent experiments. *** p < 0.005, paired Student’s t -test. (B–F) After 48 h of RANKL treatment, the differentiation markers were quantified by real-time qPCR, and normalized using β2-microglobulin expression, as the housekeeping gene. The mRNA levels of Ctr were undetectable in undifferentiated cells. Data are expressed as fold of RANKL-differentiated si–NT cells (RANKL si–NT), and are shown as means ± SEM of three independent experiments. *** p < 0.005 versus RANKL si–NT (one-way ANOVA or paired Student’s t -tests). (G,H) Osteoclast syncytium formation was determined as number of nuclei/cell, by fluorescence microscopy. Data are means ± SEM of three independent experiments performed in duplicates. ** p < 0.01 versus correspondent RANKL si–NT (paired Student’s t -tests).

Article Snippet: After 24 and 48 h, cells were double transfected with 125 pmol/well of small-interfering (si)RNAs against sPLA 2 -IIA (si–sPLA 2 -IIA) (murine group II sPLA 2 siRNA, sc-43818, from Santa Cruz Biotecnology, San Diego, CA, United States), or with non-targeting siRNAs (si–NT) (siGENOME non-targeting siRNA pool #2, cat. No. D-001206–14 from Dharmacon, Chicago, IL, United States), using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, United States), according to the manufacturer instructions.

Techniques: Expressing, Fluorescence, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Multimodal regulation of the osteoclastogenesis process by secreted group IIA phospholipase A 2

doi: 10.3389/fcell.2022.966950

Figure Lengend Snippet: Bifunctional inhibitors of sPLA 2 -IIA impair RANKL-induced osteoclastogenesis of RAW264.7 macrophages. (A–E) RAW264.7 cells were treated without (w/o) or with 30 ng/mL RANKL for 72 h, in presence of the indicated sPLA 2 -IIA inhibitors (20 μM Inhib-I, 40 μM KH064, 30 μM LY311727) or with DMSO as carrier (‒). The differentiation markers were quantified by real-time qPCR, and normalized using β2-microglobulin expression, as the housekeeping gene. Data are expressed as fold of RANKL, and are means ± SEM of three independent experiments. (F) RAW264.7 cells were treated without (w/o) or with 15–30 ng/mL RANKL for 72–96 h, in presence of the above indicated sPLA 2 -IIA inhibitors or with DMSO as carrier. Osteoclast syncytium formation was determined as number of nuclei/cell, by fluorescence microscopy. Data are means ± SE of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.005 versus correspondent RANKL (paired Student’s t -tests).

Article Snippet: After 24 and 48 h, cells were double transfected with 125 pmol/well of small-interfering (si)RNAs against sPLA 2 -IIA (si–sPLA 2 -IIA) (murine group II sPLA 2 siRNA, sc-43818, from Santa Cruz Biotecnology, San Diego, CA, United States), or with non-targeting siRNAs (si–NT) (siGENOME non-targeting siRNA pool #2, cat. No. D-001206–14 from Dharmacon, Chicago, IL, United States), using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, United States), according to the manufacturer instructions.

Techniques: Inhibition, Expressing, Fluorescence, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Multimodal regulation of the osteoclastogenesis process by secreted group IIA phospholipase A 2

doi: 10.3389/fcell.2022.966950

Figure Lengend Snippet: Murine recombinant sPLA 2 -IIA stimulates RANKL-induced osteoclastogenesis of RAW264.7 macrophages. RAW264.7 cells were treated without (w/o) or with 15 ng/mL RANKL for 72 h, in presence of the indicated recombinant proteins: 30 nM murine sPLA 2 -IIA (mGIIA), 30 nM human sPLA 2 -IIA (hGIIA), 30 nM human sPLA 2 -IIA H48Q (hGIIA H48Q), 100 nM murine sPLA 2 -V (mGV); or with PBS (‒) as carrier. The differentiation markers were quantified by real-time qPCR, and normalized using β2-microglobulin expression, as the housekeeping gene. Data are expressed as fold of RANKL, and are means ± SEM of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.005 versus RANKL (paired Student’s t -tests).

Article Snippet: After 24 and 48 h, cells were double transfected with 125 pmol/well of small-interfering (si)RNAs against sPLA 2 -IIA (si–sPLA 2 -IIA) (murine group II sPLA 2 siRNA, sc-43818, from Santa Cruz Biotecnology, San Diego, CA, United States), or with non-targeting siRNAs (si–NT) (siGENOME non-targeting siRNA pool #2, cat. No. D-001206–14 from Dharmacon, Chicago, IL, United States), using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, United States), according to the manufacturer instructions.

Techniques: Recombinant, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Multimodal regulation of the osteoclastogenesis process by secreted group IIA phospholipase A 2

doi: 10.3389/fcell.2022.966950

Figure Lengend Snippet: Murine recombinant sPLA 2 -IIA stimulates RANKL-induced osteoclast fusion of RAW264.7 macrophages. RAW264.7 cells were treated without (w/o) or with 15 ng/mL RANKL for 72 h, in presence of the indicated recombinant proteins: 30 nM murine sPLA 2 -IIA (mGIIA), 30 nM human sPLA 2 -IIA (hGIIA), 30 nM human sPLA 2 -IIA H48Q (hGIIA H48Q), 100 nM murine sPLA 2 -V (mGV); or with PBS as carrier. Osteoclast syncytium formation was determined as number of nuclei/cell, by fluorescence microscopy. Data are means ± SE of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.005 versus correspondent RANKL (paired Student’s t -tests).

Article Snippet: After 24 and 48 h, cells were double transfected with 125 pmol/well of small-interfering (si)RNAs against sPLA 2 -IIA (si–sPLA 2 -IIA) (murine group II sPLA 2 siRNA, sc-43818, from Santa Cruz Biotecnology, San Diego, CA, United States), or with non-targeting siRNAs (si–NT) (siGENOME non-targeting siRNA pool #2, cat. No. D-001206–14 from Dharmacon, Chicago, IL, United States), using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, United States), according to the manufacturer instructions.

Techniques: Recombinant, Fluorescence, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Multimodal regulation of the osteoclastogenesis process by secreted group IIA phospholipase A 2

doi: 10.3389/fcell.2022.966950

Figure Lengend Snippet: The LLKYK cyclic peptide inhibits RANKL-induced Mmp-9 transcription and osteoclast fusion of RAW264.7 macrophages. (A) Alignment of sPLA 2 -IIA sequences from mus musculus (UniProtKB - P31482), homo sapiens (UniProtKB - P14555), and C. durissus (UniProtKB - P24027). Consensus symbols indicate residue conservation, as calculated with the Clustal Omega program. The pentapeptide sequences are boxed in red, and the catalytic histidine residue in the human sPLA 2 -IIA sequence is shown in red-letter code. (B–D) RAW264.7 cells were treated without (w/o) or with 15–30 ng/mL RANKL for 72 h, in presence of the carriers (‒), or 10 nM BPB, or 250 μM of the indicated peptides (LLKYK from the murine sPLA 2 -IIA sequence, FLSYK from that of human sPLA 2 -IIA, WDIYR from that of C. durissus ). The differentiation markers were quantified by real-time qPCR analysis, and normalized using β2-microglobulin expression, as the housekeeping gene. Data are expressed as fold of RANKL, and are means ± SEM of at least three independent experiments. (E) RAW264.7 cells were treated without (w/o) or with of 15 ng/mL RANKL for 72 h, in presence of the indicated peptides (250 μM), or with PBS as carrier. Osteoclast syncytium formation was determined as number of nuclei/cell, by fluorescence microscopy. Data are means ± SE of six independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.005 versus correspondent RANKL (paired Student’s t -tests).

Article Snippet: After 24 and 48 h, cells were double transfected with 125 pmol/well of small-interfering (si)RNAs against sPLA 2 -IIA (si–sPLA 2 -IIA) (murine group II sPLA 2 siRNA, sc-43818, from Santa Cruz Biotecnology, San Diego, CA, United States), or with non-targeting siRNAs (si–NT) (siGENOME non-targeting siRNA pool #2, cat. No. D-001206–14 from Dharmacon, Chicago, IL, United States), using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, United States), according to the manufacturer instructions.

Techniques: Residue, Sequencing, Expressing, Fluorescence, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Multimodal regulation of the osteoclastogenesis process by secreted group IIA phospholipase A 2

doi: 10.3389/fcell.2022.966950

Figure Lengend Snippet: The LLKYK cyclic peptide inhibits RANKL-induced osteoclast resorbing activity of RAW264.7 macrophages. RAW264.7 cells plated on Osteo Assay Surface plates were treated without (w/o) or with 15 ng/mL RANKL for 7 days, in presence of PBS (‒) or 250 μM of the indicated cyclic pentapeptides (LLKYK from murine sPLA 2 -IIA sequence, and FLSYK from that of human sPLA 2 -IIA). Below are shown representative images of the degrading activity, acquired with the EVOS XL Core microscope at a final magnification of ×2. Data are means ± SEM of three independent experiments performed in triplicates. *** p < 0.005, paired Student’s t -test.

Article Snippet: After 24 and 48 h, cells were double transfected with 125 pmol/well of small-interfering (si)RNAs against sPLA 2 -IIA (si–sPLA 2 -IIA) (murine group II sPLA 2 siRNA, sc-43818, from Santa Cruz Biotecnology, San Diego, CA, United States), or with non-targeting siRNAs (si–NT) (siGENOME non-targeting siRNA pool #2, cat. No. D-001206–14 from Dharmacon, Chicago, IL, United States), using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, United States), according to the manufacturer instructions.

Techniques: Activity Assay, Sequencing, Microscopy