Journal: Scientific Reports

Article Title: TRPM7 and MagT1 in the osteogenic differentiation of human mesenchymal stem cells in vitro

doi: 10.1038/s41598-018-34324-8

Figure Lengend Snippet: Osteogenic differentiation associates with the upregulation of TRPM7 and MagT1. ( A ) hMSC were cultured in OM or CM for 3, 6, 10 and 14 days. Real-Time PCR was performed on RNA extracted from hMSC using primers designed on TRPM7 and MagT1 sequence. ( B ) Western blot was performed on extracts from hMSC cultured in OM or CM for 6 and 14 days using antibodies against TRPM7 or MagT1. Actin was used as a control of loading. A representative blot is shown and quantification is provided in the Supplementary information (Fig. ).

Article Snippet: Western blot was performed on hMSC induced to differentiate into osteoblasts, exposed to siRNAs targeting TRPM7 and/or MagT1 or cultured in medium containing 0.1 mM Mg. After lysis, samples (80 μg/lane) were separated on SDS-polyacrylamide gel, transferred to nitrocellulose sheets at 150 mA for 16 h, and probed with antibodies against TRPM7 (Bethyl), MagT1 (Abcam), LC3B, beclin 1 (Cell Signalling Technology) and actin (Santa Cruz Biotechnology).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Sequencing, Western Blot, Control

Journal: Scientific Reports

Article Title: TRPM7 and MagT1 in the osteogenic differentiation of human mesenchymal stem cells in vitro

doi: 10.1038/s41598-018-34324-8

Figure Lengend Snippet: Specific siRNAs silence TRPM7 or MagT1 in hMSC. ( A ) After exposure to siRNA, hMSC were cultured in CM for 3 days. Real-Time PCR was performed on RNA extracted from hMSC using primers designed on the sequence of TRPM7 and MagT1 . The controls, indicated as -, were exposed to non-silencing, scrambled sequences. ( B ) Western blot was performed on extracts from hMSC after 3 days silencing. Antibodies against TRPM7 or MagT1 were used. Actin was used as a control of loading. A representative blot is shown and quantification is provided in the Supplementary information (Fig. ). ( C ) Total Mg was measured using the fluorescent chemosensor DCHQ5 as described.

Article Snippet: Western blot was performed on hMSC induced to differentiate into osteoblasts, exposed to siRNAs targeting TRPM7 and/or MagT1 or cultured in medium containing 0.1 mM Mg. After lysis, samples (80 μg/lane) were separated on SDS-polyacrylamide gel, transferred to nitrocellulose sheets at 150 mA for 16 h, and probed with antibodies against TRPM7 (Bethyl), MagT1 (Abcam), LC3B, beclin 1 (Cell Signalling Technology) and actin (Santa Cruz Biotechnology).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Sequencing, Western Blot, Control

Journal: Scientific Reports

Article Title: TRPM7 and MagT1 in the osteogenic differentiation of human mesenchymal stem cells in vitro

doi: 10.1038/s41598-018-34324-8

Figure Lengend Snippet: siRNAs targeting TRPM7 or MagT1 enhance osteogenic differentiation. ( A ) After exposure to siRNA, hMSC were cultured in CM or OM for 3 days. Real-Time PCR was performed on RNA extracted from hMSC using primers designed on RUNX2 and COL1A1 sequence. ( B ) ELISA for RUNX2 and collagen type 1 was conducted on extracts from hMSC cultured in CM or OM for 5 days. The controls, indicated as -, were exposed to non-silencing, scrambled sequences.

Article Snippet: Western blot was performed on hMSC induced to differentiate into osteoblasts, exposed to siRNAs targeting TRPM7 and/or MagT1 or cultured in medium containing 0.1 mM Mg. After lysis, samples (80 μg/lane) were separated on SDS-polyacrylamide gel, transferred to nitrocellulose sheets at 150 mA for 16 h, and probed with antibodies against TRPM7 (Bethyl), MagT1 (Abcam), LC3B, beclin 1 (Cell Signalling Technology) and actin (Santa Cruz Biotechnology).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Sequencing, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: TRPM7 and MagT1 in the osteogenic differentiation of human mesenchymal stem cells in vitro

doi: 10.1038/s41598-018-34324-8

Figure Lengend Snippet: siRNAs targeting MagT1 or TRPM7 induce the deposition of calcium. Alizarin Red S staining was performed after exposure to CM or OM for 14 days. Whole well image (upper left panel) and photographs taken at 10X magnification (lower panel) are shown. After acid extraction the absorbance was measured at 550 nm (upper right panel).

Article Snippet: Western blot was performed on hMSC induced to differentiate into osteoblasts, exposed to siRNAs targeting TRPM7 and/or MagT1 or cultured in medium containing 0.1 mM Mg. After lysis, samples (80 μg/lane) were separated on SDS-polyacrylamide gel, transferred to nitrocellulose sheets at 150 mA for 16 h, and probed with antibodies against TRPM7 (Bethyl), MagT1 (Abcam), LC3B, beclin 1 (Cell Signalling Technology) and actin (Santa Cruz Biotechnology).

Techniques: Staining, Extraction

Journal: Scientific Reports

Article Title: TRPM7 and MagT1 in the osteogenic differentiation of human mesenchymal stem cells in vitro

doi: 10.1038/s41598-018-34324-8

Figure Lengend Snippet: siRNAs targeting TRPM7 or MagT1 induce autophagy. hMSC were exposed to siRNAs targeting TRPM7 or MagT1 , or to non-silencing sequences (−), for 3 days. ( A ) The cells were lysed and Western blot was performed using antibodies against LC3B and beclin 1. Actin was used as a control of loading. A representative blot is shown and quantification is provided in the Supplementary information (Fig. ). ( B ) Autophagic flux was detected by Tandem fluorescent-tagged LC3 assay as described in the methods. ( C ) Intracellular free Ca was measured using Fura-2-AM as described. ( D ) Real-Time PCR was performed on RNA extracted from hMSC in CM or OM, treated or untreated with bafilomycin A1 (10 nM) for 3 days. Primers designed on RUNX2 sequence were used.

Article Snippet: Western blot was performed on hMSC induced to differentiate into osteoblasts, exposed to siRNAs targeting TRPM7 and/or MagT1 or cultured in medium containing 0.1 mM Mg. After lysis, samples (80 μg/lane) were separated on SDS-polyacrylamide gel, transferred to nitrocellulose sheets at 150 mA for 16 h, and probed with antibodies against TRPM7 (Bethyl), MagT1 (Abcam), LC3B, beclin 1 (Cell Signalling Technology) and actin (Santa Cruz Biotechnology).

Techniques: Western Blot, Control, Real-time Polymerase Chain Reaction, Sequencing

Journal: Scientific Reports

Article Title: TRPM7 and MagT1 in the osteogenic differentiation of human mesenchymal stem cells in vitro

doi: 10.1038/s41598-018-34324-8

Figure Lengend Snippet: Mg deficiency accelerates osteogenic differentiation. ( A ) Alizarin Red S staining was performed on hMSC cultured in 0.1 or 1.0 mM Mg with or without the osteogenic cocktail for 14 days. Whole well image (upper left panel) and photographs taken at 10X magnification (lower panel) are shown. Absorbance was measured at 550 nm after acid extraction (upper right panel). ( B ) Real-Time PCR was performed on RNA extracted from hMSC cultured in 1.0 or 0.1 mM Mg, additioned or not with the osteogenic cocktail, treated or untreated with bafilomycin A1 (10 nM) for 3 days. Primers designed on RUNX2 sequence were used. ( C ) Total Mg was measured using the fluorescent chemosensor DCHQ5 as described. ( D ) Western blot was performed on extracts from hMSC cultured in 1.0 or 0.1 mM Mg for 3 and 6 days using antibodies against TRPM7 or MagT1. Actin was used as a control of loading. A representative blot is shown and quantification is provided in the Supplementary information (Fig. ).

Article Snippet: Western blot was performed on hMSC induced to differentiate into osteoblasts, exposed to siRNAs targeting TRPM7 and/or MagT1 or cultured in medium containing 0.1 mM Mg. After lysis, samples (80 μg/lane) were separated on SDS-polyacrylamide gel, transferred to nitrocellulose sheets at 150 mA for 16 h, and probed with antibodies against TRPM7 (Bethyl), MagT1 (Abcam), LC3B, beclin 1 (Cell Signalling Technology) and actin (Santa Cruz Biotechnology).

Techniques: Staining, Cell Culture, Extraction, Real-time Polymerase Chain Reaction, Sequencing, Western Blot, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Functional transient receptor potential melastatin 7 channels are critical for human mast cell survival.

doi: 10.4049/jimmunol.179.6.4045

Figure Lengend Snippet: FIGURE 6. The siRNA-mediated knockdown of TRPM7 mRNA. A, Gel demonstrating reduced mRNA expression for TRPM7 in HMC-1 fol- lowing transfection with three different TRPM7 siRNA oligonucleotides (A, B, and C). B, Quantification of the gel shown in A using densitometry. No knockdown of -actin was seen. C, TRPV2 mRNA was not knocked down by TRPM7 siRNA constructs.

Article Snippet: We used the following reagents: stem cell factor, IL-6, and IL-10 (R&D Systems); mouse IgG1 mAb YB5B8 (anti-CD117; Cambridge Bioscience); sheep anti-mouse IgG1 Dynabeads (Dynal Biotech); DMEM/Glutamax/ HEPES, antibiotic/antimycotic solution, MEM nonessential amino acids, and FCS (Invitrogen Life Technologies); HiPerfect transfection reagent (Qiagen); TRPM7 short interfering RNA (siRNA) and controls (Invitrogen Life Technologies); TRPM7 short hairpin RNA (shRNA) and controls (Biofocus).

Techniques: Knockdown, Expressing, Transfection, Construct

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Functional transient receptor potential melastatin 7 channels are critical for human mast cell survival.

doi: 10.4049/jimmunol.179.6.4045

Figure Lengend Snippet: FIGURE 7. The siRNA-mediated reduction in TRPM7 currents and cell survival. A, TRPM7 siRNAs induced death of HMC-1 cells over a period of 72 h from transfection. This effect was not rescued significantly by 10 mM extracellular Mg2. Mean of three experiments performed on different occasions, each in triplicate is shown. p 0.001 by ANOVA at days 1, 2, and 3. B, In viable cells, TRPM7 currents were markedly reduced at both 48 and 72 h by TRPM7 siRNA constructs but not a scrambled control siRNA. Mean data from two experiments performed on separate occasions (n 6 cells per condition).

Article Snippet: We used the following reagents: stem cell factor, IL-6, and IL-10 (R&D Systems); mouse IgG1 mAb YB5B8 (anti-CD117; Cambridge Bioscience); sheep anti-mouse IgG1 Dynabeads (Dynal Biotech); DMEM/Glutamax/ HEPES, antibiotic/antimycotic solution, MEM nonessential amino acids, and FCS (Invitrogen Life Technologies); HiPerfect transfection reagent (Qiagen); TRPM7 short interfering RNA (siRNA) and controls (Invitrogen Life Technologies); TRPM7 short hairpin RNA (shRNA) and controls (Biofocus).

Techniques: Transfection, Construct, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Functional transient receptor potential melastatin 7 channels are critical for human mast cell survival.

doi: 10.4049/jimmunol.179.6.4045

Figure Lengend Snippet: FIGURE 8. The shRNA-mediated reduction in TRPM7 currents and HLMC survival. A, Transfection of primary HLMC with the Ad5C20Att01 adenovirus containing an eGFP construct reveals 70% transfection effi- ciency. Brightfield image is overlaid with fluorescent imaging at magnifi- cation of 100. B, Adenoviral transduction of HLMC with shRNA to TRPM7 reduces cell survival, but shRNA to GFP and overexpressed eGFP have no effect on cell viability. Mean of three experiments performed on HLMC from two donors is shown. , p 0.05 compared with control. C, Adenoviral transfection of HLMC with shRNA to TRPM7 reduces the size of the TRPM7 currents in three different target sequences for human TRPM7 (referred to as v1, v2, and v3). Mean data from two HLMC donors for TRPM7 v2 and v3 (n 6 cells per condition), and from a separate HLMC donor for TRPM7 v1 (n 3 cells per condition). There was no change in the size of the currents under control conditions.

Article Snippet: We used the following reagents: stem cell factor, IL-6, and IL-10 (R&D Systems); mouse IgG1 mAb YB5B8 (anti-CD117; Cambridge Bioscience); sheep anti-mouse IgG1 Dynabeads (Dynal Biotech); DMEM/Glutamax/ HEPES, antibiotic/antimycotic solution, MEM nonessential amino acids, and FCS (Invitrogen Life Technologies); HiPerfect transfection reagent (Qiagen); TRPM7 short interfering RNA (siRNA) and controls (Invitrogen Life Technologies); TRPM7 short hairpin RNA (shRNA) and controls (Biofocus).

Techniques: shRNA, Transfection, Construct, Imaging, Transduction, Control

Journal: Scientific Reports

Article Title: TRPM7 and MagT1 in the osteogenic differentiation of human mesenchymal stem cells in vitro

doi: 10.1038/s41598-018-34324-8

Figure Lengend Snippet: siRNAs targeting TRPM7 or MagT1 induce autophagy. hMSC were exposed to siRNAs targeting TRPM7 or MagT1 , or to non-silencing sequences (−), for 3 days. ( A ) The cells were lysed and Western blot was performed using antibodies against LC3B and beclin 1. Actin was used as a control of loading. A representative blot is shown and quantification is provided in the Supplementary information (Fig. ). ( B ) Autophagic flux was detected by Tandem fluorescent-tagged LC3 assay as described in the methods. ( C ) Intracellular free Ca was measured using Fura-2-AM as described. ( D ) Real-Time PCR was performed on RNA extracted from hMSC in CM or OM, treated or untreated with bafilomycin A1 (10 nM) for 3 days. Primers designed on RUNX2 sequence were used.

Article Snippet: Western blot was performed on hMSC induced to differentiate into osteoblasts, exposed to siRNAs targeting TRPM7 and/or MagT1 or cultured in medium containing 0.1 mM Mg. After lysis, samples (80 μg/lane) were separated on SDS-polyacrylamide gel, transferred to nitrocellulose sheets at 150 mA for 16 h, and probed with antibodies against TRPM7 (Bethyl), MagT1 (Abcam), LC3B, beclin 1 (Cell Signalling Technology) and actin (Santa Cruz Biotechnology).

Techniques: Western Blot, Control, Real-time Polymerase Chain Reaction, Sequencing

Journal: Scientific Reports

Article Title: TRPM7 and MagT1 in the osteogenic differentiation of human mesenchymal stem cells in vitro

doi: 10.1038/s41598-018-34324-8

Figure Lengend Snippet: Mg deficiency induces autophagy. ( A ) hMSC were cultured in 0.1 or 1.0 mM Mg. After 3 days the cells were lysed and Western blot was performed using antibodies against LC3B and beclin 1. Actin was used as a control of loading. A representative blot is shown and quantification is provided in the Supplementary information (Fig. ). ( B ) Autophagic flux was detected by Tandem fluorescent-tagged LC3 assay as described in the methods. ( C ) Intracellular free Ca was measured using Fura-2-AM as described.

Article Snippet: Western blot was performed on hMSC induced to differentiate into osteoblasts, exposed to siRNAs targeting TRPM7 and/or MagT1 or cultured in medium containing 0.1 mM Mg. After lysis, samples (80 μg/lane) were separated on SDS-polyacrylamide gel, transferred to nitrocellulose sheets at 150 mA for 16 h, and probed with antibodies against TRPM7 (Bethyl), MagT1 (Abcam), LC3B, beclin 1 (Cell Signalling Technology) and actin (Santa Cruz Biotechnology).

Techniques: Cell Culture, Western Blot, Control