Journal: Theranostics

Article Title: Molecular hydrogen triggers TRPC4-TRPC4AP-dependent reversible calcium transients via extracellular influx

doi: 10.7150/thno.124352

Figure Lengend Snippet: [Ca 2+ i ] t induced by H 2 is broad-spectrum. A. Pseudocolor and time series images of [Ca 2+ i ] changes in H 2 -free and H 2 -medium in MSCs. B. Fluo 4 averaged F/F0 trace in imaging H 2 -free (blue) and H 2 -medium (red). C. H 2 stimulates calcium transients in various cells, including mouse bone marrow-derived mesenchymal stem cells (BMSCs,C1), mouse osteoblasts (MC3T3-E1,C2), mouse myoblasts (C2C12,C3), human skin fibroblasts (ESF,C4), mouse fibroblasts (NIH-3T3,C5), , and rat neuronal cells (PC12,C6); left panels show random field fluorescence images of Ca 2+ at 0 and 120 s, and the right panels display the relative fluorescence intensity statistics and Ca 2+ flow velocity measurements over 600 s, comparing the periods before and after the introduction of H 2 . Scale bar in A, C1, C2, C4, and C5 = 100 μm; Scale bar in C3 and C6 = 25 μm.

Article Snippet: The cells were washed gently with warm Hank's Balanced Salt Solution (HBSS) (Thermo Fisher Scientific 14175095) to remove the culture medium and incubated with ion-sensitive fluorescent probes for Ca 2+ -Fluo-4-AM (5 μM ) diluted in serum-free medium (SFM) (KeyGEN, KGAF024), K + -EPG-4-AM (5 μM) diluted in SFM (Maokang MX4521), for Na + -ENG-2-AM (5 μM ) diluted in SFM, and for Ca 2+ -Rhod-2-AM (5 μM ) diluted in HBSS (YEASEN 40776ES72) for 10-30 min at 37°C to allow probe uptake.

Techniques: Imaging, Derivative Assay, Fluorescence

Journal: Theranostics

Article Title: Molecular hydrogen triggers TRPC4-TRPC4AP-dependent reversible calcium transients via extracellular influx

doi: 10.7150/thno.124352

Figure Lengend Snippet: H 2 promotes [Ca 2+ i ] without altering cellular activity or Ca 2+ overload. A. & B . Pseudocolor (A left) and time series (A right) images and the Fluo4 averaged F/F0 trace of the [Ca 2+ i ] changes under H 2 -free, Ca 2+ -medium, H 2 +Ca 2+ free-medium, and then back to H 2 -medium in MSCs. C & D . Pseudocolor (C left) and time series (C right) images and the Fluo4 averaged F/F0 trace of the [Ca 2+ i ] changes under H 2 -free, 2-APB-medium, and then back to 2-APB+H 2 -medium in MSCs. E & F . Time series images and the averaged F/F0 trace of the [Ca 2+ i ] changes in ER (E) and Mito (F) under H 2 -free and H 2 -medium in MSCs. H .Co-localization of Mito-Ca 2+ and ER-Ca 2+ with their trackers. G .Time-lapse images and the Fluo4 averaged F/F0 trace of the [Ca 2+ i ] changes under H 2 -free, Ca 2+ free-medium, H 2 + Ca 2+ free-medium, and then back to H 2 -medium conditions in HUVEC. J . Time-lapse images and the Fluo4 averaged F/F0 trace of the [Ca 2+ i ] changes in H 2 -free, 2-APB-medium, and then back to 2-APB+ H 2 -medium in HUVEC. I & M . The cell viability of MSCs and 293T remained unaffected by H 2 exposure, in contrast to the significant cytotoxicity induced by the Ca 2+ ionophore lonomycin. K & L . Live cell imaging of 293T cell death staining following 24 h of H 2 treatment and survival rate, the ethanol-treated group served as the dead cell dye control. N & O . Time-lapse live cell imaging of H 2 -promoted [Ca 2+ i ] dynamics and F/F0 fluorescence intensity analysis. A, C, E and F indicate cells at distinct time points within the time-lapse capture, sampling rate, 10 sec. G and J indicate cells at distinct time points within the time-lapse capture, sampling rate, 20 sec. Data in I and M were processed using an unpaired T test, and were plotted as Mean ± SEM. *P value < 0.05; **P value < 0.01; ***P value < 0.001; no stars for P value > 0.05. Scale bar in A, C, E, F, and H = 100 μm; Scale bar in G and J= 25 μm. Scale bar in K and N= 200 μm.

Article Snippet: The cells were washed gently with warm Hank's Balanced Salt Solution (HBSS) (Thermo Fisher Scientific 14175095) to remove the culture medium and incubated with ion-sensitive fluorescent probes for Ca 2+ -Fluo-4-AM (5 μM ) diluted in serum-free medium (SFM) (KeyGEN, KGAF024), K + -EPG-4-AM (5 μM) diluted in SFM (Maokang MX4521), for Na + -ENG-2-AM (5 μM ) diluted in SFM, and for Ca 2+ -Rhod-2-AM (5 μM ) diluted in HBSS (YEASEN 40776ES72) for 10-30 min at 37°C to allow probe uptake.

Techniques: Activity Assay, Live Cell Imaging, Staining, Control, Fluorescence, Sampling

Journal: Theranostics

Article Title: Molecular hydrogen triggers TRPC4-TRPC4AP-dependent reversible calcium transients via extracellular influx

doi: 10.7150/thno.124352

Figure Lengend Snippet: H 2 induces calcium ion influx via the TRPC4-TRPC4AP axis, triggering calcium transients in MSCs. A-C. Bulk RNAseq analysis of MSCs reveals a comparative assessment of FPKM values for various types of Ca 2+ channels and their associated gene expression. D - G & I . Screening for Ca 2+ channels potentially targeted by H 2 , including N-, L-, T-type, TRPC, and TRPC4/5, using different inhibitors. H & J-S. Screening for the potential influences of siRNA-mediated TRPM7, TRPV2, TRPM4, TRPC5, TRPC1, Orai1, STIM1, Gα i2 , Orai2, TRPC4 and TRPC4AP knockdown in [Ca 2+ i ] triggered by H 2 . T & U. Confirmation of trpc4 and trpc4ap knockout in monoclonal 293T cell lines generated by CRISPR-Cas 9. For each sgRNA, the target sequence is shown. The mutant site is highlighted in red, and the protospacer adjacent motif (PAM) is underlined. Sequences of knockout cells were determined by next-generation sequencing. V & W. [Ca 2+ i ] induced by H 2 vanished following the knockout of trpc4 or trpc4 ap. graphs showing the Fluo4 averaged F/F0 trace of the [Ca 2+ i ] changes under different conditions. X & Y . [Ca 2+ i ] induced by H 2 recovered after trpc4 or trpc4ap overexpression in the trpc4 or trpc4ap knockout cell lines; graphs showing the Fluo4 averaged F/F0 trace of the [Ca 2+ i ] changes under different conditions. Figures A, B, and C display the FPKM values of genes associated with VGCC, TRP, and SOCE channels in MSC cells based on transcriptomic sequencing results. Relevant supporting materials are provided in the . Scale bar = 200 μm.

Article Snippet: The cells were washed gently with warm Hank's Balanced Salt Solution (HBSS) (Thermo Fisher Scientific 14175095) to remove the culture medium and incubated with ion-sensitive fluorescent probes for Ca 2+ -Fluo-4-AM (5 μM ) diluted in serum-free medium (SFM) (KeyGEN, KGAF024), K + -EPG-4-AM (5 μM) diluted in SFM (Maokang MX4521), for Na + -ENG-2-AM (5 μM ) diluted in SFM, and for Ca 2+ -Rhod-2-AM (5 μM ) diluted in HBSS (YEASEN 40776ES72) for 10-30 min at 37°C to allow probe uptake.

Techniques: RNA sequencing, Gene Expression, Knockdown, Knock-Out, Generated, CRISPR, Sequencing, Mutagenesis, Next-Generation Sequencing, Over Expression

Journal: Theranostics

Article Title: Molecular hydrogen triggers TRPC4-TRPC4AP-dependent reversible calcium transients via extracellular influx

doi: 10.7150/thno.124352

Figure Lengend Snippet: Molecular docking prediction and validation of the binding site of TRPC4-TRPC4AP protein. A. Diagram of the structure of a TRPC4 Ca 2+ channel including specific motifs for the TRPC4 family. B. Amino acid sequence alignment of TRPC1, TRPC5, and 11 subtypes of the TRPC4 family within the TM6, TRP Box, Protein 4.1, and CIRB domains. The specific arginine residues within the TRPC4 family are highlighted, and red pentagrams indicate the areas of interest (arginine residues) in the CIRB motif in the sequence alignment. C & D. Molecular docking utilizing advanced protein-docking software (C) and the AlphaFold 3 platform (D) confirmed the interaction binding sites of TRPC4 and TRPC4ap, with consistent prediction outcomes. E & F. Free energy of the docking system. G&H. Amino acid frequency analysis at various positions (G) and total count (H) from the top 61 TRPC4-TRPC4ap combination patterns with the highest scores. Double Arginine residues are indicated with red pentagram symbol. I. Schematic representation of the wild-type and two mutant forms of the TRPC4 protein at positions 730 and 731 at the C-terminus. J. Molecular docking utilizing the AlphaFold 3 predicted the binding affinity of two TRPC4 mutants with TRPC4ap at positions 730 and 731, indicating a loss of binding interaction. K - R. [Ca 2+ i ] induced by H 2 and the Fluo4 averaged F/F0 trace of the [Ca 2+ i ] changes under different conditions, including blank vehicle, trpc4-KO, trpc4-KO+Arg 730 Arg 731 , trpc4-KO+Ala 730 Arg 731 . (For E: △E vdw: van der Waals interaction energy; △E ele: electrostatic interaction energy; △E GB: polar solvation free energy; △E surf: nonpolar solvation free energy; △G gas: gas-phase binding free energy; △G solv: total solvation free energy; △G bind: calculated binding free energy.) Scale bar in K, M, O and Q= 200 μm.

Article Snippet: The cells were washed gently with warm Hank's Balanced Salt Solution (HBSS) (Thermo Fisher Scientific 14175095) to remove the culture medium and incubated with ion-sensitive fluorescent probes for Ca 2+ -Fluo-4-AM (5 μM ) diluted in serum-free medium (SFM) (KeyGEN, KGAF024), K + -EPG-4-AM (5 μM) diluted in SFM (Maokang MX4521), for Na + -ENG-2-AM (5 μM ) diluted in SFM, and for Ca 2+ -Rhod-2-AM (5 μM ) diluted in HBSS (YEASEN 40776ES72) for 10-30 min at 37°C to allow probe uptake.

Techniques: Biomarker Discovery, Binding Assay, Sequencing, Software, Mutagenesis

Journal: Theranostics

Article Title: Molecular hydrogen triggers TRPC4-TRPC4AP-dependent reversible calcium transients via extracellular influx

doi: 10.7150/thno.124352

Figure Lengend Snippet: H 2 intervention on proton extrusion, intracellular pH dynamics, calcium transients in mesenchymal stem cells and multi-cellular calcium signaling pH dynamics, and TRPC4AP expression. A. Schematic diagram of live cell NMT detection of MSCs. B. Dynamic changes in proton flux across the plasma membrane of MSCs under the influence of H 2 . C & D. Time-lapse imaging showing the effect of H 2 on intracellular pH changes and mean fluorescence intensity statistics. E . Ca 2+ relative fluorescence intensity statistics over 600 s in MSCs under H 2 treatment. F. Ca 2+ flow velocity measurements over 600 s in MSCs under the influence of H 2 . G. Intracellular pH variation in MSCs under the influence of H 2 . H. Ca 2+ relative fluorescence intensity statistics in cells. I. Intracellular pH variation in cells. J . TRPC4AP mean fluorescence intensity per unit area in cells. Data in H, I and J were processed using an unpaired t-test, and were plotted as Mean ± SEM. *P value < 0.05; **P value < 0.01; ***P value < 0.001; no stars for P value > 0.05. Scale bar in A= 200 μm; Scale bar in C= 100 μm.

Article Snippet: The cells were washed gently with warm Hank's Balanced Salt Solution (HBSS) (Thermo Fisher Scientific 14175095) to remove the culture medium and incubated with ion-sensitive fluorescent probes for Ca 2+ -Fluo-4-AM (5 μM ) diluted in serum-free medium (SFM) (KeyGEN, KGAF024), K + -EPG-4-AM (5 μM) diluted in SFM (Maokang MX4521), for Na + -ENG-2-AM (5 μM ) diluted in SFM, and for Ca 2+ -Rhod-2-AM (5 μM ) diluted in HBSS (YEASEN 40776ES72) for 10-30 min at 37°C to allow probe uptake.

Techniques: Expressing, Clinical Proteomics, Membrane, Imaging, Fluorescence

Journal: Theranostics

Article Title: Molecular hydrogen triggers TRPC4-TRPC4AP-dependent reversible calcium transients via extracellular influx

doi: 10.7150/thno.124352

Figure Lengend Snippet: In vivo validation of H 2 -induced [Ca 2+ i ]. A. Upper left, common window of mouse brain preparations for in vivo two-photon Ca 2+ imaging; upper right, six distinct functional areas of the mouse cerebral cortex, including the monitored Primary Motor Cortex (M1) region.; Schematic diagram of the animal experimental procedure. B. Randomly selected 15 neurons in the M1 region. C. Comparison between the control group (Ctrl) and H 2 inhalation group (H 2 ) in the number of peak fluctuations per hour. D. Comparison between Ctrl and H 2 groups in the maximum calcium signal value. E. Two-photon in vivo imaging of skin calcium ions. F&G. Fluorescence images of Ca 2+ at 0 and 600 s in empty vector (AAV9-K14-PS8) and GCAM plasmid-transfected group (AAV9- K14-GCAMP8f). H&I. Ca 2+ mean fluorescence intensity in mouse skin over time and Ca 2+ mean fluorescence intensity signal value in empty vector (AAV9-PS8-K14). G&K. Ca 2+ mean fluorescence intensity in mouse skin over time and Ca 2+ mean fluorescence intensity signal value in GCAM plasmid-transfected group (AAV9- K14-GCAMP8f). *P value < 0.05; **P value < 0.01; ***P value < 0.001; no stars for P value > 0.05. Scale bar in B= 200 μm; Scale bar in F and G= 200 μm.

Article Snippet: The cells were washed gently with warm Hank's Balanced Salt Solution (HBSS) (Thermo Fisher Scientific 14175095) to remove the culture medium and incubated with ion-sensitive fluorescent probes for Ca 2+ -Fluo-4-AM (5 μM ) diluted in serum-free medium (SFM) (KeyGEN, KGAF024), K + -EPG-4-AM (5 μM) diluted in SFM (Maokang MX4521), for Na + -ENG-2-AM (5 μM ) diluted in SFM, and for Ca 2+ -Rhod-2-AM (5 μM ) diluted in HBSS (YEASEN 40776ES72) for 10-30 min at 37°C to allow probe uptake.

Techniques: In Vivo, Biomarker Discovery, Imaging, Functional Assay, Comparison, Control, In Vivo Imaging, Fluorescence, Plasmid Preparation, Transfection

Journal: Theranostics

Article Title: Molecular hydrogen triggers TRPC4-TRPC4AP-dependent reversible calcium transients via extracellular influx

doi: 10.7150/thno.124352

Figure Lengend Snippet: H 2 enhances cell motility by increasing [Ca 2+ i ], thereby remodeling the cytoskeleton. A & B. RNA sequencing analysis of cells treated with H 2 for 2 h and 24 h. C. Time-lapse F-Actin probe images of MSCs in H 2 -free (Control) and H 2 -medium. D. Electron microscopy results of MSCs after treatment with H 2 for 2 h and 24 h. E. Immunofluorescence results of Vimentin and α-SMA in the Control and H 2 groups of MSCs. F. Western Blot results of the effect of H 2 on Vimentin expression in MSCs and BMSCs. G. Fluorescence co-localization results of F-Actin (red) and the TRPC4AP (green). H. Cell scratch results and healing rate (right) for MSCs treated with H 2 for 24 h after siRNA AP treatment. I & J. Time-lapse images of the [Ca 2+ i ] changes in H 2 -free and H 2 -medium (red) in HepG2 (H), MSCs(I) and Fluo 4 averaged F/F0 trace in imaging H 2 -free (blue) and H 2 -medium (red). K. Schematic diagram of H 2 enhancing the motility of MSCs. L. Detection of co-culture and corresponding migration distance of tumor cells HepG2 (red) and MSCs (yellow) in H 2 -free and H 2 -medium using a live-cell imaging system and relative motion distance. Data in H and L were processed using an unpaired T test, and were plotted as Mean ± SEM. *P value < 0.05; **P value < 0.01; ***P value < 0.001; no stars for P value > 0.05. Scale bar in C and G= 50 μm; Scale bar in E= 100 μm; Scale bar in I and J= 500 μm; Scale bar in L= 200 μm.

Article Snippet: The cells were washed gently with warm Hank's Balanced Salt Solution (HBSS) (Thermo Fisher Scientific 14175095) to remove the culture medium and incubated with ion-sensitive fluorescent probes for Ca 2+ -Fluo-4-AM (5 μM ) diluted in serum-free medium (SFM) (KeyGEN, KGAF024), K + -EPG-4-AM (5 μM) diluted in SFM (Maokang MX4521), for Na + -ENG-2-AM (5 μM ) diluted in SFM, and for Ca 2+ -Rhod-2-AM (5 μM ) diluted in HBSS (YEASEN 40776ES72) for 10-30 min at 37°C to allow probe uptake.

Techniques: RNA Sequencing, Control, Electron Microscopy, Immunofluorescence, Western Blot, Expressing, Fluorescence, Imaging, Co-Culture Assay, Migration, Live Cell Imaging