Journal: Bioengineered

Article Title: Bone mesenchymal stem cells-derived miR-223-3p-containing exosomes ameliorate lipopolysaccharide-induced acute uterine injury via interacting with endothelial progenitor cells

doi: 10.1080/21655979.2021.2001185

Figure Lengend Snippet: LPS suppressed EPCs cell viability via inducing cell pyroptosis. (a, b) NLRP3 was silenced in EPCs cells. (c) MTT assay was used to examine cell viability. (d) The expression status of Cyclin D1 and CDK2 were determined by Western blot analysis. Individual experiment repeated 3 times, and * P < 0.05

Article Snippet: Next, the PVDF membranes were incubated with the primary antibodies against NLRP3 (1:1500, Catalog MAB7578, R&D system, USA), ASC (1:1000, Catalog AF3805, R&D system, USA), N-Gasdermin D (1:1000, Catalog ab209845, Abcam, UK), GAPDH (1:2000, Catalog 2275-PC-100, R&D system, USA), cleaved Caspase-3 (1:1500, Catalog AF835, R&D system, USA), Bax (1:2000, AF820, R&D system, USA), Cyclin D1 (1:1500, Catalog MAB4314, R&D system, USA) and CDK2 (1:2000, Catalog AF4654, R&D system, USA) at 4 °C overnight.

Techniques: MTT Assay, Expressing, Western Blot

Journal: Nature Communications

Article Title: Optoribogenetic control of regulatory RNA molecules

doi: 10.1038/s41467-020-18673-5

Figure Lengend Snippet: a shRNA variants used to control cyclin B1 gene expression. Blue: aptamer domain; orange: siRNA domain. b Percentages of HEK293PAL cells in G 2 /M phase of the cell cycle when transfected with indicated shRNAs targeting cyclin B1. c shRNA variants used to control CDK1 gene expression. Blue: aptamer domain; green: siRNA domain. d Percentages of HEK293PAL cells in G 2 /M phase of the cell cycle when transfected with indicated shRNAs targeting CDK1. b N = 20 (SHCB1, SHCB1m) or 6 (SHCB2, SHCB3). d N = 10. b , d Each biologically independent experiment was performed in duplicates. b The identity of SHCB1 and SHCB1m was blinded and double-blinded in one experiment, each. d The identity of SHCDK1 and SHCDK1m was blinded and double-blinded in one experiment, each. b , d Wilcoxon two-sided signed-rank test was used for statistical analysis. e , Representative western blot image showing cyclin B1, CDK1 and GAPDH protein expression after transfection with the indicated shRNAs (for complete blots see Supplementary Fig. ). e , N = 4. e One biologically independent experiment was performed in duplicates, all others once. f , g Quantification of cyclin B1 and CDK1 protein levels using pixel densitometry. f , g N = 3. f , g All biologically independent experiments were performed once. f , g Cohen’s d effect size was used for statistical analysis. Values were normalized to non-transfected cells incubated in darkness (Untransfected). b , d , f , g Gray bars: cells incubated under light conditions, black bars: cells incubated under dark conditions. Values are means ± s.d. Source data for ( b , d , f , g ) are provided as source data file.

Article Snippet: 5 μg of protein per lane was loaded onto 10 or 12.5% SDS-PAGE gels and blotted in Transfer Buffer (2.5 mM Tris, 2% (w/v) glycine, 0.9 M urea) onto a nitrocellulose membrane (GE Healthcare Life Sciences) using a Bio-Rad Trans-Blot SD Semi-Dry Transfer Cell (BioRad) for 75 min at 20 V and 30 W. Membranes were blocked with TBS-T buffer (20 mM Tris/HCl, pH 7.6, 150 mM NaCl, 0.05% Tween 20 (v/v)) containing 5% BSA (AppliChem, Western Blot grade) under agitation at room temperature for 1 h. Blots were cut according to the protein ladder (Prestained Protein Ladder—Mid-range molecular weight (10–180 kDa), abcam) in a way that all target proteins can be individually incubated with the respective primary antibody (mouse anti -cdc2 (CDK1), Cell signaling POH1, #9116 (1:1000); mouse anti -GAPDH, Santa Cruz Biotechnology sc-47724 (1:4000); goat anti- Cyclin B1, R&D Systems AF6000 (1:1000)) at 4 °C overnight or at room temperature for 1 h in TBS-T containing 5% BSA.

Techniques: shRNA, Control, Gene Expression, Transfection, Western Blot, Expressing, Incubation

Journal: PLoS Biology

Article Title: CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

doi: 10.1371/journal.pbio.3001496

Figure Lengend Snippet: (A) HA-TRPM7 was coexpressed with FLAG-tagged CNNM1-4 in HEK-293T cells, and the channel was immunoprecipitated with HA-agarose. CNNM1 and CNNM3 strongly interacted with TRPM7. CNNM2 and CNNM4 also interacted with TRPM7, but lowered TRPM7 expression nonspecifically. CNNMs were not coimmunoprecipitated from HEK-293T cells N.T. with HA-TRPM7. Supplementation of the growth medium with 20 mM MgCl 2 boosted TRPM7 expression, revealing the channel’s capacity to interact with CNNM2 and CNNM4. (B) FLAG-tagged TRPM7 robustly coimmunoprecipitated native CNNM3 and CNNM4, whereas FLAG-TRPM2 did not. (C) A Zinc influx assay using the FluoZin-3 Zn 2 + indicator was used to monitor TRPM7 function in intact 293-TRPM7 cells (293-M7 cells), which express FLAG-tagged TRPM7 upon tetracycline treatment. Coexpression of CNNMs with TRPM7 increased intracellular free Zn 2 + more than expression of TRPM7 alone. The T-REx-293 cell line (293), which expresses the Tet Repressor protein, was used as the negative control. Shown are images taken at a time point between 5 and 10 minutes after application of 30 μM ZnCl 2 . White scale bar = 100 μM. (D) Quantification of the results from (C). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (E) Western blot showing expression of TRPM7 in the sample from (C). (F) Separate time course measurements were also acquired to further demonstrate that cells coexpressing CNNMs with TRPM7 have a higher rate of Zn 2+ influx compared to 293-TRPM7 cells expressing TRPM7 or CNNMs alone. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. Unprocessed images of blots are shown in . The underlying data for this figure can be found in . HBSS, Hanks’ balanced salt solution; N.T., not transfected.

Article Snippet: Colonies were screened for loss of protein expression using antibodies specific for CNNM3 (NBP2-32134, Novus Biologicals Centennial, Colorado, USA) and CNNM4 (ab191207; Abcam Cambridge, UK).

Techniques: Immunoprecipitation, Expressing, Negative Control, Western Blot, Fluorescence, Transfection

Journal: PLoS Biology

Article Title: CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

doi: 10.1371/journal.pbio.3001496

Figure Lengend Snippet: (A) A Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in intact cells under the same conditions described in . KO of CNNM3 , CNNM4 , and both CNNM3 and CNNM4 from 293-TRPM7 cells (293-M7-ΔCNNM3, 293-M7-ΔCNNM4, and 293-M7-ΔCNNM3/4) reduced TRPM7 channel function, which could be rescued to varying degrees by reexpression of CNNM3 and/or CNNM4. The T-REx-293 cell line (293), which expresses the Tet Repressor protein, was used as the negative control. All the cells in the assay were treated with tetracycline to induce TRPM7 expression. White scale bar = 100 μM. (B) Quantification of the results from (A). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. Images taken 5 to 10 minutes after application of 30 μM ZnCl 2 . (C) Separate time course measurements were also acquired to demonstrate that 293-TRPM7 cells lacking CNNMs have a reduced rate of Zn 2+ influx compared to 293-TRPM7 expressing TRPM7. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. (D) CRISPR/Cas-9 was used to KO CNNM3 , CNNM4 , and both CNNM3&4 from 293-TRPM7 cells, which express TRPM7. Western blotting of 2 independent clones showed specific KO of CNNM isoforms and similar expression of TRPM7 among the lines. Note that the antibody used to detected CNNM4 detects an unrelated protein as indicated. (E) Cell surface biotinylation of 293-TRPM7 WT and 293-M7-ΔCNNM3/4 cells demonstrated that surface level of TRPM7 in the 2 cell lines is similar. Shown are SA purified (SA purified) proteins, the input lysate for each cell type, and the proteins not bound to SA (SA unbound). (F) Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in intact cells. Shown are images taken at a time point between 5 and 10 minutes after application of 30 μM ZnCl 2 . Overexpression of PRL-2 stimulates TRPM7 channels function, which requires to varying degrees CNNM3 and CNNM4. White scale bar = 100 μM. (G) Western blot demonstrating expression of TRPM7 (Anti-FLAG) and PRL-2 under the conditions described in (F). (H) Quantification of the results from (F). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (I) Separate time course measurements were also acquired to demonstrate that PRL overexpression stimulates an increased rate of Zn 2+ influx compared to 293-TRPM7 expressing TRPM7 and that CNNM3 and CNNM4 are required for this activity. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. Unprocessed images of blots are shown in . The underlying data for this figure can be found in . HBSS, Hanks’ balanced salt solution; KO, knockout; PRL, phosphatase of regenerating liver; PRL-2, phosphatase of regenerating liver 2; SA, streptavidin agarose.

Article Snippet: Colonies were screened for loss of protein expression using antibodies specific for CNNM3 (NBP2-32134, Novus Biologicals Centennial, Colorado, USA) and CNNM4 (ab191207; Abcam Cambridge, UK).

Techniques: Negative Control, Expressing, Fluorescence, CRISPR, Western Blot, Clone Assay, Purification, Over Expression, Activity Assay, Knock-Out

Journal: PLoS Biology

Article Title: CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

doi: 10.1371/journal.pbio.3001496

Figure Lengend Snippet: (A) A 25 Mg uptake assay was employed to assay TRPM7 channel function and its dependence on CNNMs. Moreover, 293-TRPM7 cells have significantly higher 25 Mg uptake than the negative control T-REx-293 cell line (293), which expresses the Tet Repressor protein. Compared to 293-TRPM7 cells, 293-M7-ΔCNNM3/4 exhibited significantly lower 25 Mg uptake, which was increased by reexpression of CNNM4. * = p < 0.05 and *** = p < 0.001, 1-way ANOVA plus Tukey post hoc. (B) Similar levels of free Mg 2+ were detected in HEK-293T (293T(WT)) and HEK-293T TRPM7 KO (293T(ΔM7)) cells using the Mag-Fluo4 Mg 2+ indicator. White scale bar = 100 μM. Overexpression of CNNM2 or CNNM4 substantially reduced cytosolic Mg 2+ in both cell lines to similar degrees. (C) Quantitation of the results from (B). n = 50. *** indicates a p -value of less than 0.001. (D) Mag-Fura-2 was also employed to compare the Mg 2+ levels between 293T(WT), 293T(ΔM7), and 293T(ΔM7) transfected with CNNM2 or CNNM4. Images of the cells were taken using a 510-nm filter from excitation at 340 nm versus 380 nm on an inverted Olympus IX70 fluorescence microscope, and the ratio of fluorescence intensity of the cells from excitation at the 340 nm versus 380 nm wavelengths (30 cells) was quantified. *** indicates a p -value of less than 0.001. (E) 293T(WT), 293T(WT) transfected with CNNM4, 293T(WT) transfected with CNNM4 and treated with the TRPM7 channel inhibitor NS85903 (10 μM), and 293T(ΔM7) cells transfected with CNNM4 were loaded with Mag-Fluo4. The fluorescence intensity of the cells (mean of 50 cells) were measured for 1 minute before the extracellular solution bathing the cells was replaced with a Na + free solution (replacing NaCl with NMDG-Cl) to inhibit Na + -dependent Mg 2+ extrusion by CNNM4. Replacement of the buffer occurred between 50 and 100 seconds after imaging began as indicated. Overexpression of CNNM4 increased the Mg 2+ levels in 293T(WT) but not 293T(ΔM7) cells, and the increase in Mg 2+ in 293T(WT) cells caused by CNNM4 overexpression could be blocked by NS8593, which was continually present in the imaging medium and was initially added just prior to imaging. The underlying data for this figure can be found in . WT, wild-type.

Article Snippet: Colonies were screened for loss of protein expression using antibodies specific for CNNM3 (NBP2-32134, Novus Biologicals Centennial, Colorado, USA) and CNNM4 (ab191207; Abcam Cambridge, UK).

Techniques: Negative Control, Over Expression, Quantitation Assay, Transfection, Fluorescence, Microscopy, Imaging

Journal: PLoS Biology

Article Title: CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

doi: 10.1371/journal.pbio.3001496

Figure Lengend Snippet: TRPM7 currents were recorded in 293-TRPM7 cells ( n = 30), 293-M7-ΔCNNM3/4 cells ( n = 30), and 293-M7-ΔCNNM3/4 cells ( n = 40) with stable episomal expression of CNNM4 to keep CNNM4 protein levels low so that it would not interfere with TRPM7 protein expression. (A) Western blot demonstrating CNNM4 expression in the cell groups used in (B–D). (B) Representative TRPM7 whole-cell currents from the 3 cell lines recorded with an internal pipette solution containing 0 Mg 2+ /Mg-ATP to achieve full TRPM7 channel activity. (C) Average current density of the different groups from (A). (D) Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in cells from (B,C). White scale bar = 100 μM. Shown are images taken at a time point 5 to 10 minutes after application of 30 μM ZnCl 2 . (E) Quantification of results from (D). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (F) Native TRPM7 currents were recorded in the T-REx-293 Cell Line (293), which was the parental cell line of 293-ΔCNNM3/4 cells. Shown are representative TRPM7 whole-cell currents from the 2 cell lines recorded with an internal pipette solution containing 0 Mg 2+ /Mg-ATP to achieve full TRPM7 channel activity. (G) Average current density of the different groups from (F) ( n = 16 per group). Unprocessed images of blots are shown in . The underlying data for this figure can be found in .

Article Snippet: Colonies were screened for loss of protein expression using antibodies specific for CNNM3 (NBP2-32134, Novus Biologicals Centennial, Colorado, USA) and CNNM4 (ab191207; Abcam Cambridge, UK).

Techniques: Expressing, Western Blot, Transferring, Activity Assay