Journal: Molecular Oncology

Article Title: STIM2 is involved in the regulation of apoptosis and the cell cycle in normal and malignant monocytic cells

doi: 10.1002/1878-0261.13584

Figure Lengend Snippet: STIM2 knockdown alters store‐operated calcium entry (SOCE) and decreases cell proliferation in THP1 and OCI‐AML3. All data were obtained (western blot, calcium imaging, isoform, RT‐qPCR, and cell count) at 48 h postinfection for siRNA and 72 h for shRNA. (A) In THP1 protein expression of STIM2 cells (si#1: 0.407 ± 0.219; si#2: 0.410 ± 0.123) as determined by western blot relative to GAPDH ( n = 3). (B, C) The STIM2.2/STIM2.1 ratio was performed by quantitative PCR in THP1 (si#1: 4.54 ± 3.259; si#2: 6.120 ± 0.9966) (B) ( n = 4) and in OCI‐AML3 (sh#1: 6.98 ± 3.84; sh#2: 5.60 ± 2.55) (C) ( n = 5). (D) Representative trace of SOCE measured with the ratio F340/F380 in THP1 cells after STIM2 silencing ( n = 4) (siCONTROL n = 57; si#1 n = 72; si#2 n = 90). Cells were exposed to 1 μ m Thapsigargin in the absence of Ca 2+ which depletes the intracellular calcium (Ca 2+ ). Extracellular Ca 2+ concentration was then brought to 2 m m to induce SOCE. (E–G) Quantification of SOCE (siCONTROL: 53.63 ± 49.61; si#1: 93.33 ± 51.78; si#2: 134.2 ± 53.02) ( n = 6) (siCONTROL n = 87; si#1 n = 175; si#2 n = 112) (E), Thapsgiargin (TG)‐response (siCONTROL: 744 ± 142; si#1: 816 ± 229; si#2: 740 ± 257) ( n = 4) (F) and basal calcium (siCONTROL: 0.808 ± 0.108; si#1: 0.890 ± 0.169; si#2: 0.811 ± 0.123) ( n = 4) (G). (H) STIM2 protein expression in THP‐1 cells (sh#1: 0.08 ± 0.02; sh#2: 0.03 ± 0.01) was determined by western blot relative to GAPDH ( n = 3). (I) STIM2 protein expression in OCI‐AML3 cells (sh#1: 0.220 ± 0.0693; sh#2: 0.130 ± 0.03) was determined by western blot relative to GAPDH ( n = 3). (J, K) In THP1 cells, proliferation was assessed by trypan blue cell count and then reported to the numbers (Nb) of GFP cells at each cytometry point (Days 1 – 3 – 5 (D1 – 3 – 5)) ( n = 3) (J) and in OCI‐AML3 ( n = 3) (K). All numeric values are presented as the mean values ± standard error of the mean. P ‐values are calculated using one‐way ANOVA, *** P < 0.001; ** P < 0.01; * P < 0.05; NS, not significant.

Article Snippet: For differentiation into monocyte‐like cells, THP1 and OCI‐AML3 cells were treated with 1 μ m 1,25(OH) 2 D 3 (Calbiochem, Millipore, Burlington, MA, USA) for 72 h. To suppress the function of p53 protein, α‐pifithrin (PFT‐α) (TargetMol, Wellesley Hills, MA, USA) was used at 10 μ m in the medium.

Techniques: Knockdown, Western Blot, Imaging, Quantitative RT-PCR, Cell Characterization, shRNA, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Cytometry

Journal: Molecular Oncology

Article Title: STIM2 is involved in the regulation of apoptosis and the cell cycle in normal and malignant monocytic cells

doi: 10.1002/1878-0261.13584

Figure Lengend Snippet: STIM2 knockdown induced apoptosis in THP1 and OCI‐AML3 cells. (A, B) Cell viability study was assessed in flow cytometry by annexin V/DAPI labeling and compared with shSCRAMBLE cells (Day 1—shSCRAMBLE: 4.14 ± 0.420; sh#1: 10.9 ± 10.5; sh#2: 13.3 ± 2.16) (Day 3—shSCRAMBLE: 5.43 ± 0.48; sh#1: 46.1 ± 18.6; sh#2: 25.5 ± 18.5) (Day 5—shSCRAMBLE: 9.68 ± 0.365; sh#1: 65.4 ± 16.1; sh#2: 67.6 ± 10.4) in THP1 ( n = 3) (A) and in OCI‐AML3 (Day 1—shSCRAMBLE: 5.09 ± 0.175; sh#1: 4.49 ± 0.924; sh#2: 2.72 ± 0.655) (Day 3—shSCRAMBLE: 3.27 ± 0.667; sh#1: 26.7 ± 7.47; sh#2: 2.3 ± 0.524) (Day 5—shSCRAMBLE: 5.13 ± 0.242; sh#1: 87.2 ± 1.15; sh#2: 22.3 ± 2.52) ( n = 3) (B). (C) In THP1 cell line, quantification of antiapoptotic proteins was performed by western blot (Bcl‐2—sh#1: 0.553 ± 0.0208; sh#2: 0.330 ± 0.0361) (Bcl‐XL—sh#1: 0.497 ± 0.0971; sh#2: 0.403 ± 0.150) (MCL‐1—sh#1: 0.642 ± 0.0351; sh#2: 0.590 ± 0.123) relative to GAPDH and compared with shSCRAMBLE ( n = 3). (D) Quantification of proapoptotic proteins was performed by western blot (BAX—sh#1: 2.43 ± 0.252; sh#2: 2.6 ± 0.5) (BAD—sh#1: 2.63 ± 1.01; sh#2: 3.03 ± 0.718) relative to GAPDH and compared with shSCRAMBLE ( n = 3). (E) Membrane and mitochondrial proteins were quantified by western blot (caspase‐8—sh#1: 1.42 ± 0.393; sh#2: 1.52 ± 0.407) (caspase‐9—sh#1: 0.103 ± 0.0351; sh#2: 0.0633 ± 0.0208) (cleaved (Cl) caspase‐9—sh#1: 2.11 ± 0.431; sh#2: 2.07 ± 0.347) relative to GAPDH and compared with shSCRAMBLE ( n = 3). (F) Apoptosis effector proteins were quantified by western blot (caspase‐3—sh#1: 0.553 ± 0.185; sh#2: 0.457 ± 0.0874) (cleaved caspase‐3—sh#1: 2.71 ± 0.499; sh#2: 1.57 ± 0.248) (PARP—sh#1: 0.717 ± 0.136; sh#2: 0.593 ± 0.204) (cleaved PARP—sh#1: 2.16 ± 0.320; sh#2: 1.84 ± 0.140) relative to GAPDH and compared with shSCRAMBLE ( n = 3). All numeric values are presented as the mean values ± standard error of the mean. P ‐values are calculated using one‐way ANOVA, ***P < 0.001; ** P < 0.01; * P < 0.05.

Article Snippet: For differentiation into monocyte‐like cells, THP1 and OCI‐AML3 cells were treated with 1 μ m 1,25(OH) 2 D 3 (Calbiochem, Millipore, Burlington, MA, USA) for 72 h. To suppress the function of p53 protein, α‐pifithrin (PFT‐α) (TargetMol, Wellesley Hills, MA, USA) was used at 10 μ m in the medium.

Techniques: Knockdown, Flow Cytometry, Labeling, Western Blot, Membrane

Journal: Molecular Oncology

Article Title: STIM2 is involved in the regulation of apoptosis and the cell cycle in normal and malignant monocytic cells

doi: 10.1002/1878-0261.13584

Figure Lengend Snippet: Blockage of cell cycle induced by STIM2 knockdown in THP1 and OCI‐AML3 cells. In THP1, the cell cycle was studied by flow cytometry (FCM) and western blot. (A, B) The FCM revealed an increase in the number of cells blocked in G2/M phase (shSCRAMBLE—19.77 ± 4.398; sh#1: 35.33 ± 6.047; sh#2: 32.80 ± 5.274) ( n = 3). (C, D) In THP1, quantification of the key proteins of G2/M transition were performed by western blot (CDK1—sh#1: 0.507 ± 0.0231; sh#2: 0.617 ± 0.0493) (cyclin B1—sh#1: 0.467 ± 0.0611; sh#2: 0.460 ± 0.0721) relative to GAPDH ( n = 3) (C) and in OCI‐AML3 (CDK1—sh#1: 0.527 ± 0.0451; sh#2: 0.393 ± 0.0643) (cyclin B1—sh#1: 0.370 ± 0.145; sh#2: 0.400 ± 0.0265) ( n = 3) (D). (E, F) CDC25c was quantified by western blot in THP1 (CDC25c—sh#1: 0.350 ± 0.201; sh#2: 0.353 ± 0.216) ( n = 3) (E) and in OCI‐AML3 (CDC25c—sh#1: 0.400 ± 0.0854; sh#2: 0.450 ± 0.0800) relative to GAPDH ( n = 3) (F). All numeric values are presented as the mean values ± standard error of the mean. P ‐values are calculated using one‐way ANOVA, *** P < 0.001; ** P < 0.01; * P < 0.05.

Article Snippet: For differentiation into monocyte‐like cells, THP1 and OCI‐AML3 cells were treated with 1 μ m 1,25(OH) 2 D 3 (Calbiochem, Millipore, Burlington, MA, USA) for 72 h. To suppress the function of p53 protein, α‐pifithrin (PFT‐α) (TargetMol, Wellesley Hills, MA, USA) was used at 10 μ m in the medium.

Techniques: Knockdown, Flow Cytometry, Western Blot

Journal: Molecular Oncology

Article Title: STIM2 is involved in the regulation of apoptosis and the cell cycle in normal and malignant monocytic cells

doi: 10.1002/1878-0261.13584

Figure Lengend Snippet: Apoptosis and cell blockage induced by STIM2 KD are related to DNA double‐strand breaks and p53 induction. (A, B) Expression of p53 and p21 in THP1 leukemic line with STIM2 KD was performed by western blot (p53—sh#1: 2.35 ± 0.560; sh#2: 2.75 ± 0.542) (p21—sh#1: 1.64 ± 0.214; sh#2: 2.12 ± 0.639) ( n = 3) (A) and in OCIA‐AML3 (p53—sh#1: 2.69 ± 0.745; sh#2: 2.79 ± 0.735) (p21—sh#1: 3.52 ± 0.671; sh#2: 3.27 ± 0.439) relative to GAPDH ( n = 3) (B), P ‐values are calculated using one‐way ANOVA. (C) Cell viability was performed by Annexin/DAPI (Ann/DAPI) labeling in THP1 cells after STIM2 knockdown (KD), treated or not with pifithrin alpha (PFT‐α) (shSCRAMBLE −PFT‐α: 91.43 ± 1.582 – shSCRAMBLE +PFT‐α: 89.8 ± 1.9; sh#1 −PFT‐α: 47.27 ± 8.223 – sh#1 +PFT‐α: 65.67 ± 4.839; sh#2 −PFT‐α: 42.17 ± 6.772 – sh#2 +PFT‐α: 67.57 ± 9.097) ( n = 3), P ‐values are calculated using two‐way ANOVA. (D) Cell cycle analysis in THP1 cells after STIM2 KD, treated or not with PFT‐α was performed by flow cytometry (FCM) and compared with cells transduced with shSCRAMBLE (shSCRAMBLE −PFT‐α: 16.8 ± 3.64 – shSCRAMBLE +PFT‐α: 19.8 ± 1.69; sh#1 −PFT‐α: 47.2 ± 5.15 – sh#1 +PFT‐α: 34.7 ± 2.89; sh#2 −PFT‐α: 47.1 ± 2.95 – sh#2 +PFT‐α: 35.6 ± 3.69) ( n = 5); P ‐values are calculated using two‐way ANOVA. (E) Quantification of p53 and CDC25c at protein level was performed by western blot in THP1 cells after STIM2 KD treated or not with PFT‐α (p53—sh#1 −PFT‐α: 2.84 ± 0.9958 – sh#1 +PFT‐α: 1.237 ± 0.05508; sh#2 −PFT‐α: 2.737 ± 1.051 – sh#2 +PFT‐α: 1.297 ± 0.2196) (CDC25c—sh#1 −PFT‐α: 0.5033 ± 0.08083 – sh#1 +PFT‐α: 1.013 ± 0.1692; sh#2 ‐PFT‐α: 0.4833 ± 0.1850 – sh#2 +PFT‐α: 0.9533 ± 0.1069) ( n = 3), P ‐values are calculated using two‐way ANOVA. (F, G) p‐H2AXγ quantification was performed in THP1 by western blot (sh#1: 5.63 ± 1.38; sh#2: 3.45 ± 0.546) ( n = 3) (F) and in OCI‐AML3 (sh#1: 2.8 ± 0.725; sh#2: 3.08 ± 1.04) relative to GAPDH ( n = 3) (G), P ‐values are calculated using one‐way ANOVA. (H) In THP‐1 cell line treated or not with PFT‐α, p‐H2AXγ quantification was performed by western blot (sh#1 −PFT‐α: 4.13 ± 0.176 – sh#1 +PFT‐α: 3.8 ± 1.55; sh#2 −PFT‐α: 4.14 ± 1.14 – sh#2 +PFT‐α: 3.6 ± 1.31) relative to GAPDH ( n = 3), P ‐values are calculated using two‐way ANOVA. All numeric values are presented as the mean values ± standard error of the mean. *** P < 0.001; ** P < 0.01; * P < 0.05.

Article Snippet: For differentiation into monocyte‐like cells, THP1 and OCI‐AML3 cells were treated with 1 μ m 1,25(OH) 2 D 3 (Calbiochem, Millipore, Burlington, MA, USA) for 72 h. To suppress the function of p53 protein, α‐pifithrin (PFT‐α) (TargetMol, Wellesley Hills, MA, USA) was used at 10 μ m in the medium.

Techniques: Expressing, Western Blot, Labeling, Knockdown, Cell Cycle Assay, Flow Cytometry, Transduction

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: Mouse kidney, but not lung, demonstrate differences in ENaC α‐ and γ‐subunit expression with aldosterone. Lysate from mouse kidney and lung collected from animals on a high salt diet (HS) or with an aldosterone infusion (Aldo) were blotted for the presence of each ENaC subunit. (a) The Stressmarq anti‐α‐subunit antibody revealed an intense 80 kDa nonspecific band, denoted by ** and a 95 kDa full length α‐subunit, denoted by *. The top panel was exposed for a longer period (~7 min) to show the 95 kDa band and the bottom panel shows a quick initial exposure (~5 s) before saturation of the 80 kDa band occurred. (b) The blot was stripped and reprobed with a previously characterized antibody, produced in the Loffing laboratory. The panel shows two exposures, separated by a dashed line, to reveal both the full‐length 95 kDa α‐subunit, denoted by *, and a 30 kDa α‐subunit N‐terminal cleavage product. (c) The Stressmarq antibody directed against the β‐subunit shows the presence of a band at 90 kDa (denoted by *). (d) The Stressmarq antibody directed against the γ‐subunit revealed bands corresponding to a full‐length 80 kDa γ‐subunit and 70 kDa cleavage products, as indicated. (e) Signal from the kidney samples was quantified by densitometry, with each band normalized to total protein. Quantification is shown as a fold change from the average HS signal with p values shown for relationships that were significant ( p < 0.05) as assessed by multiple t ‐tests. (f) Mouse lung lysate was first probed with the Stressmarq α antibody, followed by a light chain only secondary antibody (LC‐only HRP). The blot was then stripped and reprobed again with the Stressmarq α antibody but followed by a whole IgG secondary antibody (H + L HRP). The blot was then stripped again and reprobed with the Loffing α antibody, followed by a whole IgG secondary antibody (H + L HRP). The Stressmarq antibody revealed an intense nonspecific band of ~80 kDa (denoted by **) and a full length α‐subunit migrating ~95 kDa (denoted by *). The Loffing antibody reveal both the full‐length 95 kDa α‐subunit and a 30 kDa N‐terminal α‐subunit cleavage product. Blots are representative of results from at least three separate experiments.

Article Snippet: As the Stressmarq α‐subunit antibody, directed against an N‐terminal epitope, does not detect the N‐terminal 30 kDa cleavage product, we encourage companies to raise antibodies against amino acids 2–21, the antigen sequence utilized by Loffing and colleagues.

Techniques: Expressing, Produced

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: Immunoprecipitation eliminates the non‐specific α‐ subunit band in mouse lung tissue and FRT cells. (a) Mouse lungs were homogenized and incubated with the α‐subunit StressMarq antibody, followed by immunoprecipitation with protein G beads. Both the pulldown (left) and the lysate (right) were run together on a gel and blotted for the α‐subunit with the same StressMarq antibody as utilized for the IP. (b) Mouse kidneys collected from animals on a high salt diet (HS) or with an aldosterone infusion (Aldo) were immunoprecipitated with the StressMarq α antibody and protein G beads. Both the IP (left) and the kidney lysate (right) were run next to each other on an 8%–16% gel to probe for the α‐subunit with the Loffing α antibody. (c) Lysate from FRT cells either mock transfected or transfected with the three ENaC subunits were run for comparison (first two lanes). The lysate was incubated with either StressMarq (SM) anti‐α‐subunit antibody, beads alone, or V5‐tagged beads and subsequent pulldown was performed. The product was run on two separate gels simultaneously, with one being probed with the StressMarq anti‐α‐subunit antibody while the other was probed with an antibody directed against the HA tag. In all panels * illustrates the α‐subunit band while ** indicates to the non‐specific band. Results are representative of three separate experiments.

Article Snippet: As the Stressmarq α‐subunit antibody, directed against an N‐terminal epitope, does not detect the N‐terminal 30 kDa cleavage product, we encourage companies to raise antibodies against amino acids 2–21, the antigen sequence utilized by Loffing and colleagues.

Techniques: Immunoprecipitation, Incubation, Transfection

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: Subunit‐specific antibodies demonstrate linearity across a wide range of protein concentrations. Decreasing amounts of lung or kidney homogenate were probed for each subunit to determine the working range of the antibody. (a) Lung lysate ranging from 80 to 2.5 μg total protein, as denoted along the top of the blot, was probed for the α‐subunit using the StressMarq antibody. (b) The band of interest, denoted by *, was quantified and normalized to the value obtained for 40 μg (the halfway value) so that the results from three separate replicates could be combined. The dashed line demonstrates perfect linearity. (c) Kidney lysate was utilized for the β‐subunit and (e) the γ‐subunit. (d, f) The quantification of each band was again performed as described for (b), with both the full‐length and cleavage product bands being quantified for the γ‐subunit. Each graph represents results obtained from three separate experiments.

Article Snippet: As the Stressmarq α‐subunit antibody, directed against an N‐terminal epitope, does not detect the N‐terminal 30 kDa cleavage product, we encourage companies to raise antibodies against amino acids 2–21, the antigen sequence utilized by Loffing and colleagues.

Techniques:

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: The α‐subunit antibody demonstrates minimal signal in AQP2‐positive cells and is mislocalized in kidney medulla. Kidney sections from mice kept on control diet (a, top) or 4 days of high K + diet (a, middle) were labeled with the StressMarq anti‐α‐subunit antibody (red on left and converted to grayscale in second column). AQP2 (green) was used as a marker of the apical lumen of principal cells. The basolateral surfaces of tubules are denoted by solid lines and the apical surface by dashed lines. A no primary control is shown for comparison (a, bottom). (b) Positive staining for the α‐subunit (red) was only observed in the inner medulla of the kidneys, with representative images displayed here. While AQP2 positive cells within the medulla (green) did show expression of ENaC (denoted by *), the majority of the signal was localized to the basolateral side of both AQP2 positive and negative tubules (shown by arrows). Scale bar represents 20 μm in all images and images are representative of three separate regions examined in three mice of each treatment.

Article Snippet: As the Stressmarq α‐subunit antibody, directed against an N‐terminal epitope, does not detect the N‐terminal 30 kDa cleavage product, we encourage companies to raise antibodies against amino acids 2–21, the antigen sequence utilized by Loffing and colleagues.

Techniques: Labeling, Marker, Staining, Expressing

Journal: PLoS ONE

Article Title: Demineralized bone matrix used for direct pulp capping in rats

doi: 10.1371/journal.pone.0172693

Figure Lengend Snippet: (A): Representative immunohistochemical images of the blank control group, the Ca(OH) 2 group and the DBM group. a: The blank control group. Pulp morphology was normal. b- f: Ca(OH) 2 group (1, 3, 7, 14, 28 days). g- k: DBM group (1, 3, 7, 14, 28 days). (B): Mean IOD value of Runx2. *means significant difference.

Article Snippet: The sections were deparaffinized with xylene, hydrated in a series of descending grades of ethanol, and then rinsed briefly with PBS for the primary antibodies; the sections were incubated overnight at 4°C with polyclonal anti- Runx2, COL I, OCN and DSP (Wuhan Boster Biological Technology, Wuhan, China).

Techniques: Immunohistochemical staining, Control

Journal: Chemical Engineering Journal

Article Title: Nanosilver-incorporated halloysite nanotubes/gelatin methacrylate hybrid hydrogel with osteoimmunomodulatory and antibacterial activity for bone regeneration

doi: 10.1016/j.cej.2019.123019

Figure Lengend Snippet: Fig. 2. Evaluation of cell viability and osteogenic differentiation in vitro. (A) MTS assay of cell culture after 1, 4, and 7 days. (B) LIVE/DEAD staining assay after 1, 4, and 7 days of cell culture (scale bar = 100 μm). (C) Osteogenic genes BMP4, RUNX2 and COL1 were assayed using RT-qPCR. (D) Osteogenic proteins detected by ELISA. *p < 0.05, **p < 0.01, NS: no statistical significance.

Article Snippet: Culture medium supernatants were collected, and the concentrations of BMP4, RUNX2 and COL1 were determined using ELISA kits (Cusabio, Wuhan, China) following the manufacturer’s instructions.

Techniques: In Vitro, MTS Assay, Cell Culture, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Chemical Engineering Journal

Article Title: Nanosilver-incorporated halloysite nanotubes/gelatin methacrylate hybrid hydrogel with osteoimmunomodulatory and antibacterial activity for bone regeneration

doi: 10.1016/j.cej.2019.123019

Figure Lengend Snippet: Fig. 4. nAg/HNTs/GelMA hybrid hydrogel enhances the osteogenic differentiation of hPDLSCs under inflammatory environment. (A) Schematic diagram showing the collection of the condition medium and hPDLSCs processing method. (B) The gene level of BMP4, RUNX2 and COL1 after osteogenic differentiation on day 14. (C) Alkaline phosphatase (ALP) staining and ALP activity after osteogenic differentiation on day 7. (D) Alizarin red staining and quantification after osteogenic dif- ferentiation on day 14. *p < 0.05, **p < 0.01, NS: no statistical significance.

Article Snippet: Culture medium supernatants were collected, and the concentrations of BMP4, RUNX2 and COL1 were determined using ELISA kits (Cusabio, Wuhan, China) following the manufacturer’s instructions.

Techniques: Staining, Activity Assay