tacrolimus (tac  (Merck & Co)

Journal: International Journal of Molecular Sciences

Article Title: An Immune-Independent Mode of Action of Tacrolimus in Promoting Human Extravillous Trophoblast Migration Involves Intracellular Calcium Release and F-Actin Cytoskeletal Reorganization

doi: 10.3390/ijms252212090

Figure Lengend Snippet: Tacrolimus spiked [Ca 2+ ]i release in live HTR8/Svneo cells. ( A – H ): Representative photomicrographs of individual live HTR8/SVneo cells visualized by intravital confocal microscopy depicting the intracellular contents of Ca 2+ in DMSO-treated control (( A ): green), ionomycin (( B ): blue), TAC-treated (( C ): red), 2-APB + TAC (( D ): yellow), U73122 + TAC (( E ): purple), Wortmannin + TAC (( F ): orange), BAPTA + TAC (( G ): turquoise) and EGTA + TAC (( H ): green), respectively. The timeframe for Ca 2+ imaging was for a total of 540 s. [Ca 2+ ]i release was spiked at the end of minute 3 of live-image tracing (depicted by the blue arrow) by the addition of ionomycin ( B ), or TAC alone ( C ) or in the presence of other inhibitors of [Ca 2+ ]i release and Ca 2+ chelators ( D – G ). Compared to the DMSO-treated ( A ) and Ionomycin-treated controls ( B ), the single use of TAC (10 ng/mL) resulted in a significant increase in [Ca 2+ ]i in the HTR8/SVneo cells (compare the intensity of red color in ( C ) before and after the addition of TAC). The inability of TAC to spike [Ca 2+ ]i-release in the presence of the IP3R antagonist 2-APB ( D ) suggests a crucial role for IP3R in mediating the [Ca 2+ ]i modulatory actions of TAC. Unexpectedly, the TAC-induced [Ca 2+ ]i-release in HTR8/SVneo cells was unaffected by inhibitory actions of the PLC inhibitor U73122 ( E ) as well as the potent and specific phosphatidylinositol 3-kinase (PI3-K) inhibitor Wortmannin ( F ). The intracellular source of the TAC-induced [Ca 2+ ]i-release is confirmed by the use of intracellular [Ca 2+ ]i chelator BPATA ( G ) and EGTA ( H ). Scale bars: ( A – H ) 30 µm; [Ca 2+ ]i is depicted in pseudocolors in ( A – H ).

Article Snippet: Tacrolimus (TAC, FK506) (10 ng/mL) (Cat# B415260, Toronto Research Chemicals, Toronto, ON, Canada) was dissolved in dimethyl sulfoxide (DMSO) HybriMax TM (Cat# D2650, Millipore Sigma) and administered to each culture according to an established protocol [ ].

Techniques: Confocal Microscopy, Control, Imaging

Journal: International Journal of Molecular Sciences

Article Title: An Immune-Independent Mode of Action of Tacrolimus in Promoting Human Extravillous Trophoblast Migration Involves Intracellular Calcium Release and F-Actin Cytoskeletal Reorganization

doi: 10.3390/ijms252212090

Figure Lengend Snippet: Tacrolimus influences F-actin cytoskeletal re-arrangement in the human-derived first-trimester extravillous trophoblast cells. ( A – C ): Single cell confocal images of control ( A ) and TAC-treated HTR8/SVneo cells ( B , C ) labeled with the CellMask Green TM Actin tracking stain. F-actin is mostly distributed in the form of stress fibers running across the cell body of untreated cells (white arrows in ( A1 , A2 )). 10 min pre-incubation with TAC resulted in a global reorganization of the F-actin filaments manifested in the formation of cortical fibers (white arrows in ( B1 , B2 )). Notably, filopodia-like structures (white arrows in ( C1 , C2 )) were observed among TAC-treated HTR8/SVneo cells evidently demonstrating a tangible outcome of the influence of TAC on F-actin cytoskeletal reorganization suggestive of cell motility. Green: CellMask Green TM -labled F-actin, Blue: DAPI-stained nuclei. Scale bars: ( A – C ) 10 µm. Nuclei were counterstained with DAPI in ( A – C ).

Article Snippet: Tacrolimus (TAC, FK506) (10 ng/mL) (Cat# B415260, Toronto Research Chemicals, Toronto, ON, Canada) was dissolved in dimethyl sulfoxide (DMSO) HybriMax TM (Cat# D2650, Millipore Sigma) and administered to each culture according to an established protocol [ ].

Techniques: Derivative Assay, Control, Labeling, Staining, Incubation

Journal: International Journal of Molecular Sciences

Article Title: An Immune-Independent Mode of Action of Tacrolimus in Promoting Human Extravillous Trophoblast Migration Involves Intracellular Calcium Release and F-Actin Cytoskeletal Reorganization

doi: 10.3390/ijms252212090

Figure Lengend Snippet: Tacrolimus negatively influenced the co-localization of IP3R and FKBP12 in HTR8/SVneo cells. ( A – E ): Representative confocal images for the immunofluorescent detection of IP3R-1 (( A1 – D1 ): red *) and FKBP12 (( A2 – D2 ): green *) and their co-localization (( A3 – D4 ); firey orange, and co-localization analysis bar graphs in ( E )) in PFA-fixed monolayers of control (DMSO-treated) and TAC-treated HTR8/SVneo cells depicting a significant reduction ( p = 0.025) in the Pearson’s correlation coefficient of mean fluorescent intensities (MFI) of the co-localization of these two protein components of the ER [Ca 2+ ]i-release channels after 1 h of exposure to TAC ( E ). Indeed, Pearson’s correlation quantification revealed that IP3R-I and FKBP12 are co-expressed in the same pixel in control cells more than in TAC-treated cells **. Note that the characteristic perinuclear distribution of these two proteins in HTR8/SVeno cells (white arrows in ( A4 – D4 ), respectively). ( F ): representative Western blot (Wb) detection of IP3R and FKBP12 in control (experimental repeats C1–C3 in lanes C1–C3) and TAC-treated (experimental repeats T1–T9 in lanes T1–T9) HTR8/SVneo cells, revealing a preservative effect of TAC in maintaining the levels of these two protein components of the ER [Ca 2+ ]i-release channels in trophoblasts as measured by the lack of a significant fold change in their protein band intensities compared to untreated control cells ( p > 0.05). The IP3R-1 bar graphs in ( F ) exclusively depict the intensities of the Wb protein bands at the 210 kDa molecular weight. Images in ( A4 – D4 ) are higher magnifications of the yellow-boxed cellular areas in ( A3 – D3 ), demonstrating the peri-nuclear distribution and co-localization of IP3R-1 and FKBP12 in control and TAC-treated HTR8/SVneo cells, respectively. ( A , B ): Representative brightfield images of control and TAC-treated HTR8/SVEneo cells depicting their general morphology and their blue-colored DAPI-stained nuclei, respectively. Scale bars: ( A – D4 ) 5 µm. Nu ( A4 – D4 ): Nuclei. TAC: Tacrolimus. ns in ( F ): Not statistically significant ( p > 0.05). *: Alexa-Fluor 790-conjugated anti-FKBP12 (anti-FKBP12-AF790; mouse anti-human) and Alexa-Fluor 680-conjugated IP3R-I (anti-IP3R-1-AF680; mouse anti-human) primary antibodies were used for the detection of their respective proteins labeled in the confocal images of HTR8/SVneo cells shown in ( A1 – D4 ). Due to current technical limitations with the wavelength detection of the 790 fluorochrome, anti-FKBP12-AF790-labeled monolayers of these cells were allowed a brief incubation with FITC-labeled goat-anti-mouse antibody suspension prior to re-incubation with the anti-IP3R-1-AF680 as described in the methods section. **: The quantification was performed by comparing all the individual frames (one cell per frame; four cells per experiment; a total of six experiments; therefore, 24 cells per treatment). Scatter blots in ( E ) represent the average rate of four cells imaged in randomly selected 5 high-power fields (HPFs) in an experiment (n = 6 plates (30 mm) per treatment).

Article Snippet: Tacrolimus (TAC, FK506) (10 ng/mL) (Cat# B415260, Toronto Research Chemicals, Toronto, ON, Canada) was dissolved in dimethyl sulfoxide (DMSO) HybriMax TM (Cat# D2650, Millipore Sigma) and administered to each culture according to an established protocol [ ].

Techniques: Control, Western Blot, Molecular Weight, Staining, Labeling, Incubation, Suspension

Journal: International Journal of Molecular Sciences

Article Title: An Immune-Independent Mode of Action of Tacrolimus in Promoting Human Extravillous Trophoblast Migration Involves Intracellular Calcium Release and F-Actin Cytoskeletal Reorganization

doi: 10.3390/ijms252212090

Figure Lengend Snippet: ( A ): Schematic depicting of the inositol triphosphate receptor (IP3R), which is an intracellular Ca 2+ -release channel located on the membrane of the endoplasmic reticulum (ER), and which belongs to the same family of the ryanodine receptors (RyRs). The conserved and widely abundant immunophilin FKBP12, which is a primary receptor for the immunosuppressant actions of TAC (FK506), has been demonstrated to physiologically interact with the inositol 1,4,5-trisphosphate receptor (IP3R) via a leucyl-prolyl dipeptide epitope that structurally resembles TAC (FK506). Here, we are postulating that TAC binding to FKBP12, likely through its structural mimicry to dipeptide epitopes on the FKBP12, sequesters this immunophilin from the IP3R, thus structurally destabilizing the channel conducive to a spiked release of [Ca 2+ ]i from ER stores (arrow). Abbreviations: TAC (FK506): tacrolimus; IP3R: inositol triphosphate receptor; ER: endoplasmic reticulum. ( B ): Schematic depicting of the inositol triphosphate receptor (IP3R) [Ca 2+ ]i-release pathway in trophoblast cells. The illustration depicts a potential mechanism through which TAC may influence [Ca 2+ ]i-release along the IP3R pathway and its putative intracellular signal transduction pathways involved in F-actin cytoskeletal reorganization in trophoblast cells. [Ca 2+ ]i-release in trophoblasts is normally a function of the G-protein-coupled receptor (GPCR)-mediated activation of phospholipase C (PLC) and the membrane-bound PI3K (which produces inositol triphosphate (IP3)). IP3 is a ligand for the intracellular IP3R channel of the internal Ca 2+ stores of the endoplasmic reticulum (ER). It is postulated that TAC influences [Ca 2+ ]i-release via its binding to the immunophilin FKBP12, plausibly resulting in the destabilization of the ER’s IP3R [Ca 2+ ]i-release channels. The observation that TAC was unable to release [Ca 2+ ]i in trophoblast cells in the presence of the IP3R inhibitor 2-APB suggests a major role for this RYR channel in the presently proposed mode of action of TAC. This notion is also supported by the ability of TAC to release [Ca 2+ ]i in the presence of the PI3K inhibitor Wortmannin, and the PLC inhibitor U73122. Moreover, PLC activation can also lead to the production of diacylglycerol (DAG), which in turn activates protein kinase C (PKC), contributing to F-actin polymerization through the phosphorylation of a large library of intermediate targets of Ca 2+ binding proteins. Based on data obtained in the present study, it is presently unclear if TAC-induced [Ca 2+ ]i-release can influence the activation of a multitude of Ca 2+ -binding proteins involved in the F-actin polymerization through the PKC signaling pathway. Abbreviations: TAC (FK506): tacrolimus; GPCR: G-coupled protein receptor; PLC: phospholipase C; IP3: inositol (1,4,5)3-phosphate; IP3R: inositol triphosphate receptor; PI3K: phosphatidylinositol 3-kinase; PKC: protein kinase C.

Article Snippet: Tacrolimus (TAC, FK506) (10 ng/mL) (Cat# B415260, Toronto Research Chemicals, Toronto, ON, Canada) was dissolved in dimethyl sulfoxide (DMSO) HybriMax TM (Cat# D2650, Millipore Sigma) and administered to each culture according to an established protocol [ ].

Techniques: Membrane, Binding Assay, Transduction, Activation Assay, Phospho-proteomics

Journal: British Journal of Cancer

Article Title: Tumorigenic role of tacrolimus through mTORC1/C2 activation in post-transplant renal cell carcinomas

doi: 10.1038/s41416-024-02597-8

Figure Lengend Snippet: Clinicopathologic characteristics of RCC patients.

Article Snippet: Cells were plated in 96-well plates (Sarstedt; Germany; 3000 cell/well) or into T25 flasks (Sarstedt; 2.5 ×10 4 cells per flask) and after 24 h, the following treatments were added in refreshed media for 72 h: tacrolimus (TAC; 10 and 50 ng/mL; Merck; USA (NJ)), rapamycin (RAPA; 10 and 50 ng/mL; Merck) and PP242 (1 μM; Tocris; UK).

Techniques:

Journal: British Journal of Cancer

Article Title: Tumorigenic role of tacrolimus through mTORC1/C2 activation in post-transplant renal cell carcinomas

doi: 10.1038/s41416-024-02597-8

Figure Lengend Snippet: Tacrolimus (TAC) elevates, while mTOR inhibitors decrease mTOR activity in vitro in normal tubular epithelial cell line (HK-2) ( a , b ) Western blot analyses of short- and long-term effect of TAC (10 ng/ml; 72 h and 21-day) on mTOR activity markers and ( c ) comparing the effects of TAC (10 ng/ml; 72 h), rapamycin (RAPA; 10 ng/ml, 72 h) and PP242 (1 μM; 72 h) on mTOR activity. Samples were derived from the same experiment, and the blots were processed in parallel. d Effects of tacrolimus, rapamycin and PP242 on proliferation of HK-2 cells (72 h). Used concentrations were indicated in the figure. Alamar Blue (AB) and Sulforhodamine B (SRB) proliferation assays were used. * p < 0.05 , ** p < 0.01 , *** p < 0.001 (one-way ANOVA with Tukey’s post hoc test, the significance compared to the control). e Long-term (21-day) effect of tacrolimus (10 ng/ml) on HK-2 cell line. Proliferation was evaluated by cell counting. No cells were discarded during the experiment.

Article Snippet: Cells were plated in 96-well plates (Sarstedt; Germany; 3000 cell/well) or into T25 flasks (Sarstedt; 2.5 ×10 4 cells per flask) and after 24 h, the following treatments were added in refreshed media for 72 h: tacrolimus (TAC; 10 and 50 ng/mL; Merck; USA (NJ)), rapamycin (RAPA; 10 and 50 ng/mL; Merck) and PP242 (1 μM; Tocris; UK).

Techniques: Activity Assay, In Vitro, Western Blot, Derivative Assay, Cell Counting

Journal: British Journal of Cancer

Article Title: Tumorigenic role of tacrolimus through mTORC1/C2 activation in post-transplant renal cell carcinomas

doi: 10.1038/s41416-024-02597-8

Figure Lengend Snippet: Tacrolimus (TAC) enhances mTOR activity and promotes the proliferation of certain renal cell carcinoma cell lines, whereas mTOR inhibitors mostly attenuate these effects. a , b Western blot analyses of short- and long-term (72 h and 21-day) effect of TAC (10 ng/ml), rapamycin (RAPA, 10 ng/ml) and PP242 (1 μM) on mTOR activity markers. Samples were derived from the same experiment, and the blots were processed in parallel. c Effects of TAC, RAPA and PP242 on proliferation of HK-2 cells (72 h). Used concentrations were indicated in the figure. * p < 0.05 , ** P < 0.01 (one-way ANOVA with Tukey’s post hoc test, the significance compared to the control). d Long-term (21-day) effect of TAC (10 ng/ml) on A498 cell line. Proliferation was evaluated by cell counting. No cells were discarded during the experiment. p = 0.07 (paired t-test).

Article Snippet: Cells were plated in 96-well plates (Sarstedt; Germany; 3000 cell/well) or into T25 flasks (Sarstedt; 2.5 ×10 4 cells per flask) and after 24 h, the following treatments were added in refreshed media for 72 h: tacrolimus (TAC; 10 and 50 ng/mL; Merck; USA (NJ)), rapamycin (RAPA; 10 and 50 ng/mL; Merck) and PP242 (1 μM; Tocris; UK).

Techniques: Activity Assay, Western Blot, Derivative Assay, Cell Counting

Journal: British Journal of Cancer

Article Title: Tumorigenic role of tacrolimus through mTORC1/C2 activation in post-transplant renal cell carcinomas

doi: 10.1038/s41416-024-02597-8

Figure Lengend Snippet: Tacrolimus (TAC) induces mTOR activation and promotes proliferation in vivo. a Tacrolimus (TAC) increases mTORC1/C2 activity, while rapamycin (RAPA) has a moderate decreasing effect in induced ischaemic kidneys of mice in vivo. IHC stainings (mTORC1: p-mTOR, p-S6; mTORC2: p-mTOR, Rictor, p-Ser473-Akt) were performed on removed kidneys (SHAM = placebo surgery; IR = induced ischaemia). DAB was used as a chromogen and haematoxylin counterstaining was performed on IHC stainings. Scale bars indicate 50 μm. b Tumour growth of TAC-treated (3 mg/kg, 3/week, 21 days) and RAPA-treated (1.5 mg/kg, 3/week, 21-day) A498 xenografts. † p < 0.1 , * p < 0.05 (paired t-test). c IHC stainings (mTORC1: p-mTOR, p-S6; mTORC2: p-mTOR, Rictor, p-Ser473-Akt) were performed on removed A498 xenograft tumours. DAB was used as a chromogen and haematoxylin counterstaining was performed on IHC stainings. Scale bars indicate 20 μm.

Article Snippet: Cells were plated in 96-well plates (Sarstedt; Germany; 3000 cell/well) or into T25 flasks (Sarstedt; 2.5 ×10 4 cells per flask) and after 24 h, the following treatments were added in refreshed media for 72 h: tacrolimus (TAC; 10 and 50 ng/mL; Merck; USA (NJ)), rapamycin (RAPA; 10 and 50 ng/mL; Merck) and PP242 (1 μM; Tocris; UK).

Techniques: Activation Assay, In Vivo, Activity Assay