Journal: International Journal of Molecular Sciences

Article Title: Antibacterial Effects of X-ray and MRI Contrast Media: An In Vitro Pilot Study

doi: 10.3390/ijms24043470

Figure Lengend Snippet: Growth of bacteria in minimal medium M9 in the presence of contrast medium.

Article Snippet: Six different bacterial strains were used in the different experiments of this study: S. aureus (ATCC 25923), P. aeruginosa (ATCC 27853), B. subtilis (DSM 618) and B. subtilis (spore suspension, Merck 110649, number of germinable spores 8 × 10 6 to 5 × 10 7 CFU/mL), M. smegmatis (ATCC 35798) and E. coli (ATCC 25922).

Techniques: Bacteria

Journal: International journal of molecular sciences

Article Title: PIP5Kγ Mediates PI(4,5)P2/Merlin/LATS1 Signaling Activation and Interplays with Hsc70 in Hippo-YAP Pathway Regulation.

doi: 10.3390/ijms241914786

Figure Lengend Snippet: Figure 1. Effects of PIP5Kγ overexpression or knockdown on LATS1 and YAP. (A–C) HEK293 cells were transfected with control vector, HA-PIP5Kγ87, or HA-PIP5Kγ90. HEK293 (D,F) or HeLa (E) cells were transfected with control siRNA (siCtrl) or PIP5Kγ siRNA (siPIP5Kγ). (G) HA-PIP5Kγ87 or HA-PIP5Kγ90 was transfected into HEK293 cells pretreated with siPIP5Kγ. Cell lysates (A,D,G) and cytosolic and nuclear fractions (B) were analyzed using WB with the indicated antibodies. (C,F) Relative quantification of CTGF, CYR61, and ANKRD1 mRNA levels analyzed by qRT-PCR (n = 3). Values in the graphs represent the mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Confocal images of PI(4,5)P2 immunostaining. Cells were visualized using nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 10 µm.

Article Snippet: For PIP5Kγ and Hsc70 knockdown, their siRNAs (sc-39137 and sc-29349, respectively, Santa Cruz Biotechnology) mixed with Lipofectamine RNAiMAX in Opti-MEM I were added to cells at a final 20 nM concentration for 48 h. Similarly, LATS1 siRNA targeting the sequence (5′-GAACCAAACUCUCAAACAAdTdT-3′) and a non-targeting siRNA (5′- UUCUCCGAACGUGUCACGUdTdT-3′) from Bioneer (Daejeon, Republic of Korea) were used for LATS1 and control knockdown, respectively [34].

Techniques: Over Expression, Knockdown, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Immunostaining, Staining

Journal: International journal of molecular sciences

Article Title: PIP5Kγ Mediates PI(4,5)P2/Merlin/LATS1 Signaling Activation and Interplays with Hsc70 in Hippo-YAP Pathway Regulation.

doi: 10.3390/ijms241914786

Figure Lengend Snippet: Figure 2. Effects of PIP5Kγ90 WT and its KD mutant on LATS1 and YAP. HEK293 (A,B) or HeLa (C,D) cells were transfected with GFP control vector, GFP-PIP5Kγ90 WT, or GFP-PIP5Kγ90 KD. (A) WB analysis of cell lysates with the indicated antibodies. Arrows indicate GFP or GFP-PIP5Kγ90. (B) Relative quantitation of CTGF, CYR61, and ANKRD1 mRNA levels as analyzed using qRT-PCR (n = 3). Values in the graphs represent the mean ± S.E.M. ** p < 0.01, *** p < 0.001. Cells were immunostained with anti-YAP (C) or anti-PI(4,5)P2 (D) antibodies, and nuclei were stained with DAPI. Representative images of GFP, YAP, or PI(4,5)P2 immunofluorescence, and DAPI-stained nuclei were captured using confocal microscopy. The arrows and arrowheads indicate transfected and non-transfected cells, respectively. Scale bars, 10 µm.

Article Snippet: For PIP5Kγ and Hsc70 knockdown, their siRNAs (sc-39137 and sc-29349, respectively, Santa Cruz Biotechnology) mixed with Lipofectamine RNAiMAX in Opti-MEM I were added to cells at a final 20 nM concentration for 48 h. Similarly, LATS1 siRNA targeting the sequence (5′-GAACCAAACUCUCAAACAAdTdT-3′) and a non-targeting siRNA (5′- UUCUCCGAACGUGUCACGUdTdT-3′) from Bioneer (Daejeon, Republic of Korea) were used for LATS1 and control knockdown, respectively [34].

Techniques: Mutagenesis, Transfection, Control, Plasmid Preparation, Quantitation Assay, Quantitative RT-PCR, Staining, Confocal Microscopy

Journal: International journal of molecular sciences

Article Title: PIP5Kγ Mediates PI(4,5)P2/Merlin/LATS1 Signaling Activation and Interplays with Hsc70 in Hippo-YAP Pathway Regulation.

doi: 10.3390/ijms241914786

Figure Lengend Snippet: Figure 4. Interaction of PIP5Kγ90 with Merlin and LATS1. HEK293 cells (A–G) or WT and Merlin KO HEK293A cells (H) were transfected in the absence and presence of the indicated expression plasmids or corresponding control vectors. HA-IP (A,B,D,E) or FLAG-IP (C,F–H) products were prepared from the resulting cell lysates (input), as indicated. (G) Cells were co-transfected 1 d after treatment with siCtrl or LATS1 siRNA (siLATS1). (A–H) Transfected and/or endogenous proteins in input samples and IP products were detected using WB analysis with the indicated antibodies.

Article Snippet: For PIP5Kγ and Hsc70 knockdown, their siRNAs (sc-39137 and sc-29349, respectively, Santa Cruz Biotechnology) mixed with Lipofectamine RNAiMAX in Opti-MEM I were added to cells at a final 20 nM concentration for 48 h. Similarly, LATS1 siRNA targeting the sequence (5′-GAACCAAACUCUCAAACAAdTdT-3′) and a non-targeting siRNA (5′- UUCUCCGAACGUGUCACGUdTdT-3′) from Bioneer (Daejeon, Republic of Korea) were used for LATS1 and control knockdown, respectively [34].

Techniques: Transfection, Expressing, Control

Journal: International journal of molecular sciences

Article Title: PIP5Kγ Mediates PI(4,5)P2/Merlin/LATS1 Signaling Activation and Interplays with Hsc70 in Hippo-YAP Pathway Regulation.

doi: 10.3390/ijms241914786

Figure Lengend Snippet: Figure 6. Effects of the PIP5Kγ90 WT and KD mutant on LATS1 co-localization with Merlin at the PM. (A–C,E,F) HeLa cells were co-transfected with GFP control vector, GFP-PIP5Kγ90 WT, or its KD mutant, together with HA-Merlin, HA-LATS1, Myc-LATS1, and/or mRFP-Tubby, as indicated. HA-Merlin, HA-LATS1, and/or Myc-LATS1 were immunostained using the respective primary antibodies followed by Alexa Fluor 594- and/or 350-labeled secondary antibodies. Representative images were captured using confocal microscopy. Arrows indicate enrichment of the transfected proteins at the PM. Scale bars, 10 µm. (D) HEK293 cells were co-transfected with GFP control vector or GFP- PIP5Kγ90 WT or its KD mutant with FLAG-Merlin. Cell lysates and FLAG-IP products were analyzed through WB using the indicated antibodies. Arrows indicate GFP-PIP5Kγ90 or GFP. (G) A schematic diagram of the results.

Article Snippet: For PIP5Kγ and Hsc70 knockdown, their siRNAs (sc-39137 and sc-29349, respectively, Santa Cruz Biotechnology) mixed with Lipofectamine RNAiMAX in Opti-MEM I were added to cells at a final 20 nM concentration for 48 h. Similarly, LATS1 siRNA targeting the sequence (5′-GAACCAAACUCUCAAACAAdTdT-3′) and a non-targeting siRNA (5′- UUCUCCGAACGUGUCACGUdTdT-3′) from Bioneer (Daejeon, Republic of Korea) were used for LATS1 and control knockdown, respectively [34].

Techniques: Mutagenesis, Transfection, Control, Plasmid Preparation, Labeling, Confocal Microscopy

Journal: International journal of molecular sciences

Article Title: PIP5Kγ Mediates PI(4,5)P2/Merlin/LATS1 Signaling Activation and Interplays with Hsc70 in Hippo-YAP Pathway Regulation.

doi: 10.3390/ijms241914786

Figure Lengend Snippet: Figure 7. Effects of Merlin KO and LATS1/2 KO on Hippo pathway regulation by PIP5Kγ. WT and Merlin KO (A,B) or LATS1/2 KO (C,D) HEK293A cells were transfected with control vector, HA-PIP5Kγ87, or HA-PIP5Kγ90. (A,C) WB analysis of cell lysates with the indicated antibodies. (B,D) CTGF, CYR61, and ANKRD1 mRNA levels as analyzed using qRT-PCR were quantified relative to those in control vector-transfected WT cells (n = 3). Values in the graphs represent the mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001. (E–G) WT, Merlin KO, or LATS1/2 KO HEK293A cells were transfected with GFP control vector, GFP-PIP5Kγ87, or GFP-PIP5Kγ90. YAP was immunostained using its primary antibody and Alexa Fluor 594-labeled secondary antibody; DAPI staining was used for nuclei visualization. Representative images were obtained using confocal microscopy. The arrows and arrowheads indicate transfected and non-transfected cells, respectively. Scale bars, 10 µm. (H) Relative abundance of cytosolic or nuclear YAP in GFP-positive cells (n = 30 each) collected from random fields in (E–G).

Article Snippet: For PIP5Kγ and Hsc70 knockdown, their siRNAs (sc-39137 and sc-29349, respectively, Santa Cruz Biotechnology) mixed with Lipofectamine RNAiMAX in Opti-MEM I were added to cells at a final 20 nM concentration for 48 h. Similarly, LATS1 siRNA targeting the sequence (5′-GAACCAAACUCUCAAACAAdTdT-3′) and a non-targeting siRNA (5′- UUCUCCGAACGUGUCACGUdTdT-3′) from Bioneer (Daejeon, Republic of Korea) were used for LATS1 and control knockdown, respectively [34].

Techniques: Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Labeling, Staining, Confocal Microscopy

Journal: International journal of molecular sciences

Article Title: PIP5Kγ Mediates PI(4,5)P2/Merlin/LATS1 Signaling Activation and Interplays with Hsc70 in Hippo-YAP Pathway Regulation.

doi: 10.3390/ijms241914786

Figure Lengend Snippet: Figure 9. Hsc70 participates in Hippo–YAP pathway regulation by PIP5Kγ90. (A,B,D) Control vector, FLAG-PIP5Kγ90, and/or V5-Hsc70 were transfected into HEK293 cells, as indicated. (C) IP products using control IgG or PIP5Kγ antibody were prepared from HEK293 cell lysates. Hsc70 was immunoblotted with short-exposure (SE) and long-exposure (LE) times. WT, Merlin KO, and LATS1/2 KO HEK293A cells (E,F), HEK293 cells (G), Hsc70 siRNA (siHsc70)-treated HeLa cells (H), or PIP5Kγ siRNA (siPIP5Kγ)-treated HEK293 cells (I) were transfected in the absence and presence of the indicated plasmids. Resulting cell lysates (A–I) and FLAG IP products (A,B,E–G) were examined using WB analysis with the indicated antibodies.

Article Snippet: For PIP5Kγ and Hsc70 knockdown, their siRNAs (sc-39137 and sc-29349, respectively, Santa Cruz Biotechnology) mixed with Lipofectamine RNAiMAX in Opti-MEM I were added to cells at a final 20 nM concentration for 48 h. Similarly, LATS1 siRNA targeting the sequence (5′-GAACCAAACUCUCAAACAAdTdT-3′) and a non-targeting siRNA (5′- UUCUCCGAACGUGUCACGUdTdT-3′) from Bioneer (Daejeon, Republic of Korea) were used for LATS1 and control knockdown, respectively [34].

Techniques: Control, Plasmid Preparation, Transfection

Journal: International journal of molecular sciences

Article Title: PIP5Kγ Mediates PI(4,5)P2/Merlin/LATS1 Signaling Activation and Interplays with Hsc70 in Hippo-YAP Pathway Regulation.

doi: 10.3390/ijms241914786

Figure Lengend Snippet: Figure 10. Proposed model for functional role of PI(4,5)P2-producing PIP5Kγ that regulates the Hippo–YAP signaling pathway by mediating a functional complex with Merlin and LATS1 in the plasma membrane, and via an interplay with Hsc70.

Article Snippet: For PIP5Kγ and Hsc70 knockdown, their siRNAs (sc-39137 and sc-29349, respectively, Santa Cruz Biotechnology) mixed with Lipofectamine RNAiMAX in Opti-MEM I were added to cells at a final 20 nM concentration for 48 h. Similarly, LATS1 siRNA targeting the sequence (5′-GAACCAAACUCUCAAACAAdTdT-3′) and a non-targeting siRNA (5′- UUCUCCGAACGUGUCACGUdTdT-3′) from Bioneer (Daejeon, Republic of Korea) were used for LATS1 and control knockdown, respectively [34].

Techniques: Functional Assay, Clinical Proteomics, Membrane