Journal: PLoS ONE

Article Title: Up-Regulation of MicroRNA-145 Associates with Lymph Node Metastasis in Colorectal Cancer

doi: 10.1371/journal.pone.0102017

Figure Lengend Snippet: (A) The expression levels of Hsp-27 were examined in HCT-8-miR-145 or HCT-8-NC cells by western blotting analysis. (B) The expression of Hsp-27 protein was detected in CRC and adjacent normal tissues by western blot assay. The relative Hsp27/actin ratios of individual bands are shown as the mean + SD of values derived from all patient samples (Non-tumor tissue, n = 47; LNM-P tumor tissue, n = 41; LNM-N tumor tissue, n = 43). (C–D) MiR-145 Enhanced Hsp-27 Stability in CRC Cells.HCT-8-miR-145 or HCT-8-NC cells were incubated with the protein synthesis inhibitor cycloheximide (CHX, 0.5 µg/µL) (C) or proteasome inhibitor MG-132 (5 µM) (D) for 24 hours. The level of total Hsp-27 was detected by western blotting analysis. The relative Hsp27/actin ratios of individual bands are shown as the mean ± SD of values normalized to beta-actin.

Article Snippet: Mouse monoclonal anti-Hsp27 antibody (G31) was purchased from Cell Signaling Technology, anti-β-actin Ab (A5411) from Sigma.

Techniques: Expressing, Western Blot, Derivative Assay, Incubation

Journal: PLoS ONE

Article Title: Up-Regulation of MicroRNA-145 Associates with Lymph Node Metastasis in Colorectal Cancer

doi: 10.1371/journal.pone.0102017

Figure Lengend Snippet: (A) HCT-8-mir-145 (mir-145) or HCT-8-NC (NC) cells were transfected with Hsp-27 siRNA (mir-145+si-hsp27) or a negative control siRNA (mir-145+mock). The expression of Hsp-27 protein was detected by western blot assay. (B) Wound-healing assay to evaluate the effect of Hsp-27 siRNA in HCT-8-miR-145 cells. (C) Knockdown of Hsp-27 by siRNA in HCT-8-miR-145 cells significantly inhibited cell invasion. The images were representatives of at least three independent experiments. Average number of invasion cell number per field from at least three independent experiments ± SD is shown by column figure. ** P <0.01.

Article Snippet: Mouse monoclonal anti-Hsp27 antibody (G31) was purchased from Cell Signaling Technology, anti-β-actin Ab (A5411) from Sigma.

Techniques: Transfection, Negative Control, Expressing, Western Blot, Wound Healing Assay, Knockdown

Journal: Cellular microbiology

Article Title: Coiled-coil domains enhance the membrane association of Salmonella type III effectors.

doi: 10.1111/j.1462-5822.2011.01635.x

Figure Lengend Snippet: Fig. 6. The CC domain directs efficient membrane association of SopB. A. HeLa cells were transfected with plasmids expressing EGFP-HuSopB, EGFP-HuSopB C460S, EGFP-HuSopBDCC or EGFP-HuSopB-L3D for 20 h. Monolayers were fixed and DNA stained with DRAQ5. Confocal microscope images show EGFP-SopB fusions in grey scale and DNA in blue. Scale bar, 10 mm. B. HeLa cells were transfected with plasmids expressing Myc-SopB, Myc-SopB C460S, Myc-SopBDCC or Myc-SopB-L3D, harvested and subject to sequential detergent fractionation. Equal volumes of the saponin-soluble (S, cytosolic proteins) and TX-100-soluble (T, membrane proteins) fractions were separated by SDS-PAGE and subject to immunoblotting with antibodies against SopB, LAMP-1 (membranes) and Hsp27 (cytosol). C. HeLa cells were infected with DsopB-sigE bacteria harbouring pWSKDE-GSK (SopB), pWSKDEDCC-GSK (DCC) or pWSKDE-L3D-GSK (L3D). At 1 h p.i., infected cells were mechanically fractionated and subject to low-speed centrifugation to remove intact bacteria, and then high-speed ultracentrifugation to separate host cell membranes (M) from cytosol (C). Equal volumes of each fraction were subject to immunoblotting with antibodies against phospho-Ser9-GSK3b (translocated SopB-GSK), LAMP-1 (membranes) and Hsp27 (cytosol).

Article Snippet: Hildreth was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa; 1:5000) and mouse monoclonal anti-Hsp27 antibodies (G31, Cell Signaling Technology, 1:20 000) as described previously (Knodler et al., 2009).

Techniques: Membrane, Transfection, Expressing, Staining, Microscopy, Fractionation, SDS Page, Western Blot, Infection, Bacteria, Centrifugation

Journal: Cellular microbiology

Article Title: Coiled-coil domains enhance the membrane association of Salmonella type III effectors.

doi: 10.1111/j.1462-5822.2011.01635.x

Figure Lengend Snippet: Fig. 7. CC domains mediate the membrane association of other Salmonella effectors. A. HeLa cells were transfected with plasmids expressing EGFP-PipB2, EGFP-PipB2 YLM3D, EGFP-SseJ, EGFP-SseJ NLV3D, EGFP-SopD2 or EGFP-SopD2 YYK3D for 24 h. Monolayers were fixed and DNA stained with DRAQ5. Confocal microscope images show EGFP-effector fusions in grey scale and DNA in blue. Scale bar, 10 mm. B. HeLa cells were transfected as described in (A). Cells were harvested and subject to sequential detergent fractionation. Equal volumes of the saponin-soluble (S, cytosolic proteins) and TX-100-soluble (T, membrane proteins) fractions were separated by SDS-PAGE and subject to immunoblotting with antibodies against GFP, LAMP-1 (membranes) and Hsp27 (cytosol). The asterisk indicates a non-specific band in saponin-soluble fractions that cross-reacts with the anti-GFP antibody.

Article Snippet: Hildreth was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa; 1:5000) and mouse monoclonal anti-Hsp27 antibodies (G31, Cell Signaling Technology, 1:20 000) as described previously (Knodler et al., 2009).

Techniques: Membrane, Transfection, Expressing, Staining, Microscopy, Fractionation, SDS Page, Western Blot

Journal: Cellular microbiology

Article Title: Coiled-coil domains enhance the membrane association of Salmonella type III effectors.

doi: 10.1111/j.1462-5822.2011.01635.x

Figure Lengend Snippet: Fig. 8. CC domains are not functionally interchangeable. A. HeLa cells were transfected with plasmids expressing EGFP-HuSopB or the following chimeras, EGFP-HuSopB-IpgDCC, EGFP- HuSopB-PipB2CC, EGFP-HuSopB-SseJCC or EGFP-HuSopB-SopD2CC for 24 h. Monolayers were fixed and DNA stained with DRAQ5. Confocal microscope images show EGFP-SopB fusions in grey scale and DNA in blue. Scale bar, 10 mm. B. HeLa cells were transfected as described in (A), harvested and subject to sequential detergent fractionation. Equal volumes of the saponin-soluble (S, cytosolic proteins) and TX-100-soluble (T, membrane proteins) fractions were separated by SDS-PAGE and subject to immunoblotting with antibodies against GFP, LAMP-1 (membranes) and Hsp27 (cytosol).

Article Snippet: Hildreth was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa; 1:5000) and mouse monoclonal anti-Hsp27 antibodies (G31, Cell Signaling Technology, 1:20 000) as described previously (Knodler et al., 2009).

Techniques: Transfection, Expressing, Staining, Microscopy, Fractionation, Membrane, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Regulation of the poly(A) Polymerase Star-PAP by a Nuclear Phosphoinositide Signalosome

doi: 10.1101/2024.07.01.601467

Figure Lengend Snippet: A, B. HeLa cells treated with 100 μM etoposide or 0.6% DEM for 4 h. 48 h later, PLA analysis was performed for Star-PAP and HSP27 (A) or ⍺B-crystallin (B) ( n = 3 images each from three independent experiments, Scale bar, 10 μM). PLA foci per cell were quantified using ImageJ ( n = 30 cells from three independent experiments). P -values are for one-sample t -test versus vehicle, *** for P < 0.001. C. HeLa cells were treated with 100 μM etoposide or 0.6% DEM for 4 h and processed for IP of Star-PAP and WB using antibodies against Star-PAP, HSP27, and ⍺B-crystallin ( n = 3 independent experiments). HSP27 and ⍺B-crystallin levels were quantified by Odyssey Imaging System (LI-COR Biosciences) and normalized to the Star-PAP level in each condition. P -values are for one-sample t -test versus vehicle, ** for P < 0.01. D. HeLa cells were transfected control siRNAd or siRNAs targeting PIPKI⍺. 48 h later, cells were processed for IP of Star-PAP and WB with antibodies against Star-PAP and HSP27 ( n = 3 independent experiments). HSP27 levels were quantified and normalized to the Star-PAP level in each condition. P -values are for one-sample t -test versus control siRNA, ** for P < 0.01.

Article Snippet: Monoclonal antibodies against HA-tag (clone C29F4, #3724, Cell Signaling) HSP27 (clone D6W5V, #95357, Cell Signaling; clone G31, #2402, Cell Signaling), αB-crystallin (clone D6S9E, #45844, Cell Signaling), HO-1 (clone D60G11, #5853, Cell Signaling), and polyclonal antibodies against β-actin (#4967, Cell Signaling), PITPα (#16613-1-AP, ThermoFisher), PITPβ (#ab127563, abcam), PIPKIα (#9693, Cell Signaling) BIK (#4592, Cell Signaling) were used in this study.

Techniques: Imaging, Transfection, Control

Journal: bioRxiv

Article Title: Regulation of the poly(A) Polymerase Star-PAP by a Nuclear Phosphoinositide Signalosome

doi: 10.1101/2024.07.01.601467

Figure Lengend Snippet: A. The binding affinity of HSP27 and ⍺B-crystallin with Star-PAP, and the effects of PIP n s on these interactions was determined by MST. A constant concentration of fluorescently labelled Star-PAP (target, 5 nM) was incubated with increasing concentrations of non- labelled ligands to calculate the binding affinity. In some experiments, 2 μM of PI, PI(4)P, PI(4,5)P 2 or PI(3,4,5)P 3 were incubated with labelled Star-PAP before adding non-labelled HSP27. The binding affinities determined by MST are shown as indicated K d values. MST was performed using a Monolith NT.115 pico, and the binding affinity was autogenerated by MO. Control v.1.6 software. Data are presented as the mean ± s.d; n = 3 independent experiments. B. The binding affinity of PIP n s and Star-PAP with HSP27, and the effects of PI(4,5)P 2 on the interaction of Star-PAP and HSP27 were examined by MST. A constant concentration of fluorescently labelled HSP27 (target, 5 nM) was incubated with increasing concentrations of non-labelled ligands to calculate the binding affinity. In some experiments, 2 μM of PI(4,5)P 2 were incubated with labelled Star-PAP before adding non- labelled HSP27. The binding affinities determined by MST are shown as indicated K d values. MST was performed as described in A. C,D. In vitro binding assay was performed by adding increasing amount of His-tagged HSP27 protein (C) or His-tagged ⍺B-crystallin protein (D) to 10 nM His-tagged Star-PAP protein. Star-PAP was then IPed and WB for Star-PAP and ⍺B-crystallin was performed ( n = 3 independent experiments). Protein levels were quantified using an Odyssey Imaging System (LI-COR Biosciences). The binding curve is drawn through the HSP27/Star-PAP or ⍺B-crystallin/Star-PAP signal ratio of each condition. E. In vitro binding assay was performed by adding increasing amounts of PI(4,5)P 2 to 5 nM Star-PAP and 200 nM HSP27. Star-PAP was then IPed and WB for Star-PAP and HSP27 was performed ( n = 3 independent experiments). HSP27 levels were quantified and normalized to the Star-PAP level of each condition. P -values are for one-sample t - test versus 0 μM PIP 2 , *** for P < 0.001. F. HeLa cells were transfected with control siRNAs or siRNAs targeting PITP⍺, PITPβ, or both PITP⍺/β . 48 h later, cells were processed for Star-PAP IP followed by WB for Star- PAP and HSP27 ( n = 3 independent experiments). HSP27 level were quantified and normalized to the Star-PAP level in each condition. P -values are for one-sample t -test versus control siRNA, * for P < 0.05, *** for P < 0.001.

Article Snippet: Monoclonal antibodies against HA-tag (clone C29F4, #3724, Cell Signaling) HSP27 (clone D6W5V, #95357, Cell Signaling; clone G31, #2402, Cell Signaling), αB-crystallin (clone D6S9E, #45844, Cell Signaling), HO-1 (clone D60G11, #5853, Cell Signaling), and polyclonal antibodies against β-actin (#4967, Cell Signaling), PITPα (#16613-1-AP, ThermoFisher), PITPβ (#ab127563, abcam), PIPKIα (#9693, Cell Signaling) BIK (#4592, Cell Signaling) were used in this study.

Techniques: Binding Assay, Concentration Assay, Incubation, Control, Software, In Vitro, Imaging, Transfection

Journal: bioRxiv

Article Title: Regulation of the poly(A) Polymerase Star-PAP by a Nuclear Phosphoinositide Signalosome

doi: 10.1101/2024.07.01.601467

Figure Lengend Snippet: A. HeLa cells were transfected with control siRNAs or siRNAs targeting both PITP⍺/β or Star-PAP. 48 h later, cells were treated with 0.6% DEM for 4 h and then processed for WB using an antibody against HO-1 ( n = 3 independent experiments). HO-1 levels were quantified by Odyssey Imaging System (LI-COR Biosciences) and normalized to the actin level in each condition. P -values are for one-sample t -test versus control siRNA, ** for P < 0.01, *** for P < 0.001. B. HEK293FT cells were transfected with control siRNAs or siRNAs targeting both PITP⍺/β or Star-PAP. 48 h later, cells were treated with 0.6% DEM for 4 h and then processed for WB with an antibody against HO-1 ( n = 3 independent experiments). HO- 1 levels were quantified and normalized to the actin level in each condition. P -values are for one-sample t -test versus control siRNA, * for P < 0.05, ** for P < 0.01, *** for P < 0.001. C-F. MDA-MB-231 cells were transfected with control siRNAs or siRNAs targeting PI4KII⍺ knockdown (C), HSP27 knockdown (D), IPMK knockdown (E), or Star-PAP knockdown (F). 48 h later, cells were treated with 1 mM tBHQ or 0.6% DEM for 4 h and then processed for WB with an antibody against HO-1 ( n = 3 independent experiments). HO-1 levels were quantified and normalized to the actin level in each condition. P -values are for one-sample t -test versus control siRNA, * for P < 0.05, ** for P < 0.01. G-J. MDA-MB-231 cells were transfected with control siRNAs or siRNAs PI4KII⍺ knockdown (G), HSP27 knockdown (H), IPMK knockdown (I), or Star-PAP knockdown (J). 24 h later, cells were treated with 30 μM Cisplatin for 24 h or 100 μM etoposide for 4 h and then processed for WB using an antibody against BIK ( n = 3 independent experiments). BIK levels were quantified and normalized to the actin level in each condition. P -values are for one-sample t -test versus control siRNA, * for P < 0.05, ** for P < 0.01. I. Model depicting the nuclear PI signalosome on Star-PAP. Created by Biorender.

Article Snippet: Monoclonal antibodies against HA-tag (clone C29F4, #3724, Cell Signaling) HSP27 (clone D6W5V, #95357, Cell Signaling; clone G31, #2402, Cell Signaling), αB-crystallin (clone D6S9E, #45844, Cell Signaling), HO-1 (clone D60G11, #5853, Cell Signaling), and polyclonal antibodies against β-actin (#4967, Cell Signaling), PITPα (#16613-1-AP, ThermoFisher), PITPβ (#ab127563, abcam), PIPKIα (#9693, Cell Signaling) BIK (#4592, Cell Signaling) were used in this study.

Techniques: Transfection, Control, Imaging, Knockdown

Journal: bioRxiv

Article Title: Regulation of the poly(A) Polymerase Star-PAP by a Nuclear Phosphoinositide Signalosome

doi: 10.1101/2024.07.01.601467

Figure Lengend Snippet: A. HeLa cells were transfected with control siRNAs or siRNAs targeting both PITP⍺/β or Star-PAP. 48 h later, cells were treated with 0.6% DEM for 4 h and analyzed by WB for HO-1 ( n = 3 independent experiments). B. HEK293FT cells were transfected with control siRNAs or siRNAs targeting both PITP⍺/β or Star-PAP. 48 h later, cells were treated with 0.6% DEM for 4 h and analyzed by WB for HO-1 ( n = 3 independent experiments). C-F. MDA-MB-231 cells were transfected with control siRNAs or siRNAs targeting PI4KII⍺ knockdown (C), HSP27 knockdown (D), IPMK knockdown (E), or Star-PAP knockdown (F). 48 h later, cells were treated with 1 mM tBHQ or 0.6% DEM for 4 h and analyzed by WB for HO-1 ( n = 3 independent experiments). G-J. MDA-MB-231 cells were transfected with control siRNAs or siRNAs targeting PI4KII⍺ knockdown (G), HSP27 knockdown (H), IPMK knockdown (I), or Star-PAP knockdown (J). 24 h later, cells were treated with 30 μM Cisplatin for 24 h or 100 μM etoposide for 4 h and analyzed by WB for BIK ( n = 3 independent experiments).

Article Snippet: Monoclonal antibodies against HA-tag (clone C29F4, #3724, Cell Signaling) HSP27 (clone D6W5V, #95357, Cell Signaling; clone G31, #2402, Cell Signaling), αB-crystallin (clone D6S9E, #45844, Cell Signaling), HO-1 (clone D60G11, #5853, Cell Signaling), and polyclonal antibodies against β-actin (#4967, Cell Signaling), PITPα (#16613-1-AP, ThermoFisher), PITPβ (#ab127563, abcam), PIPKIα (#9693, Cell Signaling) BIK (#4592, Cell Signaling) were used in this study.

Techniques: Transfection, Control, Knockdown