3044 Search Results


enterobacter cloacae  (ATCC)
93
ATCC enterobacter cloacae
Enterobacter Cloacae, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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neobasetm 2 newborn screening kit  (Revvity)
92
Revvity neobasetm 2 newborn screening kit
Neobasetm 2 Newborn Screening Kit, supplied by Revvity, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti phospho hsf1 ser230 rabbit polyclonal antibody  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology anti phospho hsf1 ser230 rabbit polyclonal antibody
( A ) H1299 or 293T cells were transfected with control vector or an IER5 expression vector. Cells were harvested 21 hrs or 27 hrs post-transfection and microarray expression analysis was performed. The table shows the HSP family genes, among the genes induced by IER5. ( B ) H1299 cells were transfected with control, IER5-Flag or mutant IER5-Flag expression vectors (representative image of mut 1 is shown in ). Cells were harvested 27 hrs post-transfection, and mRNA expressions of the HSP family genes were analyzed by Northern blotting. ( C ) H1299 and 293T cells were transfected with control vector or IER5-Flag expression vector, and cells were harvested 24 hrs post-transfection. Expressions of the HSP family proteins were analyzed by Western blotting. ( D–F ) Control or IER5-targeting siRNAs were introduced into OE33 cells. Cells were harvested 52 hrs post-transfection. Expression of IER5 ( D,F ) and HSPA1A ( E,F ) were analyzed by quantitative RT-PCR ( D,E ) and Western blotting ( F ). (**p < 0.01). ( G ) The promoter regions of HSPA1A , HSPA1B and HSPA6 were inserted into the luciferase reporter plasmid containing a minimal promoter, and assayed 24 hrs post-transfection. Experiments were run in triplicate, and data are represented as the mean-fold activation ±SD. ( H ) Serially deleted regions of the HSPA1A promoter were analyzed as in ( G ). Numbers indicate the position of the 5′ most nucleotide relative to the transcription initiation site. A heat shock element (HSE), to which <t>HSF1</t> binds, was found between positions −132 and −109.
Anti Phospho Hsf1 Ser230 Rabbit Polyclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews
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pd173074  (R&D Systems)
95
R&D Systems pd173074
( A ) H1299 or 293T cells were transfected with control vector or an IER5 expression vector. Cells were harvested 21 hrs or 27 hrs post-transfection and microarray expression analysis was performed. The table shows the HSP family genes, among the genes induced by IER5. ( B ) H1299 cells were transfected with control, IER5-Flag or mutant IER5-Flag expression vectors (representative image of mut 1 is shown in ). Cells were harvested 27 hrs post-transfection, and mRNA expressions of the HSP family genes were analyzed by Northern blotting. ( C ) H1299 and 293T cells were transfected with control vector or IER5-Flag expression vector, and cells were harvested 24 hrs post-transfection. Expressions of the HSP family proteins were analyzed by Western blotting. ( D–F ) Control or IER5-targeting siRNAs were introduced into OE33 cells. Cells were harvested 52 hrs post-transfection. Expression of IER5 ( D,F ) and HSPA1A ( E,F ) were analyzed by quantitative RT-PCR ( D,E ) and Western blotting ( F ). (**p < 0.01). ( G ) The promoter regions of HSPA1A , HSPA1B and HSPA6 were inserted into the luciferase reporter plasmid containing a minimal promoter, and assayed 24 hrs post-transfection. Experiments were run in triplicate, and data are represented as the mean-fold activation ±SD. ( H ) Serially deleted regions of the HSPA1A promoter were analyzed as in ( G ). Numbers indicate the position of the 5′ most nucleotide relative to the transcription initiation site. A heat shock element (HSE), to which <t>HSF1</t> binds, was found between positions −132 and −109.
Pd173074, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human liposarcoma cell lines 94t778  (ATCC)
94
ATCC human liposarcoma cell lines 94t778
ATR inhibition by siRNA decreased liposarcoma cell proliferation. (A and B) Cell viability of <t>94T778</t> (A) and SW872 (B) determined by MTT assays after four days of ATR siRNA and negative control (NS) siRNA transfection. The data are presented as mean ± SE of the 2 experiments carried out in triplicate. (C) Microscopy images of morphologic changes and a reduction in cell number after 72 h of ATR siRNA transfection. (D and E) The expression of proteins ATR, p-ATR, p-CHK1, and γ-H2AX in the ATR-associated signaling pathway as measured by Western blotting in the liposarcoma cell lines 94T778 (D), and SW872 (E) after 72 h of siRNA transfection. (F and G) Semiquantitative analysis of (D) and (E) by densitometry relative to tubulin. The data are mean ± SE of the experiment carried out in triplicate.
Human Liposarcoma Cell Lines 94t778, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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serum  (ATCC)
94
ATCC serum
ATR inhibition by siRNA decreased liposarcoma cell proliferation. (A and B) Cell viability of <t>94T778</t> (A) and SW872 (B) determined by MTT assays after four days of ATR siRNA and negative control (NS) siRNA transfection. The data are presented as mean ± SE of the 2 experiments carried out in triplicate. (C) Microscopy images of morphologic changes and a reduction in cell number after 72 h of ATR siRNA transfection. (D and E) The expression of proteins ATR, p-ATR, p-CHK1, and γ-H2AX in the ATR-associated signaling pathway as measured by Western blotting in the liposarcoma cell lines 94T778 (D), and SW872 (E) after 72 h of siRNA transfection. (F and G) Semiquantitative analysis of (D) and (E) by densitometry relative to tubulin. The data are mean ± SE of the experiment carried out in triplicate.
Serum, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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serum - by Bioz Stars, 2026-03
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halomonas campaniensis ls21  (DSMZ)
93
DSMZ halomonas campaniensis ls21
Preliminary electroporation protocol of <t>Halomonas</t> elongata DSM 2581. Cells were cultured in LB medium supplemented with NaCl (LB60) at 30°C and 225 rpm for 14 h. Electrocompetent cells were prepared by washing the harvested cells with CaCl 2 (50 mM) + NaCl (6%) for 5 min, followed by two sucrose (300 mM) washes. The cell pellet was then resuspended in sucrose and transferred to a 0.2‐cm gap electroporation cuvette, to which the plasmid DNA was subsequently added. Electroporation was performed using a Bio‐Rad MicroPulser, after which cells were recovered in LB60 supplemented with glucose (20 mM) at 30°C and 225 rpm for 90 min. Following recovery, electroporated cells were plated onto LB60 agar plates and incubated at 30°C for 36 h to allow for colony formation. (Created in BioRender. Rios Solis, L. (2025) https://BioRender.com/osb8xfd ).
Halomonas Campaniensis Ls21, supplied by DSMZ, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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halomonas campaniensis ls21 - by Bioz Stars, 2026-03
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xav939  (Tocris)
94
Tocris xav939
Preliminary electroporation protocol of <t>Halomonas</t> elongata DSM 2581. Cells were cultured in LB medium supplemented with NaCl (LB60) at 30°C and 225 rpm for 14 h. Electrocompetent cells were prepared by washing the harvested cells with CaCl 2 (50 mM) + NaCl (6%) for 5 min, followed by two sucrose (300 mM) washes. The cell pellet was then resuspended in sucrose and transferred to a 0.2‐cm gap electroporation cuvette, to which the plasmid DNA was subsequently added. Electroporation was performed using a Bio‐Rad MicroPulser, after which cells were recovered in LB60 supplemented with glucose (20 mM) at 30°C and 225 rpm for 90 min. Following recovery, electroporated cells were plated onto LB60 agar plates and incubated at 30°C for 36 h to allow for colony formation. (Created in BioRender. Rios Solis, L. (2025) https://BioRender.com/osb8xfd ).
Xav939, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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colchicine  (Chem Impex International)
95
Chem Impex International colchicine
Preliminary electroporation protocol of <t>Halomonas</t> elongata DSM 2581. Cells were cultured in LB medium supplemented with NaCl (LB60) at 30°C and 225 rpm for 14 h. Electrocompetent cells were prepared by washing the harvested cells with CaCl 2 (50 mM) + NaCl (6%) for 5 min, followed by two sucrose (300 mM) washes. The cell pellet was then resuspended in sucrose and transferred to a 0.2‐cm gap electroporation cuvette, to which the plasmid DNA was subsequently added. Electroporation was performed using a Bio‐Rad MicroPulser, after which cells were recovered in LB60 supplemented with glucose (20 mM) at 30°C and 225 rpm for 90 min. Following recovery, electroporated cells were plated onto LB60 agar plates and incubated at 30°C for 36 h to allow for colony formation. (Created in BioRender. Rios Solis, L. (2025) https://BioRender.com/osb8xfd ).
Colchicine, supplied by Chem Impex International, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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colchicine - by Bioz Stars, 2026-03
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recombinant human matrilin 2  (R&D Systems)
92
R&D Systems recombinant human matrilin 2
<t>Matrilin-2</t> induces the inflammatory responses in human aortic valve interstitial cells (AVICs). Human AVICs were stimulated with different concentrations of recombinant matrilin-2 for 48 h. ( A ) Recombinant matrilin-2 has a dose-dependent effect on ICAM-1 expression in human AVICs. ( B ) Recombinant matrilin-2 (2.0 μg/mL) increases ICAM-1 levels. ( C ) Recombinant matrilin-2 promotes the release of MCP-1 and IL-6. Values are means ± SE. n = 5 experiments using distinct cell isolates; * P < 0.05 vs. control.
Recombinant Human Matrilin 2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ns0  (R&D Systems)
93
R&D Systems ns0
<t>Matrilin-2</t> induces the inflammatory responses in human aortic valve interstitial cells (AVICs). Human AVICs were stimulated with different concentrations of recombinant matrilin-2 for 48 h. ( A ) Recombinant matrilin-2 has a dose-dependent effect on ICAM-1 expression in human AVICs. ( B ) Recombinant matrilin-2 (2.0 μg/mL) increases ICAM-1 levels. ( C ) Recombinant matrilin-2 promotes the release of MCP-1 and IL-6. Values are means ± SE. n = 5 experiments using distinct cell isolates; * P < 0.05 vs. control.
Ns0, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ns0 - by Bioz Stars, 2026-03
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bdp tr ceramide  (Lumiprobe)
93
Lumiprobe bdp tr ceramide
( A ) Western blot analysis of uninfected or lentivirus-infected HEK293 cells. The upper panel shows GNAO1 expression; the lower panel shows GAPDH expression. ( B ) Live confocal imaging of astrocytes expressing GNAO1-GFP variants or GFP alone. Yellow arrows indicate GNAO1 localization on the cell membrane and in the Golgi apparatus. Scale bar is 50 µm. ( C ) Colocalization of GNAO1 variants (shown in green) with BDP TR <t>ceramide</t> (shown in red) in the Golgi apparatus (live confocal imaging, maximum intensity projection). Scale bar is 20 µm.
Bdp Tr Ceramide, supplied by Lumiprobe, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


( A ) H1299 or 293T cells were transfected with control vector or an IER5 expression vector. Cells were harvested 21 hrs or 27 hrs post-transfection and microarray expression analysis was performed. The table shows the HSP family genes, among the genes induced by IER5. ( B ) H1299 cells were transfected with control, IER5-Flag or mutant IER5-Flag expression vectors (representative image of mut 1 is shown in ). Cells were harvested 27 hrs post-transfection, and mRNA expressions of the HSP family genes were analyzed by Northern blotting. ( C ) H1299 and 293T cells were transfected with control vector or IER5-Flag expression vector, and cells were harvested 24 hrs post-transfection. Expressions of the HSP family proteins were analyzed by Western blotting. ( D–F ) Control or IER5-targeting siRNAs were introduced into OE33 cells. Cells were harvested 52 hrs post-transfection. Expression of IER5 ( D,F ) and HSPA1A ( E,F ) were analyzed by quantitative RT-PCR ( D,E ) and Western blotting ( F ). (**p < 0.01). ( G ) The promoter regions of HSPA1A , HSPA1B and HSPA6 were inserted into the luciferase reporter plasmid containing a minimal promoter, and assayed 24 hrs post-transfection. Experiments were run in triplicate, and data are represented as the mean-fold activation ±SD. ( H ) Serially deleted regions of the HSPA1A promoter were analyzed as in ( G ). Numbers indicate the position of the 5′ most nucleotide relative to the transcription initiation site. A heat shock element (HSE), to which HSF1 binds, was found between positions −132 and −109.

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( A ) H1299 or 293T cells were transfected with control vector or an IER5 expression vector. Cells were harvested 21 hrs or 27 hrs post-transfection and microarray expression analysis was performed. The table shows the HSP family genes, among the genes induced by IER5. ( B ) H1299 cells were transfected with control, IER5-Flag or mutant IER5-Flag expression vectors (representative image of mut 1 is shown in ). Cells were harvested 27 hrs post-transfection, and mRNA expressions of the HSP family genes were analyzed by Northern blotting. ( C ) H1299 and 293T cells were transfected with control vector or IER5-Flag expression vector, and cells were harvested 24 hrs post-transfection. Expressions of the HSP family proteins were analyzed by Western blotting. ( D–F ) Control or IER5-targeting siRNAs were introduced into OE33 cells. Cells were harvested 52 hrs post-transfection. Expression of IER5 ( D,F ) and HSPA1A ( E,F ) were analyzed by quantitative RT-PCR ( D,E ) and Western blotting ( F ). (**p < 0.01). ( G ) The promoter regions of HSPA1A , HSPA1B and HSPA6 were inserted into the luciferase reporter plasmid containing a minimal promoter, and assayed 24 hrs post-transfection. Experiments were run in triplicate, and data are represented as the mean-fold activation ±SD. ( H ) Serially deleted regions of the HSPA1A promoter were analyzed as in ( G ). Numbers indicate the position of the 5′ most nucleotide relative to the transcription initiation site. A heat shock element (HSE), to which HSF1 binds, was found between positions −132 and −109.

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Microarray, Mutagenesis, Northern Blot, Western Blot, Quantitative RT-PCR, Luciferase, Activation Assay

( A–C ) Control or HSF1-targeting siRNAs were introduced into H1299 cells. Subsequently, cells were transfected with control vector or an IER5 expression vector 24 hrs post-siRNA transfection. Cells were harvested 24 hrs post-DNA transfection. Expression of HSPA1A ( A ), HSPA6 ( B ) and HSF1 ( C ) mRNAs were analyzed by quantitative RT-PCR. ( # p < 0.0001). ( D ) 293T cells were transfected with HA-HSF1 and control vector or IER5-Flag. Cells were harvested 24 hrs post-transfection. Cell lysates were crosslinked by DSP (1 mg/ml), and immunoprecipitated using anti-HA antibody. HSP90 binding to HA-HSF1 was detected by Western blotting. ( E ) H1299 cells were transfected with control vector or IER5-Flag expression vector. Cells were harvested 25 hrs post-transfection. Whole cell lysates were crosslinked with EGS. HSF1 and IER-Flag expression was analyzed by Western blotting. ( F ) H1299 cells were transfected with control vector, IER5-Flag, mut 1-Flag expression vector. Cells were harvested 24 hrs post-transfection. Subcellular localizations of endogenous HSF1 (detected using anti-HSF1 antibody), IER5-Flag and mut 1 (detected using anti-Flag antibody) were analyzed. HSF1 and IER5 expression levels were quantitated and shown at the bottom. ( G ) HSF1 binding to Heat Shock Elements (HSEs) was analyzed by streptavidin pull-down assay. Biotinylated control or HSE-containing DNA probes were bound to streptavidin-coated beads and mixed with control or IER5-expressing cell lysates. Bead-bound HSF1 was denatured in Laemmli buffer and analyzed by Western blotting.

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( A–C ) Control or HSF1-targeting siRNAs were introduced into H1299 cells. Subsequently, cells were transfected with control vector or an IER5 expression vector 24 hrs post-siRNA transfection. Cells were harvested 24 hrs post-DNA transfection. Expression of HSPA1A ( A ), HSPA6 ( B ) and HSF1 ( C ) mRNAs were analyzed by quantitative RT-PCR. ( # p < 0.0001). ( D ) 293T cells were transfected with HA-HSF1 and control vector or IER5-Flag. Cells were harvested 24 hrs post-transfection. Cell lysates were crosslinked by DSP (1 mg/ml), and immunoprecipitated using anti-HA antibody. HSP90 binding to HA-HSF1 was detected by Western blotting. ( E ) H1299 cells were transfected with control vector or IER5-Flag expression vector. Cells were harvested 25 hrs post-transfection. Whole cell lysates were crosslinked with EGS. HSF1 and IER-Flag expression was analyzed by Western blotting. ( F ) H1299 cells were transfected with control vector, IER5-Flag, mut 1-Flag expression vector. Cells were harvested 24 hrs post-transfection. Subcellular localizations of endogenous HSF1 (detected using anti-HSF1 antibody), IER5-Flag and mut 1 (detected using anti-Flag antibody) were analyzed. HSF1 and IER5 expression levels were quantitated and shown at the bottom. ( G ) HSF1 binding to Heat Shock Elements (HSEs) was analyzed by streptavidin pull-down assay. Biotinylated control or HSE-containing DNA probes were bound to streptavidin-coated beads and mixed with control or IER5-expressing cell lysates. Bead-bound HSF1 was denatured in Laemmli buffer and analyzed by Western blotting.

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Control, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Immunoprecipitation, Binding Assay, Western Blot, Pull Down Assay

( A–E ) Whole cell extracts and immunoprecipitated samples were analyzed by Western blotting. ( A ) 293T cells were transfected with HSF1-Flag together with control vector or an IER5 expression vector. Cells were harvested 21 hrs post-transfection. ( B ) 293T cells were transfected with control vector or HSF1-Flag expression vector, together with control or IER5-targetting siRNAs. Cells were harvested 49 hrs post-transfection. ( C ) 293T cells were transfected with HSF1-Flag and control vector or IER5. Cells were harvested 27 hrs post-transfection. Cell lysates were immunoprecipitated using anti-Flag antibody, and incubated with or without CIAP for 30 min. Total HSF1 and p-Ser320 were detected. ( D ) 293T cells were transfected with control vector or HA-HSF1 expression vector. Cells were treated with or without heat shock at 42 °C for 3 hrs, and harvested 24 hrs post-DNA transfection. ( E ) 293T cells were transfected with HA-HSF1 and control or IER5-Flag expression vector. Cells were harvested 24 hrs post-DNA transfection. ( F ) HSF1 modification was analyzed by LC-MS/MS. The experiment was performed nine times, and the numbers of analysis that detected each phosphorylation are shown. Significant reductions in phosphorylation at 5 residues (S121, S307, S314, T3232 and T367) were detected. Phosphorylation sites previously reported to be involved in the repression of HSF1 activity (S121 and S307) are shown in blue. ( G ) 293T cells were transfected with the indicated plasmids and harvested 24 hrs post-transfection. HSPA1A mRNA expression was analyzed by quantitative RT-PCR and other proteins were detected by Western Blotting. (***p < 0.001, # p < 0.0001). ( H,I ) 293T cells were transfected with control vector or IER5-Flag expression vector. Cells were treated with or without heat shock at 42 °C for 3 hrs, and harvested 24 hrs post-DNA transfection. Endogenous HSF1, IER5-Flag protein expression was analyzed by Western blotting ( H ), and HSPA1A , HSPA6 and HSPA1B mRNA expression was analyzed by quantitative RT-PCR ( I ). (*p < 0.05, **p < 0.01, ***p < 0.001, # p < 0.0001).

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( A–E ) Whole cell extracts and immunoprecipitated samples were analyzed by Western blotting. ( A ) 293T cells were transfected with HSF1-Flag together with control vector or an IER5 expression vector. Cells were harvested 21 hrs post-transfection. ( B ) 293T cells were transfected with control vector or HSF1-Flag expression vector, together with control or IER5-targetting siRNAs. Cells were harvested 49 hrs post-transfection. ( C ) 293T cells were transfected with HSF1-Flag and control vector or IER5. Cells were harvested 27 hrs post-transfection. Cell lysates were immunoprecipitated using anti-Flag antibody, and incubated with or without CIAP for 30 min. Total HSF1 and p-Ser320 were detected. ( D ) 293T cells were transfected with control vector or HA-HSF1 expression vector. Cells were treated with or without heat shock at 42 °C for 3 hrs, and harvested 24 hrs post-DNA transfection. ( E ) 293T cells were transfected with HA-HSF1 and control or IER5-Flag expression vector. Cells were harvested 24 hrs post-DNA transfection. ( F ) HSF1 modification was analyzed by LC-MS/MS. The experiment was performed nine times, and the numbers of analysis that detected each phosphorylation are shown. Significant reductions in phosphorylation at 5 residues (S121, S307, S314, T3232 and T367) were detected. Phosphorylation sites previously reported to be involved in the repression of HSF1 activity (S121 and S307) are shown in blue. ( G ) 293T cells were transfected with the indicated plasmids and harvested 24 hrs post-transfection. HSPA1A mRNA expression was analyzed by quantitative RT-PCR and other proteins were detected by Western Blotting. (***p < 0.001, # p < 0.0001). ( H,I ) 293T cells were transfected with control vector or IER5-Flag expression vector. Cells were treated with or without heat shock at 42 °C for 3 hrs, and harvested 24 hrs post-DNA transfection. Endogenous HSF1, IER5-Flag protein expression was analyzed by Western blotting ( H ), and HSPA1A , HSPA6 and HSPA1B mRNA expression was analyzed by quantitative RT-PCR ( I ). (*p < 0.05, **p < 0.01, ***p < 0.001, # p < 0.0001).

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Immunoprecipitation, Western Blot, Transfection, Control, Plasmid Preparation, Expressing, Incubation, Modification, Liquid Chromatography with Mass Spectroscopy, Phospho-proteomics, Activity Assay, Quantitative RT-PCR

( A–C ) 293T cells were transfected with HA-HSF1 together with control vector or IER5-Flag. Cells were treated with 20 mM NaF acid for 4 hrs and harvested 24 hrs post-transfection. Expression of HSPA1A and HSPA6 were analyzed by quantitative RT-PCR ( A,B ) and expression of IER5-Flag and HA-HSF1 were detected by Western Blotting ( C ). (**p < 0.01, ***p < 0.001). ( D ) 293T cells were transfected with HA-HSF1 together with control vector or IER5-Flag. Cells were treated with 500 nM okadaic acid for 8 hrs and harvested 24 hrs post-transfection. Expression of HSPA1A was analyzed by quantitative RT-PCR and expression of IER5-Flag and HA-HSF1 were detected by Western Blotting. (***p < 0.001). ( E ) 293T cells were transfected as in ( D ). Cells were treated with 250 nM okadaic acid for 4 hrs and harvested 24 hrs post-transfection. Cell lysates were immunoprecipitated using anti-HA antibody. Total HSF1 and HSF1 phosphorylated at Ser121 or p-Ser303, 307 were detected by Western Blotting. ( F–H ) Control or PPP2CA (PP2A catalytic subunit)-targeting siRNAs were introduced into 293T cells. Subsequently, cells were transfected with HA-HSF1 and control vector or IER5-Flag expression vector 72 hrs post-siRNA transfection. Cells were harvested 24 hrs post-DNA transfection. IER5, HSF1 and PPP2CA protein levels were analyzed by Western blotting, and HSPA1A ( G ) and HSPA6 ( H ) mRNAs were analyzed by quantitative RT-PCR. (**p < 0.01). ( I,J ) 293T cells were transfected with HA-HSF1 together with control vector, IER5-Flag or IER5-Flag mut 1-Flag, and harvested 24 hrs post-transfection. IER5-Flag ( I ) or HA-HSF1 ( J ) was immunoprecipitated using anti-Flag or anti-HA antibodies. Association of HSF1, PP2A catalytic subunit PPP2CA and PP2A regulatory subunit B55 with IER5 was analyzed by Western blotting in ( I ), and association of IER5, PPP2CA and B55 with HSF1 was analyzed in ( J ). ( K ) Endogenous IER5 in OE33 cells was immunoprecipitated using anti-IER5 antibody. Association of HSF1 and PPP2CA with IER5 was analyzed. ( L ) PP2A phosphatase activities were analyzed using the indicated amounts of cell lysates. 293T cells were treated with or without Okadaic acid (500 nM) for 8 hrs. Control or IER5-Flag expression vector were transfected and harvested 24 hrs post-transfection.

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( A–C ) 293T cells were transfected with HA-HSF1 together with control vector or IER5-Flag. Cells were treated with 20 mM NaF acid for 4 hrs and harvested 24 hrs post-transfection. Expression of HSPA1A and HSPA6 were analyzed by quantitative RT-PCR ( A,B ) and expression of IER5-Flag and HA-HSF1 were detected by Western Blotting ( C ). (**p < 0.01, ***p < 0.001). ( D ) 293T cells were transfected with HA-HSF1 together with control vector or IER5-Flag. Cells were treated with 500 nM okadaic acid for 8 hrs and harvested 24 hrs post-transfection. Expression of HSPA1A was analyzed by quantitative RT-PCR and expression of IER5-Flag and HA-HSF1 were detected by Western Blotting. (***p < 0.001). ( E ) 293T cells were transfected as in ( D ). Cells were treated with 250 nM okadaic acid for 4 hrs and harvested 24 hrs post-transfection. Cell lysates were immunoprecipitated using anti-HA antibody. Total HSF1 and HSF1 phosphorylated at Ser121 or p-Ser303, 307 were detected by Western Blotting. ( F–H ) Control or PPP2CA (PP2A catalytic subunit)-targeting siRNAs were introduced into 293T cells. Subsequently, cells were transfected with HA-HSF1 and control vector or IER5-Flag expression vector 72 hrs post-siRNA transfection. Cells were harvested 24 hrs post-DNA transfection. IER5, HSF1 and PPP2CA protein levels were analyzed by Western blotting, and HSPA1A ( G ) and HSPA6 ( H ) mRNAs were analyzed by quantitative RT-PCR. (**p < 0.01). ( I,J ) 293T cells were transfected with HA-HSF1 together with control vector, IER5-Flag or IER5-Flag mut 1-Flag, and harvested 24 hrs post-transfection. IER5-Flag ( I ) or HA-HSF1 ( J ) was immunoprecipitated using anti-Flag or anti-HA antibodies. Association of HSF1, PP2A catalytic subunit PPP2CA and PP2A regulatory subunit B55 with IER5 was analyzed by Western blotting in ( I ), and association of IER5, PPP2CA and B55 with HSF1 was analyzed in ( J ). ( K ) Endogenous IER5 in OE33 cells was immunoprecipitated using anti-IER5 antibody. Association of HSF1 and PPP2CA with IER5 was analyzed. ( L ) PP2A phosphatase activities were analyzed using the indicated amounts of cell lysates. 293T cells were treated with or without Okadaic acid (500 nM) for 8 hrs. Control or IER5-Flag expression vector were transfected and harvested 24 hrs post-transfection.

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Immunoprecipitation

( A–D ) OE33 cells (2 × 10 3 cells) were plated in adherent ( A ) or suspension ( B ) 96 well culture plates, and control, IER5-targeting or HSF1-targeting siRNAs were introduced. Cell growth assays were performed on the indicated days. Relative cell numbers were analyzed with CellTiter-Glo reagents from four wells and the mean cell numbers ±SD are shown. IER5 and HSF1 mRNA expression was analyzed by quantitative RT-PCR 48 hrs post-transfection ( C,D ). (**p < 0.01, # p < 0.0001). ( E ) OE33 cells were stably transfected with caHSF1, and the indicated siRNAs were introduced. Cell lysates were prepared 48 hrs post-transfection, and expression of caHSF1-Flag, IER5, HSPA1A/1B were analyzed by Western blotting. ( F ) Cell growth assays were performed as in ( B ) using OE33 cells expressing caHSF1. (**p < 0.01). ( G,H ) Expression of IER5 ( G ) and HSPA6 ( H ) and prognosis in cancer patients. Disease-specific survival of patients with bladder cancer (Transitional cell carcinoma, dataset GSE13507) was analyzed using the PrognoScan database. ( I–K ) Expression of IER5 ( I ), HSPA6 ( J ) and HSPA1A ( K ) mRNA in cells treated with Adriamycin. Cell lines carrying wild-type p53 were treated with Adriamycin (1 μM) for 24 hrs (MRC5, MCF7, U2OS) or 19 hrs (A549). (*p < 0.05, **p < 0.01). ( L ) Expression of IER5, p53 and HSPA1A protein level in U2OS cells. Control, p53 and IER5 targeting siRNAs were introduced. Cells were treated with Adriamycin (1 μM) for 24 hrs and harvested 48 hrs post siRNA-transfection. ( M ) IER5 is transiently induced downstream of p53 and activates HSF1 in stressed cells, while IER5 is overexpressed and constitutively activates HSF1 in cancer cells.

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( A–D ) OE33 cells (2 × 10 3 cells) were plated in adherent ( A ) or suspension ( B ) 96 well culture plates, and control, IER5-targeting or HSF1-targeting siRNAs were introduced. Cell growth assays were performed on the indicated days. Relative cell numbers were analyzed with CellTiter-Glo reagents from four wells and the mean cell numbers ±SD are shown. IER5 and HSF1 mRNA expression was analyzed by quantitative RT-PCR 48 hrs post-transfection ( C,D ). (**p < 0.01, # p < 0.0001). ( E ) OE33 cells were stably transfected with caHSF1, and the indicated siRNAs were introduced. Cell lysates were prepared 48 hrs post-transfection, and expression of caHSF1-Flag, IER5, HSPA1A/1B were analyzed by Western blotting. ( F ) Cell growth assays were performed as in ( B ) using OE33 cells expressing caHSF1. (**p < 0.01). ( G,H ) Expression of IER5 ( G ) and HSPA6 ( H ) and prognosis in cancer patients. Disease-specific survival of patients with bladder cancer (Transitional cell carcinoma, dataset GSE13507) was analyzed using the PrognoScan database. ( I–K ) Expression of IER5 ( I ), HSPA6 ( J ) and HSPA1A ( K ) mRNA in cells treated with Adriamycin. Cell lines carrying wild-type p53 were treated with Adriamycin (1 μM) for 24 hrs (MRC5, MCF7, U2OS) or 19 hrs (A549). (*p < 0.05, **p < 0.01). ( L ) Expression of IER5, p53 and HSPA1A protein level in U2OS cells. Control, p53 and IER5 targeting siRNAs were introduced. Cells were treated with Adriamycin (1 μM) for 24 hrs and harvested 48 hrs post siRNA-transfection. ( M ) IER5 is transiently induced downstream of p53 and activates HSF1 in stressed cells, while IER5 is overexpressed and constitutively activates HSF1 in cancer cells.

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Suspension, Control, Expressing, Quantitative RT-PCR, Transfection, Stable Transfection, Western Blot

ATR inhibition by siRNA decreased liposarcoma cell proliferation. (A and B) Cell viability of 94T778 (A) and SW872 (B) determined by MTT assays after four days of ATR siRNA and negative control (NS) siRNA transfection. The data are presented as mean ± SE of the 2 experiments carried out in triplicate. (C) Microscopy images of morphologic changes and a reduction in cell number after 72 h of ATR siRNA transfection. (D and E) The expression of proteins ATR, p-ATR, p-CHK1, and γ-H2AX in the ATR-associated signaling pathway as measured by Western blotting in the liposarcoma cell lines 94T778 (D), and SW872 (E) after 72 h of siRNA transfection. (F and G) Semiquantitative analysis of (D) and (E) by densitometry relative to tubulin. The data are mean ± SE of the experiment carried out in triplicate.

Journal: American Journal of Cancer Research

Article Title: ATR inhibition sensitizes liposarcoma to doxorubicin by increasing DNA damage

doi:

Figure Lengend Snippet: ATR inhibition by siRNA decreased liposarcoma cell proliferation. (A and B) Cell viability of 94T778 (A) and SW872 (B) determined by MTT assays after four days of ATR siRNA and negative control (NS) siRNA transfection. The data are presented as mean ± SE of the 2 experiments carried out in triplicate. (C) Microscopy images of morphologic changes and a reduction in cell number after 72 h of ATR siRNA transfection. (D and E) The expression of proteins ATR, p-ATR, p-CHK1, and γ-H2AX in the ATR-associated signaling pathway as measured by Western blotting in the liposarcoma cell lines 94T778 (D), and SW872 (E) after 72 h of siRNA transfection. (F and G) Semiquantitative analysis of (D) and (E) by densitometry relative to tubulin. The data are mean ± SE of the experiment carried out in triplicate.

Article Snippet: The human liposarcoma cell lines 94T778 and SW872 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Inhibition, Negative Control, Transfection, Microscopy, Expressing, Western Blot

ATR inhibition by VE-822 decreased liposarcoma cell proliferation. (A, B) VE-822, at the indicated concentrations, inhibited liposarcoma cell lines 94T778 (A) and SW872 (B) cell proliferation, determined by MTT assay. The data represent the mean ± SE of 2 experiments carried out in triplicate. (C) Microscopy images of morphologic changes and a reduction in cell number after 72 h of VE-822 treatment. (D and E) The expression of respective proteins ATR, p-ATR, p-CHK1, and γ-H2AX in the ATR-associated signaling pathway was measured by Western blotting in the liposarcoma cell lines 94T778 (D), and SW872 (E) after 72 h of VE-822 treatment. (F and G) Semiquantitative analysis of (D) and (E) by densitometry relative to tubulin. The data are mean ± SE of the experiment carried out in triplicate.

Journal: American Journal of Cancer Research

Article Title: ATR inhibition sensitizes liposarcoma to doxorubicin by increasing DNA damage

doi:

Figure Lengend Snippet: ATR inhibition by VE-822 decreased liposarcoma cell proliferation. (A, B) VE-822, at the indicated concentrations, inhibited liposarcoma cell lines 94T778 (A) and SW872 (B) cell proliferation, determined by MTT assay. The data represent the mean ± SE of 2 experiments carried out in triplicate. (C) Microscopy images of morphologic changes and a reduction in cell number after 72 h of VE-822 treatment. (D and E) The expression of respective proteins ATR, p-ATR, p-CHK1, and γ-H2AX in the ATR-associated signaling pathway was measured by Western blotting in the liposarcoma cell lines 94T778 (D), and SW872 (E) after 72 h of VE-822 treatment. (F and G) Semiquantitative analysis of (D) and (E) by densitometry relative to tubulin. The data are mean ± SE of the experiment carried out in triplicate.

Article Snippet: The human liposarcoma cell lines 94T778 and SW872 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Inhibition, MTT Assay, Microscopy, Expressing, Western Blot

ATR inhibition reduced liposarcoma cell migration and clonogenicity in vitro and decreased the spheroid diameter in a 3D cell culture. A. 94T778 and SW872 cell line migration distances after VE-822 treatment for 24 h, 48 h, and 72 h. B. Representative results of colony formation in 94T778 and SW872. The numbers of colonies and their sizes were markedly decreased in cells treated with VE-822. C. Spheroid formation of 94T778 and SW872 were significantly smaller than untreated cells at all observation points. Cell fluorescence images of spheroid formation were recorded after 12 days of cultivation.

Journal: American Journal of Cancer Research

Article Title: ATR inhibition sensitizes liposarcoma to doxorubicin by increasing DNA damage

doi:

Figure Lengend Snippet: ATR inhibition reduced liposarcoma cell migration and clonogenicity in vitro and decreased the spheroid diameter in a 3D cell culture. A. 94T778 and SW872 cell line migration distances after VE-822 treatment for 24 h, 48 h, and 72 h. B. Representative results of colony formation in 94T778 and SW872. The numbers of colonies and their sizes were markedly decreased in cells treated with VE-822. C. Spheroid formation of 94T778 and SW872 were significantly smaller than untreated cells at all observation points. Cell fluorescence images of spheroid formation were recorded after 12 days of cultivation.

Article Snippet: The human liposarcoma cell lines 94T778 and SW872 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Inhibition, Migration, In Vitro, Cell Culture, Fluorescence

VE-822 is synergistic with doxorubicin in liposarcoma cells. (A and E) Dose-response curve of doxorubicin sensitivity in 94T778 (A) and SW872 (E) treated with different concentrations of VE-822. Cell viability significantly decreased with increasing concentrations of VE-822. The data represent mean ± SD of the independent triple experiment. (B and F) Dose-response matrix of doxorubicin combined with VE-822 in 94T778 (B) and SW872 (F), as analyzed by SynergyFinder 2.0. (C and G) Two-dimensional synergy map showing synergistic effects of VE-822 combined with doxorubicin in 94T778 (C) and SW872 (G) with Zero Interaction potency (ZIP) scores. The most synergistic area in the interaction map was 0.1-0.5 μM of VE-822 and 0.07-0.62 μM of doxorubicin in 94T778 (C), with ZIP score 13.915; and 0.1-0.5 μM of VE-822 and 0.21-1.85 μM of doxorubicin in SW872 (G) with ZIP score 19.789. (D and H) Three-dimensional synergy illustration depicting the results from (C and G).

Journal: American Journal of Cancer Research

Article Title: ATR inhibition sensitizes liposarcoma to doxorubicin by increasing DNA damage

doi:

Figure Lengend Snippet: VE-822 is synergistic with doxorubicin in liposarcoma cells. (A and E) Dose-response curve of doxorubicin sensitivity in 94T778 (A) and SW872 (E) treated with different concentrations of VE-822. Cell viability significantly decreased with increasing concentrations of VE-822. The data represent mean ± SD of the independent triple experiment. (B and F) Dose-response matrix of doxorubicin combined with VE-822 in 94T778 (B) and SW872 (F), as analyzed by SynergyFinder 2.0. (C and G) Two-dimensional synergy map showing synergistic effects of VE-822 combined with doxorubicin in 94T778 (C) and SW872 (G) with Zero Interaction potency (ZIP) scores. The most synergistic area in the interaction map was 0.1-0.5 μM of VE-822 and 0.07-0.62 μM of doxorubicin in 94T778 (C), with ZIP score 13.915; and 0.1-0.5 μM of VE-822 and 0.21-1.85 μM of doxorubicin in SW872 (G) with ZIP score 19.789. (D and H) Three-dimensional synergy illustration depicting the results from (C and G).

Article Snippet: The human liposarcoma cell lines 94T778 and SW872 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques:

Preliminary electroporation protocol of Halomonas elongata DSM 2581. Cells were cultured in LB medium supplemented with NaCl (LB60) at 30°C and 225 rpm for 14 h. Electrocompetent cells were prepared by washing the harvested cells with CaCl 2 (50 mM) + NaCl (6%) for 5 min, followed by two sucrose (300 mM) washes. The cell pellet was then resuspended in sucrose and transferred to a 0.2‐cm gap electroporation cuvette, to which the plasmid DNA was subsequently added. Electroporation was performed using a Bio‐Rad MicroPulser, after which cells were recovered in LB60 supplemented with glucose (20 mM) at 30°C and 225 rpm for 90 min. Following recovery, electroporated cells were plated onto LB60 agar plates and incubated at 30°C for 36 h to allow for colony formation. (Created in BioRender. Rios Solis, L. (2025) https://BioRender.com/osb8xfd ).

Journal: Microbial Biotechnology

Article Title: Developing High‐Efficiency Electroporation Protocols for Hard‐To‐Transform Halomonas spp.

doi: 10.1111/1751-7915.70285

Figure Lengend Snippet: Preliminary electroporation protocol of Halomonas elongata DSM 2581. Cells were cultured in LB medium supplemented with NaCl (LB60) at 30°C and 225 rpm for 14 h. Electrocompetent cells were prepared by washing the harvested cells with CaCl 2 (50 mM) + NaCl (6%) for 5 min, followed by two sucrose (300 mM) washes. The cell pellet was then resuspended in sucrose and transferred to a 0.2‐cm gap electroporation cuvette, to which the plasmid DNA was subsequently added. Electroporation was performed using a Bio‐Rad MicroPulser, after which cells were recovered in LB60 supplemented with glucose (20 mM) at 30°C and 225 rpm for 90 min. Following recovery, electroporated cells were plated onto LB60 agar plates and incubated at 30°C for 36 h to allow for colony formation. (Created in BioRender. Rios Solis, L. (2025) https://BioRender.com/osb8xfd ).

Article Snippet: Halomonas elongata DSM 2581, Halomonas boliviensis LC1 and Halomonas campaniensis LS21 were obtained from DSMZ.

Techniques: Electroporation, Cell Culture, Plasmid Preparation, Incubation

Preliminary electroporation tests of Halomonas elongata DSM 2581. (a) Electroporation efficiencies at different voltages (1.5, 1.7, 2.1 and 2.5 kV) and pulse numbers (2× 2.1, 2× 2.5 kV). (b) Electroporation efficiencies from electrocompetent cells prepared with sucrose (300 mM) vs. glycerol (10%). (c) Electroporation efficiencies from electrocompetent cells prepared from cells harvested at 6, 14 and 24 h. Data shown represent mean ± standard deviation from three biological replicates for panels (a) and (b), and four biological replicates for panel (c). Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA241. (ns, not significant; *** p < 0.001)

Journal: Microbial Biotechnology

Article Title: Developing High‐Efficiency Electroporation Protocols for Hard‐To‐Transform Halomonas spp.

doi: 10.1111/1751-7915.70285

Figure Lengend Snippet: Preliminary electroporation tests of Halomonas elongata DSM 2581. (a) Electroporation efficiencies at different voltages (1.5, 1.7, 2.1 and 2.5 kV) and pulse numbers (2× 2.1, 2× 2.5 kV). (b) Electroporation efficiencies from electrocompetent cells prepared with sucrose (300 mM) vs. glycerol (10%). (c) Electroporation efficiencies from electrocompetent cells prepared from cells harvested at 6, 14 and 24 h. Data shown represent mean ± standard deviation from three biological replicates for panels (a) and (b), and four biological replicates for panel (c). Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA241. (ns, not significant; *** p < 0.001)

Article Snippet: Halomonas elongata DSM 2581, Halomonas boliviensis LC1 and Halomonas campaniensis LS21 were obtained from DSMZ.

Techniques: Electroporation, Standard Deviation, Negative Control, Plasmid Preparation

Effect of salinity on electroporation efficiency and cell morphology of Halomonas elongata DSM 2581. (a) Electroporation efficiencies from electrocompetent cells prepared from cultures grown in LB medium containing different concentrations of NaCl: 0.5%, 1%, 2% and 6%. Data shown represent geometric mean */ geometric SD factor from three biological replicates. Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA241. (** p < 0.01; **** p < 0.0001). (b) Transmission electron microscopy imaging of cells cultured for 24 h in LB medium containing 1% NaCl. (c) Transmission electron microscopy imaging of cells cultured for 24 h in LB medium containing 6% NaCl. (d) Transmission electron microscopy imaging of cells cultured for 14 h in LB medium containing 6% NaCl.

Journal: Microbial Biotechnology

Article Title: Developing High‐Efficiency Electroporation Protocols for Hard‐To‐Transform Halomonas spp.

doi: 10.1111/1751-7915.70285

Figure Lengend Snippet: Effect of salinity on electroporation efficiency and cell morphology of Halomonas elongata DSM 2581. (a) Electroporation efficiencies from electrocompetent cells prepared from cultures grown in LB medium containing different concentrations of NaCl: 0.5%, 1%, 2% and 6%. Data shown represent geometric mean */ geometric SD factor from three biological replicates. Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA241. (** p < 0.01; **** p < 0.0001). (b) Transmission electron microscopy imaging of cells cultured for 24 h in LB medium containing 1% NaCl. (c) Transmission electron microscopy imaging of cells cultured for 24 h in LB medium containing 6% NaCl. (d) Transmission electron microscopy imaging of cells cultured for 14 h in LB medium containing 6% NaCl.

Article Snippet: Halomonas elongata DSM 2581, Halomonas boliviensis LC1 and Halomonas campaniensis LS21 were obtained from DSMZ.

Techniques: Electroporation, Negative Control, Plasmid Preparation, Transmission Assay, Electron Microscopy, Imaging, Cell Culture

Further optimisation of electroporation parameters for Halomonas elongata DSM 2581. (a) Comparison of electroporation efficiencies using two different volumes of electrocompetent cell suspension: 40 μL vs. 200 μL. (b) Effect of cell concentration on electroporation efficiency. Electrocompetent cells were resuspended in glycerol (10%) at different optical densities: OD 600 = 10, 20, 40, 60. (c) Comparison of electroporation efficiencies using two different electroporator systems—Bio‐Rad MicroPulser vs. Bio‐Rad Gene Pulser—under different electroporation conditions: Voltage, pulse number and resistance. (d) Table summarising the eight electroporation conditions tested in panel (c), resulting from the constrained mixed‐level orthogonal array design. Data shown represent mean ± standard deviation from three biological replicates. Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA241. (ns, not significant; * p < 0.05; *** p < 0.001; **** p < 0.0001).

Journal: Microbial Biotechnology

Article Title: Developing High‐Efficiency Electroporation Protocols for Hard‐To‐Transform Halomonas spp.

doi: 10.1111/1751-7915.70285

Figure Lengend Snippet: Further optimisation of electroporation parameters for Halomonas elongata DSM 2581. (a) Comparison of electroporation efficiencies using two different volumes of electrocompetent cell suspension: 40 μL vs. 200 μL. (b) Effect of cell concentration on electroporation efficiency. Electrocompetent cells were resuspended in glycerol (10%) at different optical densities: OD 600 = 10, 20, 40, 60. (c) Comparison of electroporation efficiencies using two different electroporator systems—Bio‐Rad MicroPulser vs. Bio‐Rad Gene Pulser—under different electroporation conditions: Voltage, pulse number and resistance. (d) Table summarising the eight electroporation conditions tested in panel (c), resulting from the constrained mixed‐level orthogonal array design. Data shown represent mean ± standard deviation from three biological replicates. Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA241. (ns, not significant; * p < 0.05; *** p < 0.001; **** p < 0.0001).

Article Snippet: Halomonas elongata DSM 2581, Halomonas boliviensis LC1 and Halomonas campaniensis LS21 were obtained from DSMZ.

Techniques: Electroporation, Comparison, Suspension, Concentration Assay, Standard Deviation, Negative Control, Plasmid Preparation

Effect of plasmid source on the electroporation efficiency of Halomonas elongata DSM 2581. (a) Electroporation efficiencies of pSEVA241 purified from either E. coli 10‐beta (NEB) or H. elongata DSM 2581. (b) Electroporation efficiencies of pSEVA231 purified from either E. coli 10‐beta (NEB), E. coli C2925 (NEB) or H. elongata DSM 2581. Data shown represent mean ± standard deviation from three biological replicates. Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA241 (a) or pSEVA231 (b) (* p < 0.05; *** p < 0.001; **** p < 0.0001).

Journal: Microbial Biotechnology

Article Title: Developing High‐Efficiency Electroporation Protocols for Hard‐To‐Transform Halomonas spp.

doi: 10.1111/1751-7915.70285

Figure Lengend Snippet: Effect of plasmid source on the electroporation efficiency of Halomonas elongata DSM 2581. (a) Electroporation efficiencies of pSEVA241 purified from either E. coli 10‐beta (NEB) or H. elongata DSM 2581. (b) Electroporation efficiencies of pSEVA231 purified from either E. coli 10‐beta (NEB), E. coli C2925 (NEB) or H. elongata DSM 2581. Data shown represent mean ± standard deviation from three biological replicates. Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA241 (a) or pSEVA231 (b) (* p < 0.05; *** p < 0.001; **** p < 0.0001).

Article Snippet: Halomonas elongata DSM 2581, Halomonas boliviensis LC1 and Halomonas campaniensis LS21 were obtained from DSMZ.

Techniques: Plasmid Preparation, Electroporation, Purification, Standard Deviation, Negative Control

Electroporation of Halomonas boliviensis LC1 and Halomonas campaniensis LS21. Electroporation efficiencies of H. boliviensis LC1 and H. campaniensis LS21 transformed with pSEVA231 purified from either E. coli C2925 (NEB) (a) Comparison of electroporation efficiencies from electrocompetent cells prepared from cultures grown in LB medium containing different concentrations of NaCl: 6% vs. 1%. (b) Comparison of electroporation efficiencies using two different electroporator systems—Bio‐Rad MicroPulser vs. Bio‐Rad Gene Pulser—under varying electroporation conditions: Voltage, pulse number and resistance. Parameters for conditions C1, C4, C6 and C7 are detailed in Figure . Data shown represent mean ± standard deviation from three biological replicates. Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA231. (*** p < 0.001; **** p < 0.0001)

Journal: Microbial Biotechnology

Article Title: Developing High‐Efficiency Electroporation Protocols for Hard‐To‐Transform Halomonas spp.

doi: 10.1111/1751-7915.70285

Figure Lengend Snippet: Electroporation of Halomonas boliviensis LC1 and Halomonas campaniensis LS21. Electroporation efficiencies of H. boliviensis LC1 and H. campaniensis LS21 transformed with pSEVA231 purified from either E. coli C2925 (NEB) (a) Comparison of electroporation efficiencies from electrocompetent cells prepared from cultures grown in LB medium containing different concentrations of NaCl: 6% vs. 1%. (b) Comparison of electroporation efficiencies using two different electroporator systems—Bio‐Rad MicroPulser vs. Bio‐Rad Gene Pulser—under varying electroporation conditions: Voltage, pulse number and resistance. Parameters for conditions C1, C4, C6 and C7 are detailed in Figure . Data shown represent mean ± standard deviation from three biological replicates. Negative control experiments were performed by electroporating electrocompetent cells without the addition of plasmid pSEVA231. (*** p < 0.001; **** p < 0.0001)

Article Snippet: Halomonas elongata DSM 2581, Halomonas boliviensis LC1 and Halomonas campaniensis LS21 were obtained from DSMZ.

Techniques: Electroporation, Transformation Assay, Purification, Comparison, Standard Deviation, Negative Control, Plasmid Preparation

Matrilin-2 induces the inflammatory responses in human aortic valve interstitial cells (AVICs). Human AVICs were stimulated with different concentrations of recombinant matrilin-2 for 48 h. ( A ) Recombinant matrilin-2 has a dose-dependent effect on ICAM-1 expression in human AVICs. ( B ) Recombinant matrilin-2 (2.0 μg/mL) increases ICAM-1 levels. ( C ) Recombinant matrilin-2 promotes the release of MCP-1 and IL-6. Values are means ± SE. n = 5 experiments using distinct cell isolates; * P < 0.05 vs. control.

Journal: Cells

Article Title: Mechanistic Roles of Matrilin-2 and Klotho in Modulating the Inflammatory Activity of Human Aortic Valve Cells

doi: 10.3390/cells9020385

Figure Lengend Snippet: Matrilin-2 induces the inflammatory responses in human aortic valve interstitial cells (AVICs). Human AVICs were stimulated with different concentrations of recombinant matrilin-2 for 48 h. ( A ) Recombinant matrilin-2 has a dose-dependent effect on ICAM-1 expression in human AVICs. ( B ) Recombinant matrilin-2 (2.0 μg/mL) increases ICAM-1 levels. ( C ) Recombinant matrilin-2 promotes the release of MCP-1 and IL-6. Values are means ± SE. n = 5 experiments using distinct cell isolates; * P < 0.05 vs. control.

Article Snippet: Recombinant human matrilin-2 and Klotho proteins (expressed by mouse myeloma cell line; endotoxin free) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Recombinant, Expressing

Matrilin-2 activates PKR and NF-κB in human AVICs. Human AVICs were stimulated with recombinant matrilin-2 for varied durations. Stimulation with recombinant matrilin-2 resulted in increased levels of phospho-PKR and phospho-NF-κB. Values are means ± SE. n = 5 experiments using distinct cell isolates; * P < 0.05 vs. control.

Journal: Cells

Article Title: Mechanistic Roles of Matrilin-2 and Klotho in Modulating the Inflammatory Activity of Human Aortic Valve Cells

doi: 10.3390/cells9020385

Figure Lengend Snippet: Matrilin-2 activates PKR and NF-κB in human AVICs. Human AVICs were stimulated with recombinant matrilin-2 for varied durations. Stimulation with recombinant matrilin-2 resulted in increased levels of phospho-PKR and phospho-NF-κB. Values are means ± SE. n = 5 experiments using distinct cell isolates; * P < 0.05 vs. control.

Article Snippet: Recombinant human matrilin-2 and Klotho proteins (expressed by mouse myeloma cell line; endotoxin free) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Recombinant

Both PKR and NF-κB are critical for AVIC inflammatory responses induced by matrilin-2, and PKR is responsible for NF-κB activation. Human AVICs were treated with PKR inhibitors (C 13 H 8 N 4 OS and 2-AP) or NF-κB inhibitor (Bay 11-7082) for 1 h or left untreated, followed by stimulation with recombinant matrilin-2 for 1 h or 48 h. ( A , B ) Inhibition of PKR suppressed NF-κB phosphorylation. ( C ) Nuclear translocation of NF-κB was inhibited by PKR inhibitors. Representative images of immunofluorescence staining show NF-κB (red) in human AVICs. Alexa 488–tagged wheat germ agglutinin (WGA) was applied to outline plasma membrane (green). DAPI (4′,6-diamidino-2-phenylindole) was applied for nuclei counterstaining (blue). Original magnification, ×40 objective. ( D , E ) Inhibition of PKR or NF-κB markedly reduced ICAM-1 production following matrilin-2 stimulation. ( F , G ) PKR and NF-κB inhibitors markedly reduced MCP-1 and IL-6 release following stimulation with matrilin-2. Values are means ± SE. n = 5 experiments using distinct cell isolates; * P < 0.05 vs. control; # P < 0.05 vs. matrilin-2 alone.

Journal: Cells

Article Title: Mechanistic Roles of Matrilin-2 and Klotho in Modulating the Inflammatory Activity of Human Aortic Valve Cells

doi: 10.3390/cells9020385

Figure Lengend Snippet: Both PKR and NF-κB are critical for AVIC inflammatory responses induced by matrilin-2, and PKR is responsible for NF-κB activation. Human AVICs were treated with PKR inhibitors (C 13 H 8 N 4 OS and 2-AP) or NF-κB inhibitor (Bay 11-7082) for 1 h or left untreated, followed by stimulation with recombinant matrilin-2 for 1 h or 48 h. ( A , B ) Inhibition of PKR suppressed NF-κB phosphorylation. ( C ) Nuclear translocation of NF-κB was inhibited by PKR inhibitors. Representative images of immunofluorescence staining show NF-κB (red) in human AVICs. Alexa 488–tagged wheat germ agglutinin (WGA) was applied to outline plasma membrane (green). DAPI (4′,6-diamidino-2-phenylindole) was applied for nuclei counterstaining (blue). Original magnification, ×40 objective. ( D , E ) Inhibition of PKR or NF-κB markedly reduced ICAM-1 production following matrilin-2 stimulation. ( F , G ) PKR and NF-κB inhibitors markedly reduced MCP-1 and IL-6 release following stimulation with matrilin-2. Values are means ± SE. n = 5 experiments using distinct cell isolates; * P < 0.05 vs. control; # P < 0.05 vs. matrilin-2 alone.

Article Snippet: Recombinant human matrilin-2 and Klotho proteins (expressed by mouse myeloma cell line; endotoxin free) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Recombinant, Inhibition, Translocation Assay, Immunofluorescence, Staining, Membrane

Both toll-like receptor (TLR) 2 and 4 mediate matrilin-2-induced PKR activation in human AVICs. Human AVICs were treated with TLR2-neutralizing antibody or TLR4-neutralizing antibody for 1 h or left untreated, followed by stimulation with recombinant matrilin-2. Neutralization either TLR2 or TLR4 attenuated matrilin-2-induced PKR phosphorylation. Values are means ± SE. n = 3 separate experiments; * P < 0.05 vs. control; # P < 0.05 vs. matrilin-2 alone.

Journal: Cells

Article Title: Mechanistic Roles of Matrilin-2 and Klotho in Modulating the Inflammatory Activity of Human Aortic Valve Cells

doi: 10.3390/cells9020385

Figure Lengend Snippet: Both toll-like receptor (TLR) 2 and 4 mediate matrilin-2-induced PKR activation in human AVICs. Human AVICs were treated with TLR2-neutralizing antibody or TLR4-neutralizing antibody for 1 h or left untreated, followed by stimulation with recombinant matrilin-2. Neutralization either TLR2 or TLR4 attenuated matrilin-2-induced PKR phosphorylation. Values are means ± SE. n = 3 separate experiments; * P < 0.05 vs. control; # P < 0.05 vs. matrilin-2 alone.

Article Snippet: Recombinant human matrilin-2 and Klotho proteins (expressed by mouse myeloma cell line; endotoxin free) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Recombinant, Neutralization

Human AVICs express Klotho. ( A ) Klotho protein is present in human AVICs and is upregulated by matrilin-2 stimulation for 48 h. ( B ) Klotho mRNA is detectable in human AVICs and increases in a time-dependent manner following matrilin-2 stimulation. Values are means ± SE. n = 3 separate experiments; * P < 0.05 vs. control.

Journal: Cells

Article Title: Mechanistic Roles of Matrilin-2 and Klotho in Modulating the Inflammatory Activity of Human Aortic Valve Cells

doi: 10.3390/cells9020385

Figure Lengend Snippet: Human AVICs express Klotho. ( A ) Klotho protein is present in human AVICs and is upregulated by matrilin-2 stimulation for 48 h. ( B ) Klotho mRNA is detectable in human AVICs and increases in a time-dependent manner following matrilin-2 stimulation. Values are means ± SE. n = 3 separate experiments; * P < 0.05 vs. control.

Article Snippet: Recombinant human matrilin-2 and Klotho proteins (expressed by mouse myeloma cell line; endotoxin free) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques:

Klotho suppresses matrilin-2-induced inflammatory responses in human AVICs via inhibition of the PKR-NF-κB cascade. Human AVICs were treated with recombinant Klotho (0.5 μg/mL) for 2 h or left untreated, followed by stimulation with matrilin-2. ( A ) Recombinant Klotho suppressed matrilin-2-induced PKR activation. ( B ) Recombinant Klotho attenuated soluble matrilin-2-induced NF-κB activation. Values are means ± SE. n = 3 separate experiment; * P < 0.05 vs. control; # P < 0.05 vs. the same time point of matrilin-2 alone. ( C ) Klotho inhibited matrilin -2 -induced NF-κB nuclear translocation. Representative images of immunofluorescence staining show NF-κB (red) in human AVICs. Alexa 488-tagged WGA (green) was applied to outline plasma membrane. DAPI (blue) was applied for nuclear counterstaining. Original magnification, ×40 objective. ( D ) Klotho attenuated matrilin-2-induced ICAM-1 expression. ( E ) Klotho reduced MCP-1 and IL-6 production induced by matrilin-2. Values are means ± SE. n = 5 separate experiments using distinct cell isolates; * P < 0.05 vs. control; # P < 0.05 vs. matrilin-2 alone.

Journal: Cells

Article Title: Mechanistic Roles of Matrilin-2 and Klotho in Modulating the Inflammatory Activity of Human Aortic Valve Cells

doi: 10.3390/cells9020385

Figure Lengend Snippet: Klotho suppresses matrilin-2-induced inflammatory responses in human AVICs via inhibition of the PKR-NF-κB cascade. Human AVICs were treated with recombinant Klotho (0.5 μg/mL) for 2 h or left untreated, followed by stimulation with matrilin-2. ( A ) Recombinant Klotho suppressed matrilin-2-induced PKR activation. ( B ) Recombinant Klotho attenuated soluble matrilin-2-induced NF-κB activation. Values are means ± SE. n = 3 separate experiment; * P < 0.05 vs. control; # P < 0.05 vs. the same time point of matrilin-2 alone. ( C ) Klotho inhibited matrilin -2 -induced NF-κB nuclear translocation. Representative images of immunofluorescence staining show NF-κB (red) in human AVICs. Alexa 488-tagged WGA (green) was applied to outline plasma membrane. DAPI (blue) was applied for nuclear counterstaining. Original magnification, ×40 objective. ( D ) Klotho attenuated matrilin-2-induced ICAM-1 expression. ( E ) Klotho reduced MCP-1 and IL-6 production induced by matrilin-2. Values are means ± SE. n = 5 separate experiments using distinct cell isolates; * P < 0.05 vs. control; # P < 0.05 vs. matrilin-2 alone.

Article Snippet: Recombinant human matrilin-2 and Klotho proteins (expressed by mouse myeloma cell line; endotoxin free) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Inhibition, Recombinant, Activation Assay, Translocation Assay, Immunofluorescence, Staining, Membrane, Expressing

( A ) Western blot analysis of uninfected or lentivirus-infected HEK293 cells. The upper panel shows GNAO1 expression; the lower panel shows GAPDH expression. ( B ) Live confocal imaging of astrocytes expressing GNAO1-GFP variants or GFP alone. Yellow arrows indicate GNAO1 localization on the cell membrane and in the Golgi apparatus. Scale bar is 50 µm. ( C ) Colocalization of GNAO1 variants (shown in green) with BDP TR ceramide (shown in red) in the Golgi apparatus (live confocal imaging, maximum intensity projection). Scale bar is 20 µm.

Journal: Cells

Article Title: The GNAO1-B Splice Variant Is the Predominant Isoform in Human Astrocytes and Localizes to Retraction Fibers and Migrasomes

doi: 10.3390/cells14221755

Figure Lengend Snippet: ( A ) Western blot analysis of uninfected or lentivirus-infected HEK293 cells. The upper panel shows GNAO1 expression; the lower panel shows GAPDH expression. ( B ) Live confocal imaging of astrocytes expressing GNAO1-GFP variants or GFP alone. Yellow arrows indicate GNAO1 localization on the cell membrane and in the Golgi apparatus. Scale bar is 50 µm. ( C ) Colocalization of GNAO1 variants (shown in green) with BDP TR ceramide (shown in red) in the Golgi apparatus (live confocal imaging, maximum intensity projection). Scale bar is 20 µm.

Article Snippet: Golgi staining The Golgi apparatus in live cells was stained with BDP-TR ceramide (Lumiprobe RUS, Moscow, Russia) according to the manufacturer’s protocol.

Techniques: Western Blot, Infection, Expressing, Imaging, Membrane