Structured Review

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Effect of MPP + <t>on</t> <t>PHB2</t> protein expression and mitochondrial function in SH-SY5Y cells. SH-SY5Y cells were treated with <t>PHB2-shRNA</t> and PHB2-Over Exp, and then induced with 1 mM MPP + for 24 hours. (A, B) PHB2 protein concentration in SH-SY5Y cells after 1 mM MPP + treatment for 6, 12, 24, and 48 hours. (A) Western blots showing PHB2 protein expression in MPP + -treated cells at different times. (B) Quantitative analysis of PHB2 protein expression. (C–G) Protein expression changes of Nrf2, HO-1, NQO-1, and PHB2. (C) Western blots showing Nrf2, HO-1, NQO-1, and PHB2 protein expression in MPP + -treated and PHB2-shRNA- or PHB2-Over Exp-treated cells. (D–G) Quantitative analysis of Nrf2, HO-1, NQO-1, and PHB2 protein expression. (H, I) The effect of PHB2 expression on mitochondrial membrane potential induced by MPP + . (H) JC-1 staining of SH-SY5Y cells treated with PHB2-shRNA and PHB2-Over Exp. Original magnification 10×, scale bar: 50 μm. (I) Quantitative analysis of JC-1 staining: the green/red ratio reflects changes in the mitochondrial membrane potential. (J, K) The effect of changes in PHB2 expression on ROS production under MPP + induction. (J) Intracellular ROS fluorescence. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp and labeled with DCFH-DA (green fluorescence). Original magnification 4×, scale bar: 100 μm. (K) Quantitative analysis of ROS (DCFH-DA) in SH-SY5Y cells. (L) Electron microscopy images of morphological changes in mitochondria in PHB2-shRNA-treated and MPP + -induced cells. Red boxes show magnified mitochondria. Scale bars: 1 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05,## P < 0.01, vs . MPP + group (one-way analysis of variance with Tukey's multiple comparison test). DCFH-DA: 2′,7′-Dichlorodihydrofluorescein diacetate; HO-1: heme oxygenase-1; JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MPP + : 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; PHB2: prohibitin 2; ROS: reactive oxygen species; SH-SY5Y: human neuroblastoma cell line.
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1) Product Images from "A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease"

Article Title: A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

Journal: Neural Regeneration Research

doi: 10.4103/1673-5374.389356

Effect of MPP + on PHB2 protein expression and mitochondrial function in SH-SY5Y cells. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp, and then induced with 1 mM MPP + for 24 hours. (A, B) PHB2 protein concentration in SH-SY5Y cells after 1 mM MPP + treatment for 6, 12, 24, and 48 hours. (A) Western blots showing PHB2 protein expression in MPP + -treated cells at different times. (B) Quantitative analysis of PHB2 protein expression. (C–G) Protein expression changes of Nrf2, HO-1, NQO-1, and PHB2. (C) Western blots showing Nrf2, HO-1, NQO-1, and PHB2 protein expression in MPP + -treated and PHB2-shRNA- or PHB2-Over Exp-treated cells. (D–G) Quantitative analysis of Nrf2, HO-1, NQO-1, and PHB2 protein expression. (H, I) The effect of PHB2 expression on mitochondrial membrane potential induced by MPP + . (H) JC-1 staining of SH-SY5Y cells treated with PHB2-shRNA and PHB2-Over Exp. Original magnification 10×, scale bar: 50 μm. (I) Quantitative analysis of JC-1 staining: the green/red ratio reflects changes in the mitochondrial membrane potential. (J, K) The effect of changes in PHB2 expression on ROS production under MPP + induction. (J) Intracellular ROS fluorescence. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp and labeled with DCFH-DA (green fluorescence). Original magnification 4×, scale bar: 100 μm. (K) Quantitative analysis of ROS (DCFH-DA) in SH-SY5Y cells. (L) Electron microscopy images of morphological changes in mitochondria in PHB2-shRNA-treated and MPP + -induced cells. Red boxes show magnified mitochondria. Scale bars: 1 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05,## P < 0.01, vs . MPP + group (one-way analysis of variance with Tukey's multiple comparison test). DCFH-DA: 2′,7′-Dichlorodihydrofluorescein diacetate; HO-1: heme oxygenase-1; JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MPP + : 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; PHB2: prohibitin 2; ROS: reactive oxygen species; SH-SY5Y: human neuroblastoma cell line.
Figure Legend Snippet: Effect of MPP + on PHB2 protein expression and mitochondrial function in SH-SY5Y cells. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp, and then induced with 1 mM MPP + for 24 hours. (A, B) PHB2 protein concentration in SH-SY5Y cells after 1 mM MPP + treatment for 6, 12, 24, and 48 hours. (A) Western blots showing PHB2 protein expression in MPP + -treated cells at different times. (B) Quantitative analysis of PHB2 protein expression. (C–G) Protein expression changes of Nrf2, HO-1, NQO-1, and PHB2. (C) Western blots showing Nrf2, HO-1, NQO-1, and PHB2 protein expression in MPP + -treated and PHB2-shRNA- or PHB2-Over Exp-treated cells. (D–G) Quantitative analysis of Nrf2, HO-1, NQO-1, and PHB2 protein expression. (H, I) The effect of PHB2 expression on mitochondrial membrane potential induced by MPP + . (H) JC-1 staining of SH-SY5Y cells treated with PHB2-shRNA and PHB2-Over Exp. Original magnification 10×, scale bar: 50 μm. (I) Quantitative analysis of JC-1 staining: the green/red ratio reflects changes in the mitochondrial membrane potential. (J, K) The effect of changes in PHB2 expression on ROS production under MPP + induction. (J) Intracellular ROS fluorescence. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp and labeled with DCFH-DA (green fluorescence). Original magnification 4×, scale bar: 100 μm. (K) Quantitative analysis of ROS (DCFH-DA) in SH-SY5Y cells. (L) Electron microscopy images of morphological changes in mitochondria in PHB2-shRNA-treated and MPP + -induced cells. Red boxes show magnified mitochondria. Scale bars: 1 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05,## P < 0.01, vs . MPP + group (one-way analysis of variance with Tukey's multiple comparison test). DCFH-DA: 2′,7′-Dichlorodihydrofluorescein diacetate; HO-1: heme oxygenase-1; JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MPP + : 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; PHB2: prohibitin 2; ROS: reactive oxygen species; SH-SY5Y: human neuroblastoma cell line.

Techniques Used: Expressing, shRNA, Protein Concentration, Western Blot, Membrane, Staining, Fluorescence, Labeling, Electron Microscopy, Comparison, Over Expression

Effect of PHB2 on mitophagy in a PD cell model. (A–F) PHB2 and LC3II/LC3I protein expression in SH-SY5Y cells after treatment with PHB2-shRNA or PHB2-Over Exp and MPP + (1 mM, 24 hours). (A) Protein expression levels of PHB2 and LC3 in SH-SY5Y cells treated with PHB2-shRNA under MPP + induction. (B, C) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (D) Protein expression levels of PHB2 and LC3 in SH-SY5Y cells treated with PHB2-Over Exp under MPP + induction. (E, F) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (G) Cellular localization of LC3 and TOM20 or TIM23 in SH-SY5Y cells treated with PHB2-shRNA under MPP + induction. Immunofluorescence changes of LC3 and mitochondrial proteins (TOM20 and TIM23). Red fluorescence represents TOM20 and TIM23, green fluorescence represents LC3, and blue fluorescence represents DAPI. Original magnification 20×, Scale bars: 20 μm. Data are expressed as mean ± SEM ( n = 3). ** P < 0.01, *** P < 0.001, vs . control group (one-way analysis of variance with Tukey's multiple comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; LC3: microtubule-associated protein 1 light chain 3; MPP + : 1-methyl-4-phenylpyridinium; Over Exp: overexpression; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line; TIM23: translocase of inner mitochondrial membrane 23; TOM20: translocase of outer mitochondrial membrane 20.
Figure Legend Snippet: Effect of PHB2 on mitophagy in a PD cell model. (A–F) PHB2 and LC3II/LC3I protein expression in SH-SY5Y cells after treatment with PHB2-shRNA or PHB2-Over Exp and MPP + (1 mM, 24 hours). (A) Protein expression levels of PHB2 and LC3 in SH-SY5Y cells treated with PHB2-shRNA under MPP + induction. (B, C) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (D) Protein expression levels of PHB2 and LC3 in SH-SY5Y cells treated with PHB2-Over Exp under MPP + induction. (E, F) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (G) Cellular localization of LC3 and TOM20 or TIM23 in SH-SY5Y cells treated with PHB2-shRNA under MPP + induction. Immunofluorescence changes of LC3 and mitochondrial proteins (TOM20 and TIM23). Red fluorescence represents TOM20 and TIM23, green fluorescence represents LC3, and blue fluorescence represents DAPI. Original magnification 20×, Scale bars: 20 μm. Data are expressed as mean ± SEM ( n = 3). ** P < 0.01, *** P < 0.001, vs . control group (one-way analysis of variance with Tukey's multiple comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; LC3: microtubule-associated protein 1 light chain 3; MPP + : 1-methyl-4-phenylpyridinium; Over Exp: overexpression; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line; TIM23: translocase of inner mitochondrial membrane 23; TOM20: translocase of outer mitochondrial membrane 20.

Techniques Used: Expressing, shRNA, Immunofluorescence, Fluorescence, Comparison, Over Expression, Membrane

Parkin increases PHB2/LC3-mediated mitophagy in SH-SY5Y cells. (A–D) Protein levels of Parkin, PHB2, and autophagy marker (LC3) in SH-SY5Y cells after treatment with Parkin-shRNA, Parkin-Over Exp, and MPP + (1 mM, 24 hours). (A) Protein expression levels of Parkin, LC3, and PHB2 in SH-SY5Y cells treated with Parkin-shRNA or Parkin-Over Exp under MPP + induction. (B–D) Quantitative analysis of Parkin, and LC3II/LC3I, PHB2 protein expression. (E) Immunofluorescence analysis of PHB2 and LC3. Cellular localization of LC3 and PHB2 in SH-SY5Y cells treated with Parkin-shRNA or Parkin-Over Exp under MPP + induction. Red fluorescence represents PHB2, green fluorescence represents LC3, and blue fluorescence represents DAPI. Original magnification 40×, Scale bar: 20 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs. control group; # P < 0.05,### P < 0.001, vs . MPP + group (one-way analysis of variance with Tukey's multiple comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; LC3: microtubule-associated protein 1 light chain 3; MPP + : 1-methyl-4-phenylpyridinium; Over Exp: overexpression; Parkin: parkin RBR E3 ubiquitin-protein ligase; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line.
Figure Legend Snippet: Parkin increases PHB2/LC3-mediated mitophagy in SH-SY5Y cells. (A–D) Protein levels of Parkin, PHB2, and autophagy marker (LC3) in SH-SY5Y cells after treatment with Parkin-shRNA, Parkin-Over Exp, and MPP + (1 mM, 24 hours). (A) Protein expression levels of Parkin, LC3, and PHB2 in SH-SY5Y cells treated with Parkin-shRNA or Parkin-Over Exp under MPP + induction. (B–D) Quantitative analysis of Parkin, and LC3II/LC3I, PHB2 protein expression. (E) Immunofluorescence analysis of PHB2 and LC3. Cellular localization of LC3 and PHB2 in SH-SY5Y cells treated with Parkin-shRNA or Parkin-Over Exp under MPP + induction. Red fluorescence represents PHB2, green fluorescence represents LC3, and blue fluorescence represents DAPI. Original magnification 40×, Scale bar: 20 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs. control group; # P < 0.05,### P < 0.001, vs . MPP + group (one-way analysis of variance with Tukey's multiple comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; LC3: microtubule-associated protein 1 light chain 3; MPP + : 1-methyl-4-phenylpyridinium; Over Exp: overexpression; Parkin: parkin RBR E3 ubiquitin-protein ligase; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line.

Techniques Used: Marker, shRNA, Expressing, Immunofluorescence, Fluorescence, Comparison, Over Expression

Parkin regulates anti-oxidative stress protein expression via PHB2. (A–D) SH-SY5Y cells were treated with PHB2-shRNA, Parkin-Over Exp, and MPP + (1 mM, 24 hours). Changes in Nrf2, HO-1, and NQO-1 protein levels and quantitative analysis. (A) Protein expression levels of Nrf2, HO-1, and NQO-1 in SH-SY5Y cells treated with PHB2-shRNA or Parkin-Over Exp under MPP + induction. (B–D) Quantitative analysis of Nrf2, HO-1, and NQO-1 protein expression. Data are expressed as mean ± SEM ( n = 3). *** P < 0.001, vs. control group; # P < 0.05,### P < 0.001, vs. MPP + group (one-way analysis of variance with Tukey's multiple comparison test). HO-1: Heme oxygenase-1; MPP + : 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; Parkin: parkin RBR E3 ubiquitin-protein ligase; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line.
Figure Legend Snippet: Parkin regulates anti-oxidative stress protein expression via PHB2. (A–D) SH-SY5Y cells were treated with PHB2-shRNA, Parkin-Over Exp, and MPP + (1 mM, 24 hours). Changes in Nrf2, HO-1, and NQO-1 protein levels and quantitative analysis. (A) Protein expression levels of Nrf2, HO-1, and NQO-1 in SH-SY5Y cells treated with PHB2-shRNA or Parkin-Over Exp under MPP + induction. (B–D) Quantitative analysis of Nrf2, HO-1, and NQO-1 protein expression. Data are expressed as mean ± SEM ( n = 3). *** P < 0.001, vs. control group; # P < 0.05,### P < 0.001, vs. MPP + group (one-way analysis of variance with Tukey's multiple comparison test). HO-1: Heme oxygenase-1; MPP + : 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; Parkin: parkin RBR E3 ubiquitin-protein ligase; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line.

Techniques Used: Expressing, shRNA, Comparison, Over Expression

Silencing PHB2 inhibits mitophagy and aggravates dopaminergic neuronal loss in PD mice. (A) Schematic of PHB2-shRNA and MPTP treatment in mice. (B, C) Quantitative analysis of PHB2 protein levels in the midbrain of C57BL/6J mice after PHB2-shRNA injection. (B) Protein expression levels of PHB2 in C57BL/6J mice treated with PHB2-shRNA. (C) Quantitative analysis of PHB2 protein expression. (D–F) An acute PD model was established by intraperitoneal injection of MPTP in C57BL/6J mice after PHB2-shRNA injection. Changes and quantitative analysis of LC3II/LC3I and PHB2 protein levels in the midbrain. (D) Protein expression levels of PHB2 and LC3 after MPTP injection in PD model mice. (E, F) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (G, H) TH immunofluorescence (Alexa Fluor 488, green fluorescence) shows dopaminergic neurons in the substantia nigra in PD mice with silenced PHB2. (G) Immunofluorescence of TH-positive neurons in the substantia nigra. Mice were treated with PHB2-shRNA and MPTP. Original magnification 10×, Scale bar: 200 μm. (H) Quantitative analysis of TH-positive neurons in the substantia nigra. (I) Fluorescence co-localization of LC3 and PHB2, TIM23, or TOM20 in the substantia nigra. Mice were treated with PHB2-shRNA and MPTP. Red fluorescence (Alexa Fluor 555): PHB2, TOM20, and TIM23, green fluorescence (Alexa Fluor 488): LC3, and blue fluorescence: DAPI. Original magnification 20×, Scale bars: 20 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, *** P < 0.001, vs. control group; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, vs. MPTP group (one-way analysis of variance with Tukey's multiple comparison test). LC3: Microtubule-associated protein 1 light chain 3; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD: Parkinson's disease; PHB2: Prohibitin 2; TH: tyrosine hydroxylase; TIM23: translocase of inner mitochondrial membrane 23; TOM20: translocase of outer mitochondrial membrane 20.
Figure Legend Snippet: Silencing PHB2 inhibits mitophagy and aggravates dopaminergic neuronal loss in PD mice. (A) Schematic of PHB2-shRNA and MPTP treatment in mice. (B, C) Quantitative analysis of PHB2 protein levels in the midbrain of C57BL/6J mice after PHB2-shRNA injection. (B) Protein expression levels of PHB2 in C57BL/6J mice treated with PHB2-shRNA. (C) Quantitative analysis of PHB2 protein expression. (D–F) An acute PD model was established by intraperitoneal injection of MPTP in C57BL/6J mice after PHB2-shRNA injection. Changes and quantitative analysis of LC3II/LC3I and PHB2 protein levels in the midbrain. (D) Protein expression levels of PHB2 and LC3 after MPTP injection in PD model mice. (E, F) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (G, H) TH immunofluorescence (Alexa Fluor 488, green fluorescence) shows dopaminergic neurons in the substantia nigra in PD mice with silenced PHB2. (G) Immunofluorescence of TH-positive neurons in the substantia nigra. Mice were treated with PHB2-shRNA and MPTP. Original magnification 10×, Scale bar: 200 μm. (H) Quantitative analysis of TH-positive neurons in the substantia nigra. (I) Fluorescence co-localization of LC3 and PHB2, TIM23, or TOM20 in the substantia nigra. Mice were treated with PHB2-shRNA and MPTP. Red fluorescence (Alexa Fluor 555): PHB2, TOM20, and TIM23, green fluorescence (Alexa Fluor 488): LC3, and blue fluorescence: DAPI. Original magnification 20×, Scale bars: 20 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, *** P < 0.001, vs. control group; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, vs. MPTP group (one-way analysis of variance with Tukey's multiple comparison test). LC3: Microtubule-associated protein 1 light chain 3; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD: Parkinson's disease; PHB2: Prohibitin 2; TH: tyrosine hydroxylase; TIM23: translocase of inner mitochondrial membrane 23; TOM20: translocase of outer mitochondrial membrane 20.

Techniques Used: shRNA, Injection, Expressing, Immunofluorescence, Fluorescence, Comparison, Membrane

Silencing PHB2 reduces antioxidative stress protein expression and aggravates motor defecits in PD mice. (A–D) Changes and quantitative analysis of Nrf2, HO-1, and NQO-1 protein levels in the midbrain of PHB2-shRNA and MPTP-treated PD mice. (E) Quantitative analysis of mice in the tail suspension and rotarod tests. Data are expressed as mean ± SEM ( n = 5). * P < 0.05, ** P < 0.01, vs. control group; $ P < 0.05, $$$ P < 0.001, vs . MPTP group (one-way analysis of variance with Tukey's multiple comparison test). HO-1: Heme oxygenase-1; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; PD: Parkinson's disease; PHB2: prohibitin 2.
Figure Legend Snippet: Silencing PHB2 reduces antioxidative stress protein expression and aggravates motor defecits in PD mice. (A–D) Changes and quantitative analysis of Nrf2, HO-1, and NQO-1 protein levels in the midbrain of PHB2-shRNA and MPTP-treated PD mice. (E) Quantitative analysis of mice in the tail suspension and rotarod tests. Data are expressed as mean ± SEM ( n = 5). * P < 0.05, ** P < 0.01, vs. control group; $ P < 0.05, $$$ P < 0.001, vs . MPTP group (one-way analysis of variance with Tukey's multiple comparison test). HO-1: Heme oxygenase-1; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; PD: Parkinson's disease; PHB2: prohibitin 2.

Techniques Used: Expressing, shRNA, Suspension, Comparison


Structured Review

Santa Cruz Biotechnology control shrna
Hypoxia upregulates the expression of periostin in TC cells <t>via</t> <t>HIF-1α.</t> (A) Results of RT-qPCR analysis: the expression of HIF-1α mRNA in BCPAP cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (*P<0.05). Compared to transfection with periostin <t>shRNA</t> under normoxic conditions, the mRNA expression of HIF-1α in BCPAP cells significantly increased with periostin shRNA transfection under hypoxic conditions ( # P<0.05). n=3 biological replicates. Error bars represent standard deviation. (B) Results of RT-qPCR analysis: The mRNA expression of periostin in BCPAP cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (**P<0.01). The mRNA expression of periostin significantly decreased following transfection with periostin shRNA under normoxic conditions (*P<0.05). As compared to transfection with periostin shRNA under normoxic conditions, the mRNA expression of periostin in the BCPAP cells significantly increased following periostin shRNA transfection under hypoxic conditions ( # P<0.01). n=3 biological replicates. Error bars represent standard deviation. (C) Results of RT-qPCR analysis: The expression of HIF-1α mRNA in BCPAPs under hypoxic conditions for 24 h is significantly greater as compared normoxia (**P<0.01). The expression of HIF-1α mRNA significantly decreased following transfection with HIF-1α shRNA under normoxic conditions (*P<0.01). Compared to transfection with HIF-1α shRNA under normoxic conditions, the mRNA expression of HIF-1α in BCPAP cells increased following transfection with HIF-1α shRNA under hypoxic conditions ( # P<0.01). n=3 biological replicates. Error bars represent standard deviation. (D) Results of RT-qPCR analysis: The mRNA expression of periostin in BCPAP cells under hypoxic conditions for 24 h was significantly greater than under normoxic conditions (**P<0.01). The expression of periostin mRNA significantly decreased following the transfection of HIF-1α shRNA under normoxic conditions (*P<0.05). As compared to transfection with HIF-1α shRNA under normoxic conditions, the mRNA expression of periostin in the BCPAP cells significantly increased following transfection with HIF-1α shRNA under hypoxic conditions ( # P<0.01). n=3 biological replicates. Error bars represent standard deviation. (E) Western blot analysis of HIF-1α, N-cadherin, E-cadherin, periostin and NIS under normoxic or hypoxic conditions, with or without periostin shRNA transfection. (F) Western blot analysis of HIF-1α, N-cadherin, E-cadherin, periostin and NIS under normoxic or hypoxic conditions, with or without HIF-1α shRNA transfection. TC, thyroid cancer; HIF-1α, hypoxia inducible factor 1 subunit α; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NIS, solute carrier family 5 member 5.
Control Shrna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/control shrna/product/Santa Cruz Biotechnology
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
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1) Product Images from "Effect of hypoxia‑HIF‑1α‑periostin axis in thyroid cancer"

Article Title: Effect of hypoxia‑HIF‑1α‑periostin axis in thyroid cancer

Journal: Oncology Reports

doi: 10.3892/or.2024.8716

Hypoxia upregulates the expression of periostin in TC cells via HIF-1α. (A) Results of RT-qPCR analysis: the expression of HIF-1α mRNA in BCPAP cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (*P<0.05). Compared to transfection with periostin shRNA under normoxic conditions, the mRNA expression of HIF-1α in BCPAP cells significantly increased with periostin shRNA transfection under hypoxic conditions ( # P<0.05). n=3 biological replicates. Error bars represent standard deviation. (B) Results of RT-qPCR analysis: The mRNA expression of periostin in BCPAP cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (**P<0.01). The mRNA expression of periostin significantly decreased following transfection with periostin shRNA under normoxic conditions (*P<0.05). As compared to transfection with periostin shRNA under normoxic conditions, the mRNA expression of periostin in the BCPAP cells significantly increased following periostin shRNA transfection under hypoxic conditions ( # P<0.01). n=3 biological replicates. Error bars represent standard deviation. (C) Results of RT-qPCR analysis: The expression of HIF-1α mRNA in BCPAPs under hypoxic conditions for 24 h is significantly greater as compared normoxia (**P<0.01). The expression of HIF-1α mRNA significantly decreased following transfection with HIF-1α shRNA under normoxic conditions (*P<0.01). Compared to transfection with HIF-1α shRNA under normoxic conditions, the mRNA expression of HIF-1α in BCPAP cells increased following transfection with HIF-1α shRNA under hypoxic conditions ( # P<0.01). n=3 biological replicates. Error bars represent standard deviation. (D) Results of RT-qPCR analysis: The mRNA expression of periostin in BCPAP cells under hypoxic conditions for 24 h was significantly greater than under normoxic conditions (**P<0.01). The expression of periostin mRNA significantly decreased following the transfection of HIF-1α shRNA under normoxic conditions (*P<0.05). As compared to transfection with HIF-1α shRNA under normoxic conditions, the mRNA expression of periostin in the BCPAP cells significantly increased following transfection with HIF-1α shRNA under hypoxic conditions ( # P<0.01). n=3 biological replicates. Error bars represent standard deviation. (E) Western blot analysis of HIF-1α, N-cadherin, E-cadherin, periostin and NIS under normoxic or hypoxic conditions, with or without periostin shRNA transfection. (F) Western blot analysis of HIF-1α, N-cadherin, E-cadherin, periostin and NIS under normoxic or hypoxic conditions, with or without HIF-1α shRNA transfection. TC, thyroid cancer; HIF-1α, hypoxia inducible factor 1 subunit α; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NIS, solute carrier family 5 member 5.
Figure Legend Snippet: Hypoxia upregulates the expression of periostin in TC cells via HIF-1α. (A) Results of RT-qPCR analysis: the expression of HIF-1α mRNA in BCPAP cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (*P<0.05). Compared to transfection with periostin shRNA under normoxic conditions, the mRNA expression of HIF-1α in BCPAP cells significantly increased with periostin shRNA transfection under hypoxic conditions ( # P<0.05). n=3 biological replicates. Error bars represent standard deviation. (B) Results of RT-qPCR analysis: The mRNA expression of periostin in BCPAP cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (**P<0.01). The mRNA expression of periostin significantly decreased following transfection with periostin shRNA under normoxic conditions (*P<0.05). As compared to transfection with periostin shRNA under normoxic conditions, the mRNA expression of periostin in the BCPAP cells significantly increased following periostin shRNA transfection under hypoxic conditions ( # P<0.01). n=3 biological replicates. Error bars represent standard deviation. (C) Results of RT-qPCR analysis: The expression of HIF-1α mRNA in BCPAPs under hypoxic conditions for 24 h is significantly greater as compared normoxia (**P<0.01). The expression of HIF-1α mRNA significantly decreased following transfection with HIF-1α shRNA under normoxic conditions (*P<0.01). Compared to transfection with HIF-1α shRNA under normoxic conditions, the mRNA expression of HIF-1α in BCPAP cells increased following transfection with HIF-1α shRNA under hypoxic conditions ( # P<0.01). n=3 biological replicates. Error bars represent standard deviation. (D) Results of RT-qPCR analysis: The mRNA expression of periostin in BCPAP cells under hypoxic conditions for 24 h was significantly greater than under normoxic conditions (**P<0.01). The expression of periostin mRNA significantly decreased following the transfection of HIF-1α shRNA under normoxic conditions (*P<0.05). As compared to transfection with HIF-1α shRNA under normoxic conditions, the mRNA expression of periostin in the BCPAP cells significantly increased following transfection with HIF-1α shRNA under hypoxic conditions ( # P<0.01). n=3 biological replicates. Error bars represent standard deviation. (E) Western blot analysis of HIF-1α, N-cadherin, E-cadherin, periostin and NIS under normoxic or hypoxic conditions, with or without periostin shRNA transfection. (F) Western blot analysis of HIF-1α, N-cadherin, E-cadherin, periostin and NIS under normoxic or hypoxic conditions, with or without HIF-1α shRNA transfection. TC, thyroid cancer; HIF-1α, hypoxia inducible factor 1 subunit α; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NIS, solute carrier family 5 member 5.

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, shRNA, Standard Deviation, Western Blot, Real-time Polymerase Chain Reaction

Effects of hypoxia and periostin on the invasiveness and migratory ability of TC cells. (A and B) Transwell migration and invasion assays. BCPAP and TPC-1 cells under normoxic or hypoxic conditions plus transfection with or without periostin shRNA (c-shRNA). The number of cells that migrated or invaded through Matrigel-coated inserts was determined. Invasiveness capability of cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (*P<0.05). Transfection with periostin shRNA inhibited the invasiveness capability of cells (**P<0.01). Hypoxia combined with periostin shRNA transfection restored the invasiveness capability of cells compared to only periostin shRNA transfection ( ## P<0.01). The migratory capability of cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (*P<0.05). Transfection with periostin shRNA inhibited the migration capability of cells (**P<0.01). Hypoxia combined with periostin shRNA transfection restored the migration capability of cells compared to only periostin shRNA transfection ( ## P<0.01). Scale bars, 50 µm; n=3 biological replicates. (C and D) Wound healing assay. BCPAP and TPC-1 cells under normoxia or hypoxia plus transfection with or without periostin shRNA. Wound healing assays suggested BCPAP cells under hypoxic conditions achieved an increased healing rate as compared with that under normoxic conditions (*P<0.05); periostin knockdown significantly inhibited this effect (**P<0.05). Hypoxia combined with periostin shRNA remitted the mobility of cells ( # P<0.05). Scale bars, 100 µm; n=3 biological replicates. TC, thyroid cancer.
Figure Legend Snippet: Effects of hypoxia and periostin on the invasiveness and migratory ability of TC cells. (A and B) Transwell migration and invasion assays. BCPAP and TPC-1 cells under normoxic or hypoxic conditions plus transfection with or without periostin shRNA (c-shRNA). The number of cells that migrated or invaded through Matrigel-coated inserts was determined. Invasiveness capability of cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (*P<0.05). Transfection with periostin shRNA inhibited the invasiveness capability of cells (**P<0.01). Hypoxia combined with periostin shRNA transfection restored the invasiveness capability of cells compared to only periostin shRNA transfection ( ## P<0.01). The migratory capability of cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (*P<0.05). Transfection with periostin shRNA inhibited the migration capability of cells (**P<0.01). Hypoxia combined with periostin shRNA transfection restored the migration capability of cells compared to only periostin shRNA transfection ( ## P<0.01). Scale bars, 50 µm; n=3 biological replicates. (C and D) Wound healing assay. BCPAP and TPC-1 cells under normoxia or hypoxia plus transfection with or without periostin shRNA. Wound healing assays suggested BCPAP cells under hypoxic conditions achieved an increased healing rate as compared with that under normoxic conditions (*P<0.05); periostin knockdown significantly inhibited this effect (**P<0.05). Hypoxia combined with periostin shRNA remitted the mobility of cells ( # P<0.05). Scale bars, 100 µm; n=3 biological replicates. TC, thyroid cancer.

Techniques Used: Migration, Transfection, shRNA, Wound Healing Assay

Hypoxia induced the Warburg effect by upregulating the expression of periostin. (A) Results of phosphomolybdic acid colorimetry. ATP expression in BCPAP cells under hypoxic conditions was significantly lower than that under normoxic conditions (*P<0.05). Transfection with periostin shRNA under normoxic conditions increased the ATP expression in BCPAPs (**P<0.01). As compared to the hypoxic group, transfection using periostin shRNA under hypoxic conditions significantly increased the ATP expression in BCPAP cells ( ## P<0.01). (B) Results of the LDH test. The LDH expression in BCPAPs under hypoxic conditions was significantly higher than that under normoxic conditions (*P<0.05). Transfection with periostin shRNA under normoxic conditions decreased LDH expression in BCPAPs (**P<0.01). In comparison with the hypoxic group, transfection with periostin shRNA under hypoxic conditions significantly decreased LDH expression in BCPAP cells ( ## P<0.01). (C and D) The ECAR and OCR of BCPAP cells under different conditions were measured using the Seahorse analyzer. The ECAR of BCPAP cells under hypoxic conditions or with HIF-1α overexpression was significantly higher than that under nomoxia (***P<0.001). Transfection with periostin shRNA under normoxic conditions significantly decreased the ECAR of BCPAP cells (**P<0.01). Transfection using periostin shRNA under hypoxic conditions or co-transfection with HIF-1α overexpression plasmids increased the ECAR of BCPAP cells ( ### P<0.001). By contrast, the OCR of BCPAPs with HIF-1α overexpression was significantly decreased (**P<0.01). Transfection with periostin shRNA significantly elevated the OCR of BCPAP cells (*P<0.05). Transfection with periostin shRNA under hypoxic conditions or co-transfection with HIF-1α overexpression plasmids decreased the OCR of BCPAP cells ( ## P<0.01 and ### P<0.001, respectively). LDH, lactate dehydrogenase; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; HIF-1α, hypoxia inducible factor 1 subunit α.
Figure Legend Snippet: Hypoxia induced the Warburg effect by upregulating the expression of periostin. (A) Results of phosphomolybdic acid colorimetry. ATP expression in BCPAP cells under hypoxic conditions was significantly lower than that under normoxic conditions (*P<0.05). Transfection with periostin shRNA under normoxic conditions increased the ATP expression in BCPAPs (**P<0.01). As compared to the hypoxic group, transfection using periostin shRNA under hypoxic conditions significantly increased the ATP expression in BCPAP cells ( ## P<0.01). (B) Results of the LDH test. The LDH expression in BCPAPs under hypoxic conditions was significantly higher than that under normoxic conditions (*P<0.05). Transfection with periostin shRNA under normoxic conditions decreased LDH expression in BCPAPs (**P<0.01). In comparison with the hypoxic group, transfection with periostin shRNA under hypoxic conditions significantly decreased LDH expression in BCPAP cells ( ## P<0.01). (C and D) The ECAR and OCR of BCPAP cells under different conditions were measured using the Seahorse analyzer. The ECAR of BCPAP cells under hypoxic conditions or with HIF-1α overexpression was significantly higher than that under nomoxia (***P<0.001). Transfection with periostin shRNA under normoxic conditions significantly decreased the ECAR of BCPAP cells (**P<0.01). Transfection using periostin shRNA under hypoxic conditions or co-transfection with HIF-1α overexpression plasmids increased the ECAR of BCPAP cells ( ### P<0.001). By contrast, the OCR of BCPAPs with HIF-1α overexpression was significantly decreased (**P<0.01). Transfection with periostin shRNA significantly elevated the OCR of BCPAP cells (*P<0.05). Transfection with periostin shRNA under hypoxic conditions or co-transfection with HIF-1α overexpression plasmids decreased the OCR of BCPAP cells ( ## P<0.01 and ### P<0.001, respectively). LDH, lactate dehydrogenase; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; HIF-1α, hypoxia inducible factor 1 subunit α.

Techniques Used: Expressing, Colorimetric Assay, Transfection, shRNA, Comparison, Over Expression, Cotransfection


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Santa Cruz Biotechnology negative control nc sirna
Overexpressing TMEM120 accelerated breast cancer stemness by <t>activating</t> <t>TAZ-mTOR</t> signaling axis. ( A ) KEGG analysis was conducted to detect the signaling pathway significantly correlated with deletion of TMEM120B in MDA-453 cells. ( B ) Phosphorylation antibodies array kit was used to explore the key signaling pathway involved in TMEM120B overexpression in SK-BR-3 cells. ( C ) Immunoblotting of Myc-tag, TMEM120B, AKT, p-AKT, mTOR, p-mTOR, YAP, TAZ, and GAPDH after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. ( D ) qPCR assay was used to investigate the alteration of the target genes of YAP/TAZ within ectopic or deleted TMEM120B in SK-BR-3 or MDA-231 cells. ( E ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without mTOR signaling pathway inhibitor rapamycin in SK-BR-3 cells. ( F ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without knocking down TAZ by <t>siRNA</t> in SK-BR-3 cells. Subcellular localization of TAZ was evaluated by immunofluorescence assay ( G ) or western blotting assay ( H ) within ectopic TMEM120B in SK-BR-3 cells. Scale bar = 20 μm. ( I ) After being treated with CHX at indicated time points, the expression of TAZ was evaluated by western blotting after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. Quantification data are expressed as mean ± SD of three independent experiments (t-test, two-sided, ***, P < 0.001)
Negative Control Nc Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "TMEM120B strengthens breast cancer cell stemness and accelerates chemotherapy resistance via β1-integrin/FAK-TAZ-mTOR signaling axis by binding to MYH9"

Article Title: TMEM120B strengthens breast cancer cell stemness and accelerates chemotherapy resistance via β1-integrin/FAK-TAZ-mTOR signaling axis by binding to MYH9

Journal: Breast Cancer Research : BCR

doi: 10.1186/s13058-024-01802-z

Overexpressing TMEM120 accelerated breast cancer stemness by activating TAZ-mTOR signaling axis. ( A ) KEGG analysis was conducted to detect the signaling pathway significantly correlated with deletion of TMEM120B in MDA-453 cells. ( B ) Phosphorylation antibodies array kit was used to explore the key signaling pathway involved in TMEM120B overexpression in SK-BR-3 cells. ( C ) Immunoblotting of Myc-tag, TMEM120B, AKT, p-AKT, mTOR, p-mTOR, YAP, TAZ, and GAPDH after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. ( D ) qPCR assay was used to investigate the alteration of the target genes of YAP/TAZ within ectopic or deleted TMEM120B in SK-BR-3 or MDA-231 cells. ( E ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without mTOR signaling pathway inhibitor rapamycin in SK-BR-3 cells. ( F ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without knocking down TAZ by siRNA in SK-BR-3 cells. Subcellular localization of TAZ was evaluated by immunofluorescence assay ( G ) or western blotting assay ( H ) within ectopic TMEM120B in SK-BR-3 cells. Scale bar = 20 μm. ( I ) After being treated with CHX at indicated time points, the expression of TAZ was evaluated by western blotting after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. Quantification data are expressed as mean ± SD of three independent experiments (t-test, two-sided, ***, P < 0.001)
Figure Legend Snippet: Overexpressing TMEM120 accelerated breast cancer stemness by activating TAZ-mTOR signaling axis. ( A ) KEGG analysis was conducted to detect the signaling pathway significantly correlated with deletion of TMEM120B in MDA-453 cells. ( B ) Phosphorylation antibodies array kit was used to explore the key signaling pathway involved in TMEM120B overexpression in SK-BR-3 cells. ( C ) Immunoblotting of Myc-tag, TMEM120B, AKT, p-AKT, mTOR, p-mTOR, YAP, TAZ, and GAPDH after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. ( D ) qPCR assay was used to investigate the alteration of the target genes of YAP/TAZ within ectopic or deleted TMEM120B in SK-BR-3 or MDA-231 cells. ( E ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without mTOR signaling pathway inhibitor rapamycin in SK-BR-3 cells. ( F ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without knocking down TAZ by siRNA in SK-BR-3 cells. Subcellular localization of TAZ was evaluated by immunofluorescence assay ( G ) or western blotting assay ( H ) within ectopic TMEM120B in SK-BR-3 cells. Scale bar = 20 μm. ( I ) After being treated with CHX at indicated time points, the expression of TAZ was evaluated by western blotting after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. Quantification data are expressed as mean ± SD of three independent experiments (t-test, two-sided, ***, P < 0.001)

Techniques Used: Over Expression, Western Blot, Immunofluorescence, Expressing


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Santa Cruz Biotechnology control shrna
Control Shrna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology control shrna plasmid a
Control Shrna Plasmid A, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology control shrna lentiviral particles
KEY RESOURCES TABLE
Control Shrna Lentiviral Particles, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/control shrna lentiviral particles/product/Santa Cruz Biotechnology
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1) Product Images from "Integration of transcriptomes of senescent cell models with multi-tissue patient samples reveals reduced COL6A3 as an inducer of senescence"

Article Title: Integration of transcriptomes of senescent cell models with multi-tissue patient samples reveals reduced COL6A3 as an inducer of senescence

Journal: Cell reports

doi: 10.1016/j.celrep.2023.113371

KEY RESOURCES TABLE
Figure Legend Snippet: KEY RESOURCES TABLE

Techniques Used: Imaging, Recombinant, SYBR Green Assay, shRNA, Plasmid Preparation, Fluorescence, Software, Expressing


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Santa Cruz Biotechnology control shrna lentiviral particles
KEY RESOURCES TABLE
Control Shrna Lentiviral Particles, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/control shrna lentiviral particles/product/Santa Cruz Biotechnology
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1) Product Images from "Integration of transcriptomes of senescent cell models with multi-tissue patient samples reveals reduced COL6A3 as an inducer of senescence"

Article Title: Integration of transcriptomes of senescent cell models with multi-tissue patient samples reveals reduced COL6A3 as an inducer of senescence

Journal: Cell reports

doi: 10.1016/j.celrep.2023.113371

KEY RESOURCES TABLE
Figure Legend Snippet: KEY RESOURCES TABLE

Techniques Used: Imaging, Recombinant, SYBR Green Assay, shRNA, Plasmid Preparation, Fluorescence, Software, Expressing


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Santa Cruz Biotechnology control shrna plasmid
Control Shrna Plasmid, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/control shrna plasmid/product/Santa Cruz Biotechnology
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Santa Cruz Biotechnology negative control shrna
Negative Control Shrna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology control shrna lentiviral particles
Control Shrna Lentiviral Particles, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology control shrna
    Effect of MPP + <t>on</t> <t>PHB2</t> protein expression and mitochondrial function in SH-SY5Y cells. SH-SY5Y cells were treated with <t>PHB2-shRNA</t> and PHB2-Over Exp, and then induced with 1 mM MPP + for 24 hours. (A, B) PHB2 protein concentration in SH-SY5Y cells after 1 mM MPP + treatment for 6, 12, 24, and 48 hours. (A) Western blots showing PHB2 protein expression in MPP + -treated cells at different times. (B) Quantitative analysis of PHB2 protein expression. (C–G) Protein expression changes of Nrf2, HO-1, NQO-1, and PHB2. (C) Western blots showing Nrf2, HO-1, NQO-1, and PHB2 protein expression in MPP + -treated and PHB2-shRNA- or PHB2-Over Exp-treated cells. (D–G) Quantitative analysis of Nrf2, HO-1, NQO-1, and PHB2 protein expression. (H, I) The effect of PHB2 expression on mitochondrial membrane potential induced by MPP + . (H) JC-1 staining of SH-SY5Y cells treated with PHB2-shRNA and PHB2-Over Exp. Original magnification 10×, scale bar: 50 μm. (I) Quantitative analysis of JC-1 staining: the green/red ratio reflects changes in the mitochondrial membrane potential. (J, K) The effect of changes in PHB2 expression on ROS production under MPP + induction. (J) Intracellular ROS fluorescence. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp and labeled with DCFH-DA (green fluorescence). Original magnification 4×, scale bar: 100 μm. (K) Quantitative analysis of ROS (DCFH-DA) in SH-SY5Y cells. (L) Electron microscopy images of morphological changes in mitochondria in PHB2-shRNA-treated and MPP + -induced cells. Red boxes show magnified mitochondria. Scale bars: 1 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05,## P < 0.01, vs . MPP + group (one-way analysis of variance with Tukey's multiple comparison test). DCFH-DA: 2′,7′-Dichlorodihydrofluorescein diacetate; HO-1: heme oxygenase-1; JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MPP + : 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; PHB2: prohibitin 2; ROS: reactive oxygen species; SH-SY5Y: human neuroblastoma cell line.
    Control Shrna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology negative control nc sirna
    Overexpressing TMEM120 accelerated breast cancer stemness by <t>activating</t> <t>TAZ-mTOR</t> signaling axis. ( A ) KEGG analysis was conducted to detect the signaling pathway significantly correlated with deletion of TMEM120B in MDA-453 cells. ( B ) Phosphorylation antibodies array kit was used to explore the key signaling pathway involved in TMEM120B overexpression in SK-BR-3 cells. ( C ) Immunoblotting of Myc-tag, TMEM120B, AKT, p-AKT, mTOR, p-mTOR, YAP, TAZ, and GAPDH after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. ( D ) qPCR assay was used to investigate the alteration of the target genes of YAP/TAZ within ectopic or deleted TMEM120B in SK-BR-3 or MDA-231 cells. ( E ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without mTOR signaling pathway inhibitor rapamycin in SK-BR-3 cells. ( F ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without knocking down TAZ by <t>siRNA</t> in SK-BR-3 cells. Subcellular localization of TAZ was evaluated by immunofluorescence assay ( G ) or western blotting assay ( H ) within ectopic TMEM120B in SK-BR-3 cells. Scale bar = 20 μm. ( I ) After being treated with CHX at indicated time points, the expression of TAZ was evaluated by western blotting after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. Quantification data are expressed as mean ± SD of three independent experiments (t-test, two-sided, ***, P < 0.001)
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    Santa Cruz Biotechnology control shrna plasmid a
    Overexpressing TMEM120 accelerated breast cancer stemness by <t>activating</t> <t>TAZ-mTOR</t> signaling axis. ( A ) KEGG analysis was conducted to detect the signaling pathway significantly correlated with deletion of TMEM120B in MDA-453 cells. ( B ) Phosphorylation antibodies array kit was used to explore the key signaling pathway involved in TMEM120B overexpression in SK-BR-3 cells. ( C ) Immunoblotting of Myc-tag, TMEM120B, AKT, p-AKT, mTOR, p-mTOR, YAP, TAZ, and GAPDH after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. ( D ) qPCR assay was used to investigate the alteration of the target genes of YAP/TAZ within ectopic or deleted TMEM120B in SK-BR-3 or MDA-231 cells. ( E ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without mTOR signaling pathway inhibitor rapamycin in SK-BR-3 cells. ( F ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without knocking down TAZ by <t>siRNA</t> in SK-BR-3 cells. Subcellular localization of TAZ was evaluated by immunofluorescence assay ( G ) or western blotting assay ( H ) within ectopic TMEM120B in SK-BR-3 cells. Scale bar = 20 μm. ( I ) After being treated with CHX at indicated time points, the expression of TAZ was evaluated by western blotting after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. Quantification data are expressed as mean ± SD of three independent experiments (t-test, two-sided, ***, P < 0.001)
    Control Shrna Plasmid A, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology control shrna lentiviral particles
    KEY RESOURCES TABLE
    Control Shrna Lentiviral Particles, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology control shrna plasmid
    KEY RESOURCES TABLE
    Control Shrna Plasmid, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology negative control shrna
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    Negative Control Shrna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    negative control shrna - by Bioz Stars, 2024-04
    86/100 stars
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    Effect of MPP + on PHB2 protein expression and mitochondrial function in SH-SY5Y cells. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp, and then induced with 1 mM MPP + for 24 hours. (A, B) PHB2 protein concentration in SH-SY5Y cells after 1 mM MPP + treatment for 6, 12, 24, and 48 hours. (A) Western blots showing PHB2 protein expression in MPP + -treated cells at different times. (B) Quantitative analysis of PHB2 protein expression. (C–G) Protein expression changes of Nrf2, HO-1, NQO-1, and PHB2. (C) Western blots showing Nrf2, HO-1, NQO-1, and PHB2 protein expression in MPP + -treated and PHB2-shRNA- or PHB2-Over Exp-treated cells. (D–G) Quantitative analysis of Nrf2, HO-1, NQO-1, and PHB2 protein expression. (H, I) The effect of PHB2 expression on mitochondrial membrane potential induced by MPP + . (H) JC-1 staining of SH-SY5Y cells treated with PHB2-shRNA and PHB2-Over Exp. Original magnification 10×, scale bar: 50 μm. (I) Quantitative analysis of JC-1 staining: the green/red ratio reflects changes in the mitochondrial membrane potential. (J, K) The effect of changes in PHB2 expression on ROS production under MPP + induction. (J) Intracellular ROS fluorescence. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp and labeled with DCFH-DA (green fluorescence). Original magnification 4×, scale bar: 100 μm. (K) Quantitative analysis of ROS (DCFH-DA) in SH-SY5Y cells. (L) Electron microscopy images of morphological changes in mitochondria in PHB2-shRNA-treated and MPP + -induced cells. Red boxes show magnified mitochondria. Scale bars: 1 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05,## P < 0.01, vs . MPP + group (one-way analysis of variance with Tukey's multiple comparison test). DCFH-DA: 2′,7′-Dichlorodihydrofluorescein diacetate; HO-1: heme oxygenase-1; JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MPP + : 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; PHB2: prohibitin 2; ROS: reactive oxygen species; SH-SY5Y: human neuroblastoma cell line.

    Journal: Neural Regeneration Research

    Article Title: A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

    doi: 10.4103/1673-5374.389356

    Figure Lengend Snippet: Effect of MPP + on PHB2 protein expression and mitochondrial function in SH-SY5Y cells. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp, and then induced with 1 mM MPP + for 24 hours. (A, B) PHB2 protein concentration in SH-SY5Y cells after 1 mM MPP + treatment for 6, 12, 24, and 48 hours. (A) Western blots showing PHB2 protein expression in MPP + -treated cells at different times. (B) Quantitative analysis of PHB2 protein expression. (C–G) Protein expression changes of Nrf2, HO-1, NQO-1, and PHB2. (C) Western blots showing Nrf2, HO-1, NQO-1, and PHB2 protein expression in MPP + -treated and PHB2-shRNA- or PHB2-Over Exp-treated cells. (D–G) Quantitative analysis of Nrf2, HO-1, NQO-1, and PHB2 protein expression. (H, I) The effect of PHB2 expression on mitochondrial membrane potential induced by MPP + . (H) JC-1 staining of SH-SY5Y cells treated with PHB2-shRNA and PHB2-Over Exp. Original magnification 10×, scale bar: 50 μm. (I) Quantitative analysis of JC-1 staining: the green/red ratio reflects changes in the mitochondrial membrane potential. (J, K) The effect of changes in PHB2 expression on ROS production under MPP + induction. (J) Intracellular ROS fluorescence. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp and labeled with DCFH-DA (green fluorescence). Original magnification 4×, scale bar: 100 μm. (K) Quantitative analysis of ROS (DCFH-DA) in SH-SY5Y cells. (L) Electron microscopy images of morphological changes in mitochondria in PHB2-shRNA-treated and MPP + -induced cells. Red boxes show magnified mitochondria. Scale bars: 1 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05,## P < 0.01, vs . MPP + group (one-way analysis of variance with Tukey's multiple comparison test). DCFH-DA: 2′,7′-Dichlorodihydrofluorescein diacetate; HO-1: heme oxygenase-1; JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MPP + : 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; PHB2: prohibitin 2; ROS: reactive oxygen species; SH-SY5Y: human neuroblastoma cell line.

    Article Snippet: Parkin-short hairpin RNA (sh-RNA) (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-42158-V) resistant to puromycin, PHB2-shRNA (Santa Cruz, Cat# sc-45849-V), and control shRNA (Santa Cruz Biotechnology, Cat# sc-108080) lentiviral particles were used.

    Techniques: Expressing, shRNA, Protein Concentration, Western Blot, Membrane, Staining, Fluorescence, Labeling, Electron Microscopy, Comparison, Over Expression

    Effect of PHB2 on mitophagy in a PD cell model. (A–F) PHB2 and LC3II/LC3I protein expression in SH-SY5Y cells after treatment with PHB2-shRNA or PHB2-Over Exp and MPP + (1 mM, 24 hours). (A) Protein expression levels of PHB2 and LC3 in SH-SY5Y cells treated with PHB2-shRNA under MPP + induction. (B, C) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (D) Protein expression levels of PHB2 and LC3 in SH-SY5Y cells treated with PHB2-Over Exp under MPP + induction. (E, F) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (G) Cellular localization of LC3 and TOM20 or TIM23 in SH-SY5Y cells treated with PHB2-shRNA under MPP + induction. Immunofluorescence changes of LC3 and mitochondrial proteins (TOM20 and TIM23). Red fluorescence represents TOM20 and TIM23, green fluorescence represents LC3, and blue fluorescence represents DAPI. Original magnification 20×, Scale bars: 20 μm. Data are expressed as mean ± SEM ( n = 3). ** P < 0.01, *** P < 0.001, vs . control group (one-way analysis of variance with Tukey's multiple comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; LC3: microtubule-associated protein 1 light chain 3; MPP + : 1-methyl-4-phenylpyridinium; Over Exp: overexpression; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line; TIM23: translocase of inner mitochondrial membrane 23; TOM20: translocase of outer mitochondrial membrane 20.

    Journal: Neural Regeneration Research

    Article Title: A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

    doi: 10.4103/1673-5374.389356

    Figure Lengend Snippet: Effect of PHB2 on mitophagy in a PD cell model. (A–F) PHB2 and LC3II/LC3I protein expression in SH-SY5Y cells after treatment with PHB2-shRNA or PHB2-Over Exp and MPP + (1 mM, 24 hours). (A) Protein expression levels of PHB2 and LC3 in SH-SY5Y cells treated with PHB2-shRNA under MPP + induction. (B, C) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (D) Protein expression levels of PHB2 and LC3 in SH-SY5Y cells treated with PHB2-Over Exp under MPP + induction. (E, F) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (G) Cellular localization of LC3 and TOM20 or TIM23 in SH-SY5Y cells treated with PHB2-shRNA under MPP + induction. Immunofluorescence changes of LC3 and mitochondrial proteins (TOM20 and TIM23). Red fluorescence represents TOM20 and TIM23, green fluorescence represents LC3, and blue fluorescence represents DAPI. Original magnification 20×, Scale bars: 20 μm. Data are expressed as mean ± SEM ( n = 3). ** P < 0.01, *** P < 0.001, vs . control group (one-way analysis of variance with Tukey's multiple comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; LC3: microtubule-associated protein 1 light chain 3; MPP + : 1-methyl-4-phenylpyridinium; Over Exp: overexpression; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line; TIM23: translocase of inner mitochondrial membrane 23; TOM20: translocase of outer mitochondrial membrane 20.

    Article Snippet: Parkin-short hairpin RNA (sh-RNA) (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-42158-V) resistant to puromycin, PHB2-shRNA (Santa Cruz, Cat# sc-45849-V), and control shRNA (Santa Cruz Biotechnology, Cat# sc-108080) lentiviral particles were used.

    Techniques: Expressing, shRNA, Immunofluorescence, Fluorescence, Comparison, Over Expression, Membrane

    Parkin increases PHB2/LC3-mediated mitophagy in SH-SY5Y cells. (A–D) Protein levels of Parkin, PHB2, and autophagy marker (LC3) in SH-SY5Y cells after treatment with Parkin-shRNA, Parkin-Over Exp, and MPP + (1 mM, 24 hours). (A) Protein expression levels of Parkin, LC3, and PHB2 in SH-SY5Y cells treated with Parkin-shRNA or Parkin-Over Exp under MPP + induction. (B–D) Quantitative analysis of Parkin, and LC3II/LC3I, PHB2 protein expression. (E) Immunofluorescence analysis of PHB2 and LC3. Cellular localization of LC3 and PHB2 in SH-SY5Y cells treated with Parkin-shRNA or Parkin-Over Exp under MPP + induction. Red fluorescence represents PHB2, green fluorescence represents LC3, and blue fluorescence represents DAPI. Original magnification 40×, Scale bar: 20 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs. control group; # P < 0.05,### P < 0.001, vs . MPP + group (one-way analysis of variance with Tukey's multiple comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; LC3: microtubule-associated protein 1 light chain 3; MPP + : 1-methyl-4-phenylpyridinium; Over Exp: overexpression; Parkin: parkin RBR E3 ubiquitin-protein ligase; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line.

    Journal: Neural Regeneration Research

    Article Title: A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

    doi: 10.4103/1673-5374.389356

    Figure Lengend Snippet: Parkin increases PHB2/LC3-mediated mitophagy in SH-SY5Y cells. (A–D) Protein levels of Parkin, PHB2, and autophagy marker (LC3) in SH-SY5Y cells after treatment with Parkin-shRNA, Parkin-Over Exp, and MPP + (1 mM, 24 hours). (A) Protein expression levels of Parkin, LC3, and PHB2 in SH-SY5Y cells treated with Parkin-shRNA or Parkin-Over Exp under MPP + induction. (B–D) Quantitative analysis of Parkin, and LC3II/LC3I, PHB2 protein expression. (E) Immunofluorescence analysis of PHB2 and LC3. Cellular localization of LC3 and PHB2 in SH-SY5Y cells treated with Parkin-shRNA or Parkin-Over Exp under MPP + induction. Red fluorescence represents PHB2, green fluorescence represents LC3, and blue fluorescence represents DAPI. Original magnification 40×, Scale bar: 20 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs. control group; # P < 0.05,### P < 0.001, vs . MPP + group (one-way analysis of variance with Tukey's multiple comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; LC3: microtubule-associated protein 1 light chain 3; MPP + : 1-methyl-4-phenylpyridinium; Over Exp: overexpression; Parkin: parkin RBR E3 ubiquitin-protein ligase; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line.

    Article Snippet: Parkin-short hairpin RNA (sh-RNA) (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-42158-V) resistant to puromycin, PHB2-shRNA (Santa Cruz, Cat# sc-45849-V), and control shRNA (Santa Cruz Biotechnology, Cat# sc-108080) lentiviral particles were used.

    Techniques: Marker, shRNA, Expressing, Immunofluorescence, Fluorescence, Comparison, Over Expression

    Parkin regulates anti-oxidative stress protein expression via PHB2. (A–D) SH-SY5Y cells were treated with PHB2-shRNA, Parkin-Over Exp, and MPP + (1 mM, 24 hours). Changes in Nrf2, HO-1, and NQO-1 protein levels and quantitative analysis. (A) Protein expression levels of Nrf2, HO-1, and NQO-1 in SH-SY5Y cells treated with PHB2-shRNA or Parkin-Over Exp under MPP + induction. (B–D) Quantitative analysis of Nrf2, HO-1, and NQO-1 protein expression. Data are expressed as mean ± SEM ( n = 3). *** P < 0.001, vs. control group; # P < 0.05,### P < 0.001, vs. MPP + group (one-way analysis of variance with Tukey's multiple comparison test). HO-1: Heme oxygenase-1; MPP + : 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; Parkin: parkin RBR E3 ubiquitin-protein ligase; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line.

    Journal: Neural Regeneration Research

    Article Title: A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

    doi: 10.4103/1673-5374.389356

    Figure Lengend Snippet: Parkin regulates anti-oxidative stress protein expression via PHB2. (A–D) SH-SY5Y cells were treated with PHB2-shRNA, Parkin-Over Exp, and MPP + (1 mM, 24 hours). Changes in Nrf2, HO-1, and NQO-1 protein levels and quantitative analysis. (A) Protein expression levels of Nrf2, HO-1, and NQO-1 in SH-SY5Y cells treated with PHB2-shRNA or Parkin-Over Exp under MPP + induction. (B–D) Quantitative analysis of Nrf2, HO-1, and NQO-1 protein expression. Data are expressed as mean ± SEM ( n = 3). *** P < 0.001, vs. control group; # P < 0.05,### P < 0.001, vs. MPP + group (one-way analysis of variance with Tukey's multiple comparison test). HO-1: Heme oxygenase-1; MPP + : 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; Parkin: parkin RBR E3 ubiquitin-protein ligase; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line.

    Article Snippet: Parkin-short hairpin RNA (sh-RNA) (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-42158-V) resistant to puromycin, PHB2-shRNA (Santa Cruz, Cat# sc-45849-V), and control shRNA (Santa Cruz Biotechnology, Cat# sc-108080) lentiviral particles were used.

    Techniques: Expressing, shRNA, Comparison, Over Expression

    Silencing PHB2 inhibits mitophagy and aggravates dopaminergic neuronal loss in PD mice. (A) Schematic of PHB2-shRNA and MPTP treatment in mice. (B, C) Quantitative analysis of PHB2 protein levels in the midbrain of C57BL/6J mice after PHB2-shRNA injection. (B) Protein expression levels of PHB2 in C57BL/6J mice treated with PHB2-shRNA. (C) Quantitative analysis of PHB2 protein expression. (D–F) An acute PD model was established by intraperitoneal injection of MPTP in C57BL/6J mice after PHB2-shRNA injection. Changes and quantitative analysis of LC3II/LC3I and PHB2 protein levels in the midbrain. (D) Protein expression levels of PHB2 and LC3 after MPTP injection in PD model mice. (E, F) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (G, H) TH immunofluorescence (Alexa Fluor 488, green fluorescence) shows dopaminergic neurons in the substantia nigra in PD mice with silenced PHB2. (G) Immunofluorescence of TH-positive neurons in the substantia nigra. Mice were treated with PHB2-shRNA and MPTP. Original magnification 10×, Scale bar: 200 μm. (H) Quantitative analysis of TH-positive neurons in the substantia nigra. (I) Fluorescence co-localization of LC3 and PHB2, TIM23, or TOM20 in the substantia nigra. Mice were treated with PHB2-shRNA and MPTP. Red fluorescence (Alexa Fluor 555): PHB2, TOM20, and TIM23, green fluorescence (Alexa Fluor 488): LC3, and blue fluorescence: DAPI. Original magnification 20×, Scale bars: 20 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, *** P < 0.001, vs. control group; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, vs. MPTP group (one-way analysis of variance with Tukey's multiple comparison test). LC3: Microtubule-associated protein 1 light chain 3; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD: Parkinson's disease; PHB2: Prohibitin 2; TH: tyrosine hydroxylase; TIM23: translocase of inner mitochondrial membrane 23; TOM20: translocase of outer mitochondrial membrane 20.

    Journal: Neural Regeneration Research

    Article Title: A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

    doi: 10.4103/1673-5374.389356

    Figure Lengend Snippet: Silencing PHB2 inhibits mitophagy and aggravates dopaminergic neuronal loss in PD mice. (A) Schematic of PHB2-shRNA and MPTP treatment in mice. (B, C) Quantitative analysis of PHB2 protein levels in the midbrain of C57BL/6J mice after PHB2-shRNA injection. (B) Protein expression levels of PHB2 in C57BL/6J mice treated with PHB2-shRNA. (C) Quantitative analysis of PHB2 protein expression. (D–F) An acute PD model was established by intraperitoneal injection of MPTP in C57BL/6J mice after PHB2-shRNA injection. Changes and quantitative analysis of LC3II/LC3I and PHB2 protein levels in the midbrain. (D) Protein expression levels of PHB2 and LC3 after MPTP injection in PD model mice. (E, F) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (G, H) TH immunofluorescence (Alexa Fluor 488, green fluorescence) shows dopaminergic neurons in the substantia nigra in PD mice with silenced PHB2. (G) Immunofluorescence of TH-positive neurons in the substantia nigra. Mice were treated with PHB2-shRNA and MPTP. Original magnification 10×, Scale bar: 200 μm. (H) Quantitative analysis of TH-positive neurons in the substantia nigra. (I) Fluorescence co-localization of LC3 and PHB2, TIM23, or TOM20 in the substantia nigra. Mice were treated with PHB2-shRNA and MPTP. Red fluorescence (Alexa Fluor 555): PHB2, TOM20, and TIM23, green fluorescence (Alexa Fluor 488): LC3, and blue fluorescence: DAPI. Original magnification 20×, Scale bars: 20 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, *** P < 0.001, vs. control group; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, vs. MPTP group (one-way analysis of variance with Tukey's multiple comparison test). LC3: Microtubule-associated protein 1 light chain 3; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD: Parkinson's disease; PHB2: Prohibitin 2; TH: tyrosine hydroxylase; TIM23: translocase of inner mitochondrial membrane 23; TOM20: translocase of outer mitochondrial membrane 20.

    Article Snippet: Parkin-short hairpin RNA (sh-RNA) (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-42158-V) resistant to puromycin, PHB2-shRNA (Santa Cruz, Cat# sc-45849-V), and control shRNA (Santa Cruz Biotechnology, Cat# sc-108080) lentiviral particles were used.

    Techniques: shRNA, Injection, Expressing, Immunofluorescence, Fluorescence, Comparison, Membrane

    Silencing PHB2 reduces antioxidative stress protein expression and aggravates motor defecits in PD mice. (A–D) Changes and quantitative analysis of Nrf2, HO-1, and NQO-1 protein levels in the midbrain of PHB2-shRNA and MPTP-treated PD mice. (E) Quantitative analysis of mice in the tail suspension and rotarod tests. Data are expressed as mean ± SEM ( n = 5). * P < 0.05, ** P < 0.01, vs. control group; $ P < 0.05, $$$ P < 0.001, vs . MPTP group (one-way analysis of variance with Tukey's multiple comparison test). HO-1: Heme oxygenase-1; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; PD: Parkinson's disease; PHB2: prohibitin 2.

    Journal: Neural Regeneration Research

    Article Title: A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

    doi: 10.4103/1673-5374.389356

    Figure Lengend Snippet: Silencing PHB2 reduces antioxidative stress protein expression and aggravates motor defecits in PD mice. (A–D) Changes and quantitative analysis of Nrf2, HO-1, and NQO-1 protein levels in the midbrain of PHB2-shRNA and MPTP-treated PD mice. (E) Quantitative analysis of mice in the tail suspension and rotarod tests. Data are expressed as mean ± SEM ( n = 5). * P < 0.05, ** P < 0.01, vs. control group; $ P < 0.05, $$$ P < 0.001, vs . MPTP group (one-way analysis of variance with Tukey's multiple comparison test). HO-1: Heme oxygenase-1; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; PD: Parkinson's disease; PHB2: prohibitin 2.

    Article Snippet: Parkin-short hairpin RNA (sh-RNA) (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-42158-V) resistant to puromycin, PHB2-shRNA (Santa Cruz, Cat# sc-45849-V), and control shRNA (Santa Cruz Biotechnology, Cat# sc-108080) lentiviral particles were used.

    Techniques: Expressing, shRNA, Suspension, Comparison

    Overexpressing TMEM120 accelerated breast cancer stemness by activating TAZ-mTOR signaling axis. ( A ) KEGG analysis was conducted to detect the signaling pathway significantly correlated with deletion of TMEM120B in MDA-453 cells. ( B ) Phosphorylation antibodies array kit was used to explore the key signaling pathway involved in TMEM120B overexpression in SK-BR-3 cells. ( C ) Immunoblotting of Myc-tag, TMEM120B, AKT, p-AKT, mTOR, p-mTOR, YAP, TAZ, and GAPDH after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. ( D ) qPCR assay was used to investigate the alteration of the target genes of YAP/TAZ within ectopic or deleted TMEM120B in SK-BR-3 or MDA-231 cells. ( E ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without mTOR signaling pathway inhibitor rapamycin in SK-BR-3 cells. ( F ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without knocking down TAZ by siRNA in SK-BR-3 cells. Subcellular localization of TAZ was evaluated by immunofluorescence assay ( G ) or western blotting assay ( H ) within ectopic TMEM120B in SK-BR-3 cells. Scale bar = 20 μm. ( I ) After being treated with CHX at indicated time points, the expression of TAZ was evaluated by western blotting after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. Quantification data are expressed as mean ± SD of three independent experiments (t-test, two-sided, ***, P < 0.001)

    Journal: Breast Cancer Research : BCR

    Article Title: TMEM120B strengthens breast cancer cell stemness and accelerates chemotherapy resistance via β1-integrin/FAK-TAZ-mTOR signaling axis by binding to MYH9

    doi: 10.1186/s13058-024-01802-z

    Figure Lengend Snippet: Overexpressing TMEM120 accelerated breast cancer stemness by activating TAZ-mTOR signaling axis. ( A ) KEGG analysis was conducted to detect the signaling pathway significantly correlated with deletion of TMEM120B in MDA-453 cells. ( B ) Phosphorylation antibodies array kit was used to explore the key signaling pathway involved in TMEM120B overexpression in SK-BR-3 cells. ( C ) Immunoblotting of Myc-tag, TMEM120B, AKT, p-AKT, mTOR, p-mTOR, YAP, TAZ, and GAPDH after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. ( D ) qPCR assay was used to investigate the alteration of the target genes of YAP/TAZ within ectopic or deleted TMEM120B in SK-BR-3 or MDA-231 cells. ( E ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without mTOR signaling pathway inhibitor rapamycin in SK-BR-3 cells. ( F ) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without knocking down TAZ by siRNA in SK-BR-3 cells. Subcellular localization of TAZ was evaluated by immunofluorescence assay ( G ) or western blotting assay ( H ) within ectopic TMEM120B in SK-BR-3 cells. Scale bar = 20 μm. ( I ) After being treated with CHX at indicated time points, the expression of TAZ was evaluated by western blotting after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. Quantification data are expressed as mean ± SD of three independent experiments (t-test, two-sided, ***, P < 0.001)

    Article Snippet: TAZ siRNA (sc-38,568) and negative control (NC) siRNA (sc-37,007) were obtained from Santa Cruz Biotechnology.

    Techniques: Over Expression, Western Blot, Immunofluorescence, Expressing

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Integration of transcriptomes of senescent cell models with multi-tissue patient samples reveals reduced COL6A3 as an inducer of senescence

    doi: 10.1016/j.celrep.2023.113371

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Control shRNA lentiviral particles , Santa Cruz , sc-108080.

    Techniques: Imaging, Recombinant, SYBR Green Assay, shRNA, Plasmid Preparation, Fluorescence, Software, Expressing