Structured Review

Abcam rvlm neurons
Methamphetamine induced necrotic cell death in <t>RVLM.</t> Representative photomicrographs of tissues stained by hematoxylin and eosin (H E) showing nuclear pyknosis and chromatolysis in RVLM neurons of rats that received saline (A–C) or died of <t>METH</t> (48 mg/kg, i.v.) (D–F). These results are typical of 4–5 animals from each experimental group. Scale bar, 5 µm. NA, nucleus ambiguus; n, nucleus; yellow arrow, chromatolysis in RVLM neuron; white arrow, karyorrhexis in RVLM neurons; green symbol (*), karyolysis in RVLM neuron.
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Images

1) Product Images from "Bioenergetics Failure and Oxidative Stress in Brain Stem Mediates Cardiovascular Collapse Associated with Fatal Methamphetamine Intoxication"

Article Title: Bioenergetics Failure and Oxidative Stress in Brain Stem Mediates Cardiovascular Collapse Associated with Fatal Methamphetamine Intoxication

Journal: PLoS ONE

doi: 10.1371/journal.pone.0030589

Methamphetamine induced necrotic cell death in RVLM. Representative photomicrographs of tissues stained by hematoxylin and eosin (H E) showing nuclear pyknosis and chromatolysis in RVLM neurons of rats that received saline (A–C) or died of METH (48 mg/kg, i.v.) (D–F). These results are typical of 4–5 animals from each experimental group. Scale bar, 5 µm. NA, nucleus ambiguus; n, nucleus; yellow arrow, chromatolysis in RVLM neuron; white arrow, karyorrhexis in RVLM neurons; green symbol (*), karyolysis in RVLM neuron.
Figure Legend Snippet: Methamphetamine induced necrotic cell death in RVLM. Representative photomicrographs of tissues stained by hematoxylin and eosin (H E) showing nuclear pyknosis and chromatolysis in RVLM neurons of rats that received saline (A–C) or died of METH (48 mg/kg, i.v.) (D–F). These results are typical of 4–5 animals from each experimental group. Scale bar, 5 µm. NA, nucleus ambiguus; n, nucleus; yellow arrow, chromatolysis in RVLM neuron; white arrow, karyorrhexis in RVLM neurons; green symbol (*), karyolysis in RVLM neuron.

Techniques Used: Staining

Presence of methamphetamine in RVLM neurons after intravenous administration. (A–C). Changes in concentration of METH in serum (A), ventrolateral medulla (B) or extracellular fluid collected from RVLM (C) of rats that died of intravenous administration of METH (at arrow). Values are mean ± SEM, n = 4–9 animals per experimental group. * P
Figure Legend Snippet: Presence of methamphetamine in RVLM neurons after intravenous administration. (A–C). Changes in concentration of METH in serum (A), ventrolateral medulla (B) or extracellular fluid collected from RVLM (C) of rats that died of intravenous administration of METH (at arrow). Values are mean ± SEM, n = 4–9 animals per experimental group. * P

Techniques Used: Concentration Assay

Methamphetamine induced bioenergetics failure in RVLM. (A). Fold changes relative to sham-control group in the level of ATP, ADP or ADP/ATP ratio in samples collected from RVLM of rats that died of METH (24 mg/kg, i.v.) or survived with pretreatment by microinjection of Mito-TEMPO (500 pmol) or coenzyme Q10 (CoQ10; 7 nmol) into RVLM prior to METH administration. Values are mean ± SEM, n = 3–5 animals per experimental group. * P
Figure Legend Snippet: Methamphetamine induced bioenergetics failure in RVLM. (A). Fold changes relative to sham-control group in the level of ATP, ADP or ADP/ATP ratio in samples collected from RVLM of rats that died of METH (24 mg/kg, i.v.) or survived with pretreatment by microinjection of Mito-TEMPO (500 pmol) or coenzyme Q10 (CoQ10; 7 nmol) into RVLM prior to METH administration. Values are mean ± SEM, n = 3–5 animals per experimental group. * P

Techniques Used:

Methamphetamine induced mitochondrial dysfunction in RVLM. Fold changes relative to sham-control group in the activity of Complex I (A), II (B), III (C), IV (D) or V (E) and electron transfer capacity between Complexes I and III (NCCR; F)or II and III (SCCR; G) in the mitochondrial respiratory chain in samples collected from RVLM of rats died of METH (24 mg/kg, i.v.) or survived with pretreatment by microinjection of Mito-TEMPO (500 pmol) or coenzyme Q10 (CoQ10; 7 nmol) into bilateral RVLM prior to METH administration. Values are mean ± SEM, n = 4–5 animals per experimental group. * P
Figure Legend Snippet: Methamphetamine induced mitochondrial dysfunction in RVLM. Fold changes relative to sham-control group in the activity of Complex I (A), II (B), III (C), IV (D) or V (E) and electron transfer capacity between Complexes I and III (NCCR; F)or II and III (SCCR; G) in the mitochondrial respiratory chain in samples collected from RVLM of rats died of METH (24 mg/kg, i.v.) or survived with pretreatment by microinjection of Mito-TEMPO (500 pmol) or coenzyme Q10 (CoQ10; 7 nmol) into bilateral RVLM prior to METH administration. Values are mean ± SEM, n = 4–5 animals per experimental group. * P

Techniques Used: Activity Assay

Methamphetamine did not induce apoptotic cell death in RVLM. Changes of activated caspase-3 or histone-associated DNA fragments in fold relative to sham-control group in samples collected from RVLM of rats that received saline or died of intravenous administration of METH. Values are mean ± SEM, n = 5–7 animals per experimental group. P > 0.05 among all groups in one-way ANOVA.
Figure Legend Snippet: Methamphetamine did not induce apoptotic cell death in RVLM. Changes of activated caspase-3 or histone-associated DNA fragments in fold relative to sham-control group in samples collected from RVLM of rats that received saline or died of intravenous administration of METH. Values are mean ± SEM, n = 5–7 animals per experimental group. P > 0.05 among all groups in one-way ANOVA.

Techniques Used:

Methamphetamine increased production of mitochondrial superoxide anions in RVLM. (A). Fold changes relative to sham-control group in mitochondrial superoxide anion in sample collected from RVLM of rats died of METH (24 mg/kg, i.v.) or survived with pretreatment by microinjection of Mito-TEMPO (500 pmol) or coenzyme Q10 (CoQ10; 7 nmol) into RVLM prior to METH administration. Values are mean ± SEM, n = 3–6 animals per experimental group. * P
Figure Legend Snippet: Methamphetamine increased production of mitochondrial superoxide anions in RVLM. (A). Fold changes relative to sham-control group in mitochondrial superoxide anion in sample collected from RVLM of rats died of METH (24 mg/kg, i.v.) or survived with pretreatment by microinjection of Mito-TEMPO (500 pmol) or coenzyme Q10 (CoQ10; 7 nmol) into RVLM prior to METH administration. Values are mean ± SEM, n = 3–6 animals per experimental group. * P

Techniques Used:

Oxidative stress in RVLM underlied methamphetamine-elicited cardiovascular collapse. Temporal changes in MAP, HR or power density of the LF component of systolic blood pressure signals in rats that received saline, died of METH (24 mg/kg, i.v.) or survived with pretreatment by microinjection of CoQ10 (7 nmol), Mito-TEMPO (500 pmol) or IM-54 (3 pmol) into bilateral RVLM prior to METH administration. Values are mean ± SEM, n = 6–37 animals per experimental group. * P
Figure Legend Snippet: Oxidative stress in RVLM underlied methamphetamine-elicited cardiovascular collapse. Temporal changes in MAP, HR or power density of the LF component of systolic blood pressure signals in rats that received saline, died of METH (24 mg/kg, i.v.) or survived with pretreatment by microinjection of CoQ10 (7 nmol), Mito-TEMPO (500 pmol) or IM-54 (3 pmol) into bilateral RVLM prior to METH administration. Values are mean ± SEM, n = 6–37 animals per experimental group. * P

Techniques Used:

Proposed mechanisms in RVLM that underlies METH-induced cardiovascular collapse. Intravenous administration of METH rapidly reaches RVLM to induce anoxia and cessation of tissue perfusion, followed by bioenergetics failure and oxidative stress because of mitochondrial dysfunction that lead to necrotic cell death. The loss of functionality in RVLM results in cessation of central cardiovascular regulation and the eventual cardiovascular collapse.
Figure Legend Snippet: Proposed mechanisms in RVLM that underlies METH-induced cardiovascular collapse. Intravenous administration of METH rapidly reaches RVLM to induce anoxia and cessation of tissue perfusion, followed by bioenergetics failure and oxidative stress because of mitochondrial dysfunction that lead to necrotic cell death. The loss of functionality in RVLM results in cessation of central cardiovascular regulation and the eventual cardiovascular collapse.

Techniques Used:

2) Product Images from "ADAMTS-4 promotes neurodegeneration in a mouse model of amyotrophic lateral sclerosis"

Article Title: ADAMTS-4 promotes neurodegeneration in a mouse model of amyotrophic lateral sclerosis

Journal: Molecular Neurodegeneration

doi: 10.1186/s13024-016-0078-3

ADAMTS-4 modulates the synthesis/release of neurotrophic factors by glial cells in vitro . a - c Quantitative RT-PCR for NGF ( a ) GDNF ( b ) and BDNF ( c ) expression in mouse adult cortical astrocyte cultures treated or not for 48 h with a human recombinant ADAMTS-4 (20, 100, 200 ng/ml). Values plotted are mean ± SEM. Mann–Whitney U -tests: * P
Figure Legend Snippet: ADAMTS-4 modulates the synthesis/release of neurotrophic factors by glial cells in vitro . a - c Quantitative RT-PCR for NGF ( a ) GDNF ( b ) and BDNF ( c ) expression in mouse adult cortical astrocyte cultures treated or not for 48 h with a human recombinant ADAMTS-4 (20, 100, 200 ng/ml). Values plotted are mean ± SEM. Mann–Whitney U -tests: * P

Techniques Used: In Vitro, Quantitative RT-PCR, Expressing, Recombinant, MANN-WHITNEY

rADAMTS-4 is not toxic to cortical neurons in vitro . Neuronal viability assessed by MTT assay in primary cortical neuron cultures treated or not with a human recombinant ADAMTS-4 ( a - b ) or ADAMTS-1 ( c - d ) at different doses (20, 100, 200, 500 ng/ml) 30 min before exposure ( b , d ) or not ( a , c ) to glutamate 400 μM (Glu) during 24 h. Values plotted are mean ± SEM. Mann–Whitney U -tests: P > 0.05 control Vs ADAMTS, *** P
Figure Legend Snippet: rADAMTS-4 is not toxic to cortical neurons in vitro . Neuronal viability assessed by MTT assay in primary cortical neuron cultures treated or not with a human recombinant ADAMTS-4 ( a - b ) or ADAMTS-1 ( c - d ) at different doses (20, 100, 200, 500 ng/ml) 30 min before exposure ( b , d ) or not ( a , c ) to glutamate 400 μM (Glu) during 24 h. Values plotted are mean ± SEM. Mann–Whitney U -tests: P > 0.05 control Vs ADAMTS, *** P

Techniques Used: In Vitro, MTT Assay, Recombinant, MANN-WHITNEY

Presymptomatic treatment with rADAMTS-4 worsens the prognosis of SOD1 G93A mice. a Kaplan-Meier graph showing the probability of symptom onset in SOD1 G93A males treated with saline (Control; black line) or recombinant ADAMTS-4 (ADAMTS-4; gray line) at early presymptomatic stage. Log-rank (Mantel-Cox) Test: * P
Figure Legend Snippet: Presymptomatic treatment with rADAMTS-4 worsens the prognosis of SOD1 G93A mice. a Kaplan-Meier graph showing the probability of symptom onset in SOD1 G93A males treated with saline (Control; black line) or recombinant ADAMTS-4 (ADAMTS-4; gray line) at early presymptomatic stage. Log-rank (Mantel-Cox) Test: * P

Techniques Used: Mouse Assay, Recombinant

rADAMTS-4 increases astrogliosis in the lumbar spinal cord of female SOD1 G93A mice. a Representative photomicrographs of ventral horns in lumbar spinal cord sections from WT and control or ADAMTS-4-treated SOD1 G93A mice stained with GFAP. Scale bar: 250 μm. b Quantification of GFAP immunoreactivity per area from male mice ( a ). Values plotted are mean ± SEM. Two-way ANOVA: *** P
Figure Legend Snippet: rADAMTS-4 increases astrogliosis in the lumbar spinal cord of female SOD1 G93A mice. a Representative photomicrographs of ventral horns in lumbar spinal cord sections from WT and control or ADAMTS-4-treated SOD1 G93A mice stained with GFAP. Scale bar: 250 μm. b Quantification of GFAP immunoreactivity per area from male mice ( a ). Values plotted are mean ± SEM. Two-way ANOVA: *** P

Techniques Used: Mouse Assay, Staining

rADAMTS-4 accelerates neurodegeneration in the lumbar spinal cord of SOD1 G93A mice. a Representative photomicrographs of ventral horns in lumbar spinal cord sections from WT and control or ADAMTS-4-treated SOD1 G93A male mice stained with ChAT. Scale bar: 500 or 250 μm. b-c Quantification of average spinal motoneuron number ( b ) and size ( c ) in male mice from ( a ). Values plotted are mean ± SEM. Two-way ANOVA: *** P
Figure Legend Snippet: rADAMTS-4 accelerates neurodegeneration in the lumbar spinal cord of SOD1 G93A mice. a Representative photomicrographs of ventral horns in lumbar spinal cord sections from WT and control or ADAMTS-4-treated SOD1 G93A male mice stained with ChAT. Scale bar: 500 or 250 μm. b-c Quantification of average spinal motoneuron number ( b ) and size ( c ) in male mice from ( a ). Values plotted are mean ± SEM. Two-way ANOVA: *** P

Techniques Used: Mouse Assay, Staining

rADAMTS-4 decreases NGF expression in the lumbar spinal cord of male SOD1 G93A mice. a Representative photomicrographs of ventral horns in lumbar spinal cord sections from control or ADAMTS-4-treated SOD1 G93A male mice stained with NGF. Scale bar: 125 μm. b Quantification of NGF immunoreactivity per area from male mice ( a ). Values plotted are m ean ± SEM. Unpaired two-tailed t-Test: $ P
Figure Legend Snippet: rADAMTS-4 decreases NGF expression in the lumbar spinal cord of male SOD1 G93A mice. a Representative photomicrographs of ventral horns in lumbar spinal cord sections from control or ADAMTS-4-treated SOD1 G93A male mice stained with NGF. Scale bar: 125 μm. b Quantification of NGF immunoreactivity per area from male mice ( a ). Values plotted are m ean ± SEM. Unpaired two-tailed t-Test: $ P

Techniques Used: Expressing, Mouse Assay, Staining, Two Tailed Test

rADAMTS-4 reduces perineuronal nets enwrapping motoneurons in the lumbar spinal cord of SOD1 G93A mice. a Representative photomicrographs of ventral horns in lumbar spinal cord sections from WT and control or ADAMTS-4-treated SOD1 G93A male mice stained with WFA, a marker of perineuronal nets. Scale bar: 500 or 250 μm. b Quantification of WFA immunoreactivity per area from male mice ( a ). Values plotted are mean ± SEM. Two-way ANOVA: *** P
Figure Legend Snippet: rADAMTS-4 reduces perineuronal nets enwrapping motoneurons in the lumbar spinal cord of SOD1 G93A mice. a Representative photomicrographs of ventral horns in lumbar spinal cord sections from WT and control or ADAMTS-4-treated SOD1 G93A male mice stained with WFA, a marker of perineuronal nets. Scale bar: 500 or 250 μm. b Quantification of WFA immunoreactivity per area from male mice ( a ). Values plotted are mean ± SEM. Two-way ANOVA: *** P

Techniques Used: Mouse Assay, Staining, Marker

ADAMTS-4 expression in the central nervous system. a Differential mRNA expression of ADAMTS proteoglycanases (eg. ADAMTS-1, −4, −5 and −9) in the lumbar spinal cord (SC) and in the cortex of 3-month-old WT male (♂) and female (♀) mice. Ct values are indicated in the histograms. Values plotted are mean ± SEM. Mann–Whitney U -tests: * P
Figure Legend Snippet: ADAMTS-4 expression in the central nervous system. a Differential mRNA expression of ADAMTS proteoglycanases (eg. ADAMTS-1, −4, −5 and −9) in the lumbar spinal cord (SC) and in the cortex of 3-month-old WT male (♂) and female (♀) mice. Ct values are indicated in the histograms. Values plotted are mean ± SEM. Mann–Whitney U -tests: * P

Techniques Used: Expressing, Mouse Assay, MANN-WHITNEY

Decrease of ADAMTS-4 activity in the lumbar spinal cord of SOD1 G93A mice at disease end-stage. a - h Quantitative RT-PCR for ADAMTS-4 ( a , e ) ADAMTS-1 ( b , f ) −5 ( c , g ) and −9 ( d , h ) expression in the lumbar spinal cord (SC) of WT (blank bar) and SOD1 G93A (black bar) male (♂; a , b , c , d ) or female (♀; e , f , g , h ) mice at presymptomatic (PS), symptomatic (SS) and end (ES) stages. Values plotted are mean ± SEM. Mann–Whitney U -tests: * P
Figure Legend Snippet: Decrease of ADAMTS-4 activity in the lumbar spinal cord of SOD1 G93A mice at disease end-stage. a - h Quantitative RT-PCR for ADAMTS-4 ( a , e ) ADAMTS-1 ( b , f ) −5 ( c , g ) and −9 ( d , h ) expression in the lumbar spinal cord (SC) of WT (blank bar) and SOD1 G93A (black bar) male (♂; a , b , c , d ) or female (♀; e , f , g , h ) mice at presymptomatic (PS), symptomatic (SS) and end (ES) stages. Values plotted are mean ± SEM. Mann–Whitney U -tests: * P

Techniques Used: Activity Assay, Mouse Assay, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Gender similarities and differences in the effect of ADAMTS-4 treatment on ALS. a Schematic representation of ADAMTS-4 treatment promoting the decline of NGF production and ALS-induced perineuronal net degradation which contribute to the degeneration and even death of motoneurons in the ventral horn of the lumbar spinal cord of SOD1 G93A mice. b A table describing the similarities and differences observed in behavioral and anatomical effects of ADAMTS-4 treatment in SOD1 G93A male and female mice
Figure Legend Snippet: Gender similarities and differences in the effect of ADAMTS-4 treatment on ALS. a Schematic representation of ADAMTS-4 treatment promoting the decline of NGF production and ALS-induced perineuronal net degradation which contribute to the degeneration and even death of motoneurons in the ventral horn of the lumbar spinal cord of SOD1 G93A mice. b A table describing the similarities and differences observed in behavioral and anatomical effects of ADAMTS-4 treatment in SOD1 G93A male and female mice

Techniques Used: Mouse Assay

Spinal cord-specific decrease of ADAMTS-4 expression in SOD1 G93A mice at disease end-stage. a-f Immunoblot for ADAMTS-4 in the cervical or thoracic spinal cord (SC) and in the cortex of WT (blank bar) and SOD1 G93A (black bar) male (♂; a , b , c ) or female (♀; d , e , f ) mice at disease end-stage (ES). Values plotted are mean ± SEM. Mann–Whitney U -tests: * P
Figure Legend Snippet: Spinal cord-specific decrease of ADAMTS-4 expression in SOD1 G93A mice at disease end-stage. a-f Immunoblot for ADAMTS-4 in the cervical or thoracic spinal cord (SC) and in the cortex of WT (blank bar) and SOD1 G93A (black bar) male (♂; a , b , c ) or female (♀; d , e , f ) mice at disease end-stage (ES). Values plotted are mean ± SEM. Mann–Whitney U -tests: * P

Techniques Used: Expressing, Mouse Assay, MANN-WHITNEY

3) Product Images from "Murine Cytomegalovirus Displays Selective Infection of Cells within Hours after Systemic Administration"

Article Title: Murine Cytomegalovirus Displays Selective Infection of Cells within Hours after Systemic Administration

Journal: The Journal of general virology

doi: 10.1099/vir.0.006668-0

MCMV-GFP infected reticular fibroblasts at 8h and DCs at 48h in the spleen. (A) GFP + cells were in close proximity to CD169 + cells, but did not co-localize. Instead, GFP + cells co-localized with ER-TR7, a marker for reticular fibroblasts. A higher magnification
Figure Legend Snippet: MCMV-GFP infected reticular fibroblasts at 8h and DCs at 48h in the spleen. (A) GFP + cells were in close proximity to CD169 + cells, but did not co-localize. Instead, GFP + cells co-localized with ER-TR7, a marker for reticular fibroblasts. A higher magnification

Techniques Used: Infection, Marker

MCMV infected stromal cells in the spleen. Infected cells in the spleen co-localized with ER-TR7 and CD29 at both 8 and 17h p.i. In addition, a few cells appeared to be MADCAM + .
Figure Legend Snippet: MCMV infected stromal cells in the spleen. Infected cells in the spleen co-localized with ER-TR7 and CD29 at both 8 and 17h p.i. In addition, a few cells appeared to be MADCAM + .

Techniques Used: Infection

Changes in splenic populations were seen at 48h p.i. but not at 8h p.i. At 48h, there was disorganization of T and B cell areas, an influx of CD11b + macrophages in the red pulp, and loss of NK cells. In addition, ER-TR7 was no longer expressed on many
Figure Legend Snippet: Changes in splenic populations were seen at 48h p.i. but not at 8h p.i. At 48h, there was disorganization of T and B cell areas, an influx of CD11b + macrophages in the red pulp, and loss of NK cells. In addition, ER-TR7 was no longer expressed on many

Techniques Used:

4) Product Images from "Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion"

Article Title: Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion

Journal: mBio

doi: 10.1128/mBio.01802-14

(A) Fibers containing SEPT2, -9, and -11 encase the chlamydial inclusion. HeLa cells infected for 48 h were fixed and stained for SEPT2 or SEPT9 or infected for 30 h, fixed, and stained for SEPT11. Chlamydial inclusions are indicated by arrows. Cell nuclei are marked with asterisks. Scale bar, 10 µm. Images are representative of at least 3 independent experiments. Proteomic analysis found the four septins SEPT2, -7, -9, and -11 on purified inclusions (see Materials and Methods for details; the following numbers of peptides were identified by mass spectrometry in infected/uninfected cells: SEPT2, 64/29; SEPT7, 6/11; SEPT9, 13/5; SEPT11, 7/11). (B) Infection increases the amount of SEPT9 in SEPT2-containing complexes. Uninfected HeLa cells or HeLa cells infected with C. trachomatis for 30 h were lysed, and proteins in the lysate supernatants (Input) were immunoprecipitated with anti-SEPT2 antibodies. Input, unbound, and IP fractions from the immunoprecipitations (IPs) were analyzed by SDS-PAGE, followed by immunoblotting with anti-SEPT2, -7, -9, or -11 or anti-GAPDH antibody. The smaller band for SEPT2 is consistent with cleavage by CPAF during sample preparation. A number of isoforms exist for SEPT9; one isoform especially appears to be recruited to septin fibers during infection.
Figure Legend Snippet: (A) Fibers containing SEPT2, -9, and -11 encase the chlamydial inclusion. HeLa cells infected for 48 h were fixed and stained for SEPT2 or SEPT9 or infected for 30 h, fixed, and stained for SEPT11. Chlamydial inclusions are indicated by arrows. Cell nuclei are marked with asterisks. Scale bar, 10 µm. Images are representative of at least 3 independent experiments. Proteomic analysis found the four septins SEPT2, -7, -9, and -11 on purified inclusions (see Materials and Methods for details; the following numbers of peptides were identified by mass spectrometry in infected/uninfected cells: SEPT2, 64/29; SEPT7, 6/11; SEPT9, 13/5; SEPT11, 7/11). (B) Infection increases the amount of SEPT9 in SEPT2-containing complexes. Uninfected HeLa cells or HeLa cells infected with C. trachomatis for 30 h were lysed, and proteins in the lysate supernatants (Input) were immunoprecipitated with anti-SEPT2 antibodies. Input, unbound, and IP fractions from the immunoprecipitations (IPs) were analyzed by SDS-PAGE, followed by immunoblotting with anti-SEPT2, -7, -9, or -11 or anti-GAPDH antibody. The smaller band for SEPT2 is consistent with cleavage by CPAF during sample preparation. A number of isoforms exist for SEPT9; one isoform especially appears to be recruited to septin fibers during infection.

Techniques Used: Infection, Staining, Purification, Mass Spectrometry, Immunoprecipitation, SDS Page, Sample Prep

5) Product Images from "Human Dental Pulp Stem Cells Grown in Neurogenic Media Differentiate Into Endothelial Cells and Promote Neovasculogenesis in the Mouse Brain"

Article Title: Human Dental Pulp Stem Cells Grown in Neurogenic Media Differentiate Into Endothelial Cells and Promote Neovasculogenesis in the Mouse Brain

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2019.00347

Human DPSCs are able to commit toward differentiation to neuronal-like and glial-like lineages. One week of culture of both human DPSCs and murine (control) NSCs in Neurocult differentiation media is sufficient to induce them express markers for (A) neuronal lineage differentiation: doublecortin (DCX) and NeuN staining, for immature and mature neurons, respectively, and (C) Astroglial lineage differentiation: glial fibrillary acidic protein (GFAP) and S-100β immunostaining, for immature and mature astrocytes, respectively. (B,D) Graphs showing quantifications (mean ± SEM, n = 360 cells) of three independent experiments. (E) After 1 week of growth in neural differentiation conditions, both NSCs and DPSCs still express VEGF but downregulate CD31. (F) Quantification of the proportion of CD31 positive cells ( n = 571). (G) Control with no 1 ary antibodies. Scale bar 20 μm.
Figure Legend Snippet: Human DPSCs are able to commit toward differentiation to neuronal-like and glial-like lineages. One week of culture of both human DPSCs and murine (control) NSCs in Neurocult differentiation media is sufficient to induce them express markers for (A) neuronal lineage differentiation: doublecortin (DCX) and NeuN staining, for immature and mature neurons, respectively, and (C) Astroglial lineage differentiation: glial fibrillary acidic protein (GFAP) and S-100β immunostaining, for immature and mature astrocytes, respectively. (B,D) Graphs showing quantifications (mean ± SEM, n = 360 cells) of three independent experiments. (E) After 1 week of growth in neural differentiation conditions, both NSCs and DPSCs still express VEGF but downregulate CD31. (F) Quantification of the proportion of CD31 positive cells ( n = 571). (G) Control with no 1 ary antibodies. Scale bar 20 μm.

Techniques Used: Staining, Immunostaining

6) Product Images from "Cyclin D1 integrates G9a-mediated histone methylation"

Article Title: Cyclin D1 integrates G9a-mediated histone methylation

Journal: Oncogene

doi: 10.1038/s41388-019-0723-8

G9a and cyclin D1 bind common regulatory regions of genes in chromatin immunoprecipitation (ChIP)-Seq. a Venn diagram depicting the overlapping intervals shared between cyclin D1 ChIP-Seq and G9a ChIP-Seq. b Gene Ontology (GO) biological function enrichment scores for overlapping terms for 744 genes common between cyclin D1 ChIP-Seq and G9a ChIP-Seq. c – k Three genes ( Mdm4 , Pttg1 , and Myc ) were selected from the “oncogene” GO biological term and eight genes were selected from the “neuronal activities” GO term (Supplemental Figs. 4 , 5 ). c , f , i Depicted are tag density profiles for cyclin D1 intervals (red) and G9a intervals (blue) with respect to the identified genes. Profiles generated by Integrated Genome Browser are depicted for enriched regions binding G9a and the same region of cyclin D1 ChIP-Seq. Enriched intervals are designated by an * for cyclin D1 and a * for G9a. Tag density profiles are not drawn to scale ( c , f , i ). d , g , j Individual ChIP-qPCR analysis of target genes identified in ChIP-Seq. FLAG (FLAG-cyclin D1) ChIP-qPCR analysis of target genes in cyclin D1 −/− plus GFP vector vs cyclin D1 −/− plus cyclin D1 WT rescue mouse embryonic fibroblasts (MEFs), and e , h , k G9a ChIP-qPCR of the same target genes in G9a −/− plus vector vs G9a −/− plus G9a WT rescued MEFs. H3K9me2 ChIP-qPCR is conducted in each cell type with IgG as control. Data are shown as mean ± SEM for ChIP-qPCR of FLAG (FLAG-cyclin D1) and H3K9me2 for target genes identified in ChIP-Seq. Significant difference are shown as ** P
Figure Legend Snippet: G9a and cyclin D1 bind common regulatory regions of genes in chromatin immunoprecipitation (ChIP)-Seq. a Venn diagram depicting the overlapping intervals shared between cyclin D1 ChIP-Seq and G9a ChIP-Seq. b Gene Ontology (GO) biological function enrichment scores for overlapping terms for 744 genes common between cyclin D1 ChIP-Seq and G9a ChIP-Seq. c – k Three genes ( Mdm4 , Pttg1 , and Myc ) were selected from the “oncogene” GO biological term and eight genes were selected from the “neuronal activities” GO term (Supplemental Figs. 4 , 5 ). c , f , i Depicted are tag density profiles for cyclin D1 intervals (red) and G9a intervals (blue) with respect to the identified genes. Profiles generated by Integrated Genome Browser are depicted for enriched regions binding G9a and the same region of cyclin D1 ChIP-Seq. Enriched intervals are designated by an * for cyclin D1 and a * for G9a. Tag density profiles are not drawn to scale ( c , f , i ). d , g , j Individual ChIP-qPCR analysis of target genes identified in ChIP-Seq. FLAG (FLAG-cyclin D1) ChIP-qPCR analysis of target genes in cyclin D1 −/− plus GFP vector vs cyclin D1 −/− plus cyclin D1 WT rescue mouse embryonic fibroblasts (MEFs), and e , h , k G9a ChIP-qPCR of the same target genes in G9a −/− plus vector vs G9a −/− plus G9a WT rescued MEFs. H3K9me2 ChIP-qPCR is conducted in each cell type with IgG as control. Data are shown as mean ± SEM for ChIP-qPCR of FLAG (FLAG-cyclin D1) and H3K9me2 for target genes identified in ChIP-Seq. Significant difference are shown as ** P

Techniques Used: Chromatin Immunoprecipitation, Generated, Binding Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation

Cyclin D1 augments H3K9me2. a Confocal microscopy of immunofluorescence for H3K9me2 (red) and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI; blue) in cyclin D1 wild-type and knockout mouse embryonic fibroblasts (MEFs), and cyclin D1 −/− MEFs rescued with MSCV-cyclin D1-IRES-GFP or vector control. Images demonstrate the reduction in H3K9me2 in cyclin D1 −/− cells. Scale bar, 20 μm with ( b ) quantitation of mean fluorescence shown as mean ± SEM. c Schematic representation of transgenic paradigm. d Immunohistochemical staining for H3K9me2 in the mammary gland of transgenic mice in which the cyclin D1 gene was deleted through Cre excision in the adult mammary glands. e The quantitation of H3K9me2 is shown as mean ± SEM for n = 10 separate mammary glands from two cyclin D1 WT (tamoxifen-treated cyclin D1 wt/wt ;-Rosa26 CreERT2/CreERT2 transgenic mice) and three cyclin D1 −/− mice (tamoxifen-treated cyclin D1 fl/fl ;-Rosa26 CreERT2/CreERT2 transgenic mice)
Figure Legend Snippet: Cyclin D1 augments H3K9me2. a Confocal microscopy of immunofluorescence for H3K9me2 (red) and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI; blue) in cyclin D1 wild-type and knockout mouse embryonic fibroblasts (MEFs), and cyclin D1 −/− MEFs rescued with MSCV-cyclin D1-IRES-GFP or vector control. Images demonstrate the reduction in H3K9me2 in cyclin D1 −/− cells. Scale bar, 20 μm with ( b ) quantitation of mean fluorescence shown as mean ± SEM. c Schematic representation of transgenic paradigm. d Immunohistochemical staining for H3K9me2 in the mammary gland of transgenic mice in which the cyclin D1 gene was deleted through Cre excision in the adult mammary glands. e The quantitation of H3K9me2 is shown as mean ± SEM for n = 10 separate mammary glands from two cyclin D1 WT (tamoxifen-treated cyclin D1 wt/wt ;-Rosa26 CreERT2/CreERT2 transgenic mice) and three cyclin D1 −/− mice (tamoxifen-treated cyclin D1 fl/fl ;-Rosa26 CreERT2/CreERT2 transgenic mice)

Techniques Used: Confocal Microscopy, Immunofluorescence, Staining, Knock-Out, Plasmid Preparation, Quantitation Assay, Fluorescence, Transgenic Assay, Immunohistochemistry, Mouse Assay

G9a dimethylation of H3K9 requires endogenous cyclin D1. a , b Confocal microscopy of immunofluorescence for H3K9me2 (red) and nuclear staining with DAPI (blue) in G9a fl/fl and G9a −/− mouse embryonic fibroblasts (MEFs), and G9a −/− MEFs rescued with G9a WT or vector control. Images show the reduction in H3K9me2 in G9a −/− cells ( a ) and quantitative analysis was shown as mean ± SEM ( b ). c Western blot for H3K9me2 and G9a in G9a fl/fl and G9a −/− MEFs. The G9a fl/fl , G9a −/− , and G9a −/− MEFs rescued with G9a and vector control were assessed by western blot for H3K9me2. Lamin B1 was used as a protein loading control. S.E. shorter exposure, L.E. longer exposure. d Quantitation of H3K9me2 is shown as mean ± SEM for N = 3. e MCF-7 cells transduced with two individual shG9a and shGFP control were assessed by western blot for H3K9me2, cyclin D1, and G9a. Lamin B1 was used as a protein loading control. f , g Confocal microscopy of immunofluorescence for H3K9me2 (green) and nuclear staining with DAPI (blue) in G9a −/− MEFs rescued with G9a WT or vector control treated with cyclin D1 small interfering RNA. Images show the reduction in H3K9me2 by cyclin D1 siRNA in G9a −/− plus G9a cells but not in G9a −/− plus vector cells. Scale bar, 20 μm ( f ) and quantitative analysis was shown as mean ± SEM ( g )
Figure Legend Snippet: G9a dimethylation of H3K9 requires endogenous cyclin D1. a , b Confocal microscopy of immunofluorescence for H3K9me2 (red) and nuclear staining with DAPI (blue) in G9a fl/fl and G9a −/− mouse embryonic fibroblasts (MEFs), and G9a −/− MEFs rescued with G9a WT or vector control. Images show the reduction in H3K9me2 in G9a −/− cells ( a ) and quantitative analysis was shown as mean ± SEM ( b ). c Western blot for H3K9me2 and G9a in G9a fl/fl and G9a −/− MEFs. The G9a fl/fl , G9a −/− , and G9a −/− MEFs rescued with G9a and vector control were assessed by western blot for H3K9me2. Lamin B1 was used as a protein loading control. S.E. shorter exposure, L.E. longer exposure. d Quantitation of H3K9me2 is shown as mean ± SEM for N = 3. e MCF-7 cells transduced with two individual shG9a and shGFP control were assessed by western blot for H3K9me2, cyclin D1, and G9a. Lamin B1 was used as a protein loading control. f , g Confocal microscopy of immunofluorescence for H3K9me2 (green) and nuclear staining with DAPI (blue) in G9a −/− MEFs rescued with G9a WT or vector control treated with cyclin D1 small interfering RNA. Images show the reduction in H3K9me2 by cyclin D1 siRNA in G9a −/− plus G9a cells but not in G9a −/− plus vector cells. Scale bar, 20 μm ( f ) and quantitative analysis was shown as mean ± SEM ( g )

Techniques Used: Confocal Microscopy, Immunofluorescence, Staining, Plasmid Preparation, Western Blot, Quantitation Assay, Transduction, Small Interfering RNA

The G9a-binding defective mutant of cyclin D1 fails to augment H3K9me2. a Confocal microscopy of immunofluorescence for H3K9me2 (red) and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI; blue) in cyclin D1 wild-type and knockout mouse embryonic fibroblasts (MEFs), and cyclin D1 −/− MEFs rescued with MSCV-cyclin D1 WT -IRES-GFP, MSCV-cyclin D1 C2 -IRES-GFP, or vector control. Images demonstrate the reduction in H3K9me2 in cyclin D1 −/− cells and rescue with cyclin D1 WT . Scale bar, 40 μm with ( b ) quantitation of mean fluorescence shown as mean ± SEM. c Western blot of cyclin D1 −/− MEFs rescued with MSCV-cyclin D1 WT -IRES-GFP, MSCV-cyclin D1 C2 -IRES-GFP, or vector control, with antibodies as indicated
Figure Legend Snippet: The G9a-binding defective mutant of cyclin D1 fails to augment H3K9me2. a Confocal microscopy of immunofluorescence for H3K9me2 (red) and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI; blue) in cyclin D1 wild-type and knockout mouse embryonic fibroblasts (MEFs), and cyclin D1 −/− MEFs rescued with MSCV-cyclin D1 WT -IRES-GFP, MSCV-cyclin D1 C2 -IRES-GFP, or vector control. Images demonstrate the reduction in H3K9me2 in cyclin D1 −/− cells and rescue with cyclin D1 WT . Scale bar, 40 μm with ( b ) quantitation of mean fluorescence shown as mean ± SEM. c Western blot of cyclin D1 −/− MEFs rescued with MSCV-cyclin D1 WT -IRES-GFP, MSCV-cyclin D1 C2 -IRES-GFP, or vector control, with antibodies as indicated

Techniques Used: Binding Assay, Mutagenesis, Confocal Microscopy, Immunofluorescence, Staining, Knock-Out, Plasmid Preparation, Quantitation Assay, Fluorescence, Western Blot

7) Product Images from "tomm22 Knockdown-Mediated Hepatocyte Damages Elicit Both the Formation of Hybrid Hepatocytes and Biliary Conversion to Hepatocytes in Zebrafish Larvae"

Article Title: tomm22 Knockdown-Mediated Hepatocyte Damages Elicit Both the Formation of Hybrid Hepatocytes and Biliary Conversion to Hepatocytes in Zebrafish Larvae

Journal: Gene Expression

doi: 10.3727/105221617X695195

Surviving hepatocytes become hybrid hepatocytes. (A) Confocal single-optical section images showing the expression of Hnf4a (green), Tp1: H2B-mCherry (red), and Bhmt (gray) in the liver. Arrows point to hepatocytes that express Tp1: H2B-mCherry; arrowheads point to BECs negative for Hnf4a and Bhmt. (B) Confocal projection images showing Tp1 :H2B-mCherry (red) and Anxa4 (gray) expression in the liver. Arrows point to H2B-mCherry/Anxa4 double-positive cells. (C) Confocal single-optical section images showing the hepatic expression of ubb :mCherry (red, Cre-labeled cells) and Hnf4a (gray) in tomm22 MO-injected larvae. Arrows point to mCherry/Hnf4a double-positive cells. (D) Graph showing the percentage of ubb: mCherry + cells among Hnf4a + cells, which were derived from BECs. Red dots indicate the larvae shown in (C); n indicates the number of larvae examined. Scale bars: 20 μm; error bars: ±SEM.
Figure Legend Snippet: Surviving hepatocytes become hybrid hepatocytes. (A) Confocal single-optical section images showing the expression of Hnf4a (green), Tp1: H2B-mCherry (red), and Bhmt (gray) in the liver. Arrows point to hepatocytes that express Tp1: H2B-mCherry; arrowheads point to BECs negative for Hnf4a and Bhmt. (B) Confocal projection images showing Tp1 :H2B-mCherry (red) and Anxa4 (gray) expression in the liver. Arrows point to H2B-mCherry/Anxa4 double-positive cells. (C) Confocal single-optical section images showing the hepatic expression of ubb :mCherry (red, Cre-labeled cells) and Hnf4a (gray) in tomm22 MO-injected larvae. Arrows point to mCherry/Hnf4a double-positive cells. (D) Graph showing the percentage of ubb: mCherry + cells among Hnf4a + cells, which were derived from BECs. Red dots indicate the larvae shown in (C); n indicates the number of larvae examined. Scale bars: 20 μm; error bars: ±SEM.

Techniques Used: Expressing, Labeling, Injection, Derivative Assay

8) Product Images from "Oxidative stress induces protein and DNA radical formation in follicular dendritic cells (FDCs) of the germinal center and modulates its cell death patterns in late sepsis"

Article Title: Oxidative stress induces protein and DNA radical formation in follicular dendritic cells (FDCs) of the germinal center and modulates its cell death patterns in late sepsis

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2010.12.037

Oxidative stress in late sepsis modulates B cell differentiation following death of FDCs in septic mice. A. B cell differentiation into plasma cells is affected by oxidative stress induced FDC death. Spleen cells from sham-treated, LPS, LPS+allopurinol
Figure Legend Snippet: Oxidative stress in late sepsis modulates B cell differentiation following death of FDCs in septic mice. A. B cell differentiation into plasma cells is affected by oxidative stress induced FDC death. Spleen cells from sham-treated, LPS, LPS+allopurinol

Techniques Used: Cell Differentiation, Mouse Assay

9) Product Images from "Acute hepatocyte growth factor treatment induces long-term neuroprotection and stroke recovery via mechanisms involving neural precursor cell proliferation and differentiation"

Article Title: Acute hepatocyte growth factor treatment induces long-term neuroprotection and stroke recovery via mechanisms involving neural precursor cell proliferation and differentiation

Journal: Journal of Cerebral Blood Flow & Metabolism

doi: 10.1038/jcbfm.2010.211

Hepatocyte growth factor (HGF) promotes postischemic cell proliferation. Postischemic treatment with HGF resulted in increased cell proliferation as assessed by 5-bromo-2-deoxyuridine (BrdU) ( A ) and Ki-67 ( B ) staining. Cell proliferation was increased
Figure Legend Snippet: Hepatocyte growth factor (HGF) promotes postischemic cell proliferation. Postischemic treatment with HGF resulted in increased cell proliferation as assessed by 5-bromo-2-deoxyuridine (BrdU) ( A ) and Ki-67 ( B ) staining. Cell proliferation was increased

Techniques Used: Staining

10) Product Images from "Bone marrow-derived cells are the major source of MMP-9 contributing to blood-brain barrier dysfunction and infarct formation after ischemic stroke in mice"

Article Title: Bone marrow-derived cells are the major source of MMP-9 contributing to blood-brain barrier dysfunction and infarct formation after ischemic stroke in mice

Journal: Brain research

doi: 10.1016/j.brainres.2009.07.070

2.1. BMDC contribute to MMP-9 activity in ischemic brain
Figure Legend Snippet: 2.1. BMDC contribute to MMP-9 activity in ischemic brain

Techniques Used: Activity Assay

2.3. BMDC-derived MMP-9 contributes to brain tissue injury
Figure Legend Snippet: 2.3. BMDC-derived MMP-9 contributes to brain tissue injury

Techniques Used: Derivative Assay

MMP-9 from BMDC contributes to BBB dysfunction and infarct formation. Graphs showing relative protein levels of albumin in the brain ( A, B, and C ), Evans blue extravasation ( D ), and infarct size ( E, F ). The brains were collected from either naïve
Figure Legend Snippet: MMP-9 from BMDC contributes to BBB dysfunction and infarct formation. Graphs showing relative protein levels of albumin in the brain ( A, B, and C ), Evans blue extravasation ( D ), and infarct size ( E, F ). The brains were collected from either naïve

Techniques Used:

Four types of chimeric mice after irradiation (9 Gy) and bone marrow transplantation with various combinations of wild-type (WT) and MMP-9 knockout (KO) as donor and recipient.
Figure Legend Snippet: Four types of chimeric mice after irradiation (9 Gy) and bone marrow transplantation with various combinations of wild-type (WT) and MMP-9 knockout (KO) as donor and recipient.

Techniques Used: Mouse Assay, Irradiation, Transplantation Assay, Knock-Out

Zymograms showing gelatinase activity in brain ( A ), and spleen ( B ) from mice subjected to 2 h of MCAO followed by 22 h of reperfusion. Compared with irradiation to the head only, whole body irradiation markedly reduced MMP-9 activity in the spleen. Note
Figure Legend Snippet: Zymograms showing gelatinase activity in brain ( A ), and spleen ( B ) from mice subjected to 2 h of MCAO followed by 22 h of reperfusion. Compared with irradiation to the head only, whole body irradiation markedly reduced MMP-9 activity in the spleen. Note

Techniques Used: Activity Assay, Mouse Assay, Irradiation

Immunocytochemical detection of MMP-9 in nucleated blood cells from chimeric mice. Nucleated blood cells were counter stained with methyl green. Blood cells from WT/KO ( C ) are MMP-9 positive as seen in WT ( A ) whereas blood cells from KO ( B ) and KO/WT
Figure Legend Snippet: Immunocytochemical detection of MMP-9 in nucleated blood cells from chimeric mice. Nucleated blood cells were counter stained with methyl green. Blood cells from WT/KO ( C ) are MMP-9 positive as seen in WT ( A ) whereas blood cells from KO ( B ) and KO/WT

Techniques Used: Mouse Assay, Staining

Gel zymograms ( A–C ) and Western blot ( D ) showing MMP-9 in spleen and brain in mice that were subjected to 90 min MCAO followed by either 1 h ( A and B ) or 24 h ( C and D ) of reperfusion. Tissue homogenates were treated with gelatin Sepharose 4B
Figure Legend Snippet: Gel zymograms ( A–C ) and Western blot ( D ) showing MMP-9 in spleen and brain in mice that were subjected to 90 min MCAO followed by either 1 h ( A and B ) or 24 h ( C and D ) of reperfusion. Tissue homogenates were treated with gelatin Sepharose 4B

Techniques Used: Western Blot, Mouse Assay

2.1. BMDC contribute to MMP-9 activity in ischemic brain
Figure Legend Snippet: 2.1. BMDC contribute to MMP-9 activity in ischemic brain

Techniques Used: Activity Assay

Fluorescent photomicrograph showing in situ gelatinase activity in four types of chimeric animal. A and B, Radiation and bone marrow transplantation from WT to MMP-9 KO mice ( B ) restored gelatinase activity (green) comparable to WT/WT ( A ) in the brain
Figure Legend Snippet: Fluorescent photomicrograph showing in situ gelatinase activity in four types of chimeric animal. A and B, Radiation and bone marrow transplantation from WT to MMP-9 KO mice ( B ) restored gelatinase activity (green) comparable to WT/WT ( A ) in the brain

Techniques Used: In Situ, Activity Assay, Transplantation Assay, Mouse Assay

11) Product Images from "Subcutaneous Inoculation of 3D Pancreatic Cancer Spheroids Results in Development of Reproducible Stroma-Rich Tumors"

Article Title: Subcutaneous Inoculation of 3D Pancreatic Cancer Spheroids Results in Development of Reproducible Stroma-Rich Tumors

Journal: Translational Oncology

doi: 10.1016/j.tranon.2018.10.003

Characterization of different types of PANC-1 tumor xenografts. (A) Growth rates of tumor xenografts inoculated subcutaneously. Experimental data (dot scatters) were fitted with Gompertz functions (solid curves) of tumor growth. Number of animals is 4 per group. Data are shown as means ± SD. (B) Representative images of frozen tumor tissue sections stained with antibodies against different ECM components including collagen I (green), fibronectin (red), hyaluronic acid (purple), and laminin (yellow). All images have a size of 635 × 635 microns. (C) Quantitative estimation of fluorescent positive areas of ECM components in xenograft and patient PDAC tumors. Values are represented as means ± SD. Western blotting expression analysis of PDAC tumor hallmarks such as α-smooth muscle actin (α-SMA), transforming growth factor β (TGFβ), β1-integrin receptors, and activated AKT signaling pathway (pAKT) (D), (E).
Figure Legend Snippet: Characterization of different types of PANC-1 tumor xenografts. (A) Growth rates of tumor xenografts inoculated subcutaneously. Experimental data (dot scatters) were fitted with Gompertz functions (solid curves) of tumor growth. Number of animals is 4 per group. Data are shown as means ± SD. (B) Representative images of frozen tumor tissue sections stained with antibodies against different ECM components including collagen I (green), fibronectin (red), hyaluronic acid (purple), and laminin (yellow). All images have a size of 635 × 635 microns. (C) Quantitative estimation of fluorescent positive areas of ECM components in xenograft and patient PDAC tumors. Values are represented as means ± SD. Western blotting expression analysis of PDAC tumor hallmarks such as α-smooth muscle actin (α-SMA), transforming growth factor β (TGFβ), β1-integrin receptors, and activated AKT signaling pathway (pAKT) (D), (E).

Techniques Used: Staining, Western Blot, Expressing

Expression of ECM components in pancreatic cancer spheroids. (A) Visualization of collagen I in PANC-1 and PANC-1/NIH3T3 spheroids. Collagen I was defined in 10 μm-thick spheroid frozen section with anti-collagen I antibodies (green). Collagen I fibrils in heterospheroids are indicated with white arrows. (B) Visualization of fibronectin network (red) in PANC-1/NIH3T3 microtumors and its absence in PANC-1 spheroids. (C) Representative images of spheroid sections stained with antibodies against hyaluronan (purple) and laminin (yellow). Sections were obtained from spheroids incubated for 2 weeks in a presence or absence of 10 ng mL −1 human bFGF in growth medium. All spheroid images in (A), (B) and (C) have a size of 635 × 635 microns. (D) Positive fluorescence area of spheroid sections stained with antibodies to different ECM components. For quantification the positive area of binary images in fluorescent channel was normalized on total spheroid area. * P
Figure Legend Snippet: Expression of ECM components in pancreatic cancer spheroids. (A) Visualization of collagen I in PANC-1 and PANC-1/NIH3T3 spheroids. Collagen I was defined in 10 μm-thick spheroid frozen section with anti-collagen I antibodies (green). Collagen I fibrils in heterospheroids are indicated with white arrows. (B) Visualization of fibronectin network (red) in PANC-1/NIH3T3 microtumors and its absence in PANC-1 spheroids. (C) Representative images of spheroid sections stained with antibodies against hyaluronan (purple) and laminin (yellow). Sections were obtained from spheroids incubated for 2 weeks in a presence or absence of 10 ng mL −1 human bFGF in growth medium. All spheroid images in (A), (B) and (C) have a size of 635 × 635 microns. (D) Positive fluorescence area of spheroid sections stained with antibodies to different ECM components. For quantification the positive area of binary images in fluorescent channel was normalized on total spheroid area. * P

Techniques Used: Expressing, Staining, Incubation, Fluorescence

12) Product Images from "Adenylyl cyclase 1 as a major isoform to generate cAMP signaling for apoA-1-mediated cholesterol efflux pathway"

Article Title: Adenylyl cyclase 1 as a major isoform to generate cAMP signaling for apoA-1-mediated cholesterol efflux pathway

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M082297

Effects of AC1 shRNA interference on cAMP levels and cholesterol efflux. All macrophages except the control were loaded with ac-LDL cholesterol and incubated with apoA-1 for the experiment. A: AC1 mRNA levels determined by qRT-PCR. ***, P
Figure Legend Snippet: Effects of AC1 shRNA interference on cAMP levels and cholesterol efflux. All macrophages except the control were loaded with ac-LDL cholesterol and incubated with apoA-1 for the experiment. A: AC1 mRNA levels determined by qRT-PCR. ***, P

Techniques Used: shRNA, Incubation, Quantitative RT-PCR

AC4 shRNA interference on cAMP production and cholesterol efflux. All macrophages except the control were loaded with ac-LDL cholesterol and incubated with apoA-1 for the experiment. A: AC4 mRNA levels. The symbol *** represents P
Figure Legend Snippet: AC4 shRNA interference on cAMP production and cholesterol efflux. All macrophages except the control were loaded with ac-LDL cholesterol and incubated with apoA-1 for the experiment. A: AC4 mRNA levels. The symbol *** represents P

Techniques Used: shRNA, Incubation

Localization of cholesterol and apoA-1 in macrophages observed by confocal microscopy. Macrophages were prepared in glass cover slips, and all cells except the control were loaded with ac-LDL cholesterol for confocal microscopy study. Blue filipin fluorescence represents cellular cholesterol and red fluorescence indicates apoA-1 binding to the cell surface. Control panel: control macrophages. Ac-LDL panel: macrophages loaded with ac-LDL cholesterol. ApoA-1 panel: macrophages incubated with apoA-1. Red arrows indicate cholesterol and apoA-1 complexes (protrusions) on the cell surface in which cholesterol (blue fluorescence) was encircled by apoA-1 (red fluorescence). AC1 shRNA + apoA-1 panel: macrophages with AC1 shRNA plus apoA-1. ApoA-1 was localized on relatively smooth cell surface without the cholesterol complex. Scrambled shRNA + apoA-1: macrophages with scrambled shRNA plus apoA-1. Cholesterol and apoA-1 complex are visible on the cell surface. Scale bar represents 5 µm in all images. The experiments were repeated three times and representative images are shown. Fluorescence intensity panel: quantification of filipin fluorescence. Each bar represents mean of the corrected cell filipin fluorescence from 80 to 100 cells from the three separate experiments. ***, P
Figure Legend Snippet: Localization of cholesterol and apoA-1 in macrophages observed by confocal microscopy. Macrophages were prepared in glass cover slips, and all cells except the control were loaded with ac-LDL cholesterol for confocal microscopy study. Blue filipin fluorescence represents cellular cholesterol and red fluorescence indicates apoA-1 binding to the cell surface. Control panel: control macrophages. Ac-LDL panel: macrophages loaded with ac-LDL cholesterol. ApoA-1 panel: macrophages incubated with apoA-1. Red arrows indicate cholesterol and apoA-1 complexes (protrusions) on the cell surface in which cholesterol (blue fluorescence) was encircled by apoA-1 (red fluorescence). AC1 shRNA + apoA-1 panel: macrophages with AC1 shRNA plus apoA-1. ApoA-1 was localized on relatively smooth cell surface without the cholesterol complex. Scrambled shRNA + apoA-1: macrophages with scrambled shRNA plus apoA-1. Cholesterol and apoA-1 complex are visible on the cell surface. Scale bar represents 5 µm in all images. The experiments were repeated three times and representative images are shown. Fluorescence intensity panel: quantification of filipin fluorescence. Each bar represents mean of the corrected cell filipin fluorescence from 80 to 100 cells from the three separate experiments. ***, P

Techniques Used: Confocal Microscopy, Fluorescence, Binding Assay, Incubation, shRNA

13) Product Images from "Overactivation of Intestinal SREBP2 in Mice Increases Serum Cholesterol"

Article Title: Overactivation of Intestinal SREBP2 in Mice Increases Serum Cholesterol

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084221

Generation of intestine-specific active SREBP2 mice. The coding sequence for the N-terminal of SREBP2 (representing the active transcription factor) was cloned down-stream of villin promoter. A : A schematic representation of the transgene and the location of the G1 and G2 primers used for genotyping and the identification of positive transgenic mice (designated as ISR2) B : A representative of genotyping results showing the expected amplified PCR fragment from genomic DNA extracted from ISR2 mice (+) but not their wild type littermates (−).
Figure Legend Snippet: Generation of intestine-specific active SREBP2 mice. The coding sequence for the N-terminal of SREBP2 (representing the active transcription factor) was cloned down-stream of villin promoter. A : A schematic representation of the transgene and the location of the G1 and G2 primers used for genotyping and the identification of positive transgenic mice (designated as ISR2) B : A representative of genotyping results showing the expected amplified PCR fragment from genomic DNA extracted from ISR2 mice (+) but not their wild type littermates (−).

Techniques Used: Mouse Assay, Sequencing, Clone Assay, Transgenic Assay, Amplification, Polymerase Chain Reaction

Distribution of active SREBP2 in ISR2 mice. Total protein lysates were prepared form intestinal mucosal scraping as mentioned in Materials and Methods . A : A representative blot depicting the bands for active SREBP2 in ISR2 mice and their wild type littermates. Villin was used as a loading control. B : Villin staining (green) of the jejunum showing similar epithelial structure in ISR2 and wild type mice. C : immuno fluorescence staining of SREBP2 in jejunum of ISR2 and wild type mice. SREBP2 is stained with red and the nuclei with blue. The figure shows predominant cytoplasmic staining in wild type mice and increased colocalization of SREBP2 with the nuclei in ISR2 mice (white arrow).
Figure Legend Snippet: Distribution of active SREBP2 in ISR2 mice. Total protein lysates were prepared form intestinal mucosal scraping as mentioned in Materials and Methods . A : A representative blot depicting the bands for active SREBP2 in ISR2 mice and their wild type littermates. Villin was used as a loading control. B : Villin staining (green) of the jejunum showing similar epithelial structure in ISR2 and wild type mice. C : immuno fluorescence staining of SREBP2 in jejunum of ISR2 and wild type mice. SREBP2 is stained with red and the nuclei with blue. The figure shows predominant cytoplasmic staining in wild type mice and increased colocalization of SREBP2 with the nuclei in ISR2 mice (white arrow).

Techniques Used: Mouse Assay, Staining, Fluorescence

14) Product Images from "Mature oligodendrocytes actively increase in vivo cytoskeletal plasticity following CNS damage"

Article Title: Mature oligodendrocytes actively increase in vivo cytoskeletal plasticity following CNS damage

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-015-0271-2

The oLucR mouse model shows CNS-specific in vivo bioluminescence. (a) ODC-specific expression of luciferase is achieved by crossing a Cre-inducible luciferase reporter mouse (left) to the MOGi-cre strain. (b) EYFP + cells were sorted and stained with CC1-, GFP-, Iba1-, and PLP-specific antibodies following cytospin. Microglia/macrophage cells were excluded by positive CD45 and CD11b staining. Dead cells were excluded by Aqua Live/Dead staining reagent (Life Technologies). (c) Kinetics of photon emission acquired with an IVIS camera in anesthetized oLucR animals following intraperitoneal injection of 150 ng/kg of D-luciferin (mean ± SEM, n = 8). (d) In vivo bioluminescence recorded in a representative oLucR mouse (left) and a control LucR animal where the STOP codon impedes luciferase expression (right). Shown in red are the specific regions of interest (ROIs) for signal acquisition. (e) The CNS from oLucR mice of the indicated ages were homogenized and analyzed in a luminometer assay. Photon emission of the lysates is shown (mean ± SEM, n = 3). (f) oLucR mice were injected with luciferin every 3 days and bioluminescence recorded from specific brain and spinal cord ROIs over the course of 36 days (mean ± SEM, n = 4).
Figure Legend Snippet: The oLucR mouse model shows CNS-specific in vivo bioluminescence. (a) ODC-specific expression of luciferase is achieved by crossing a Cre-inducible luciferase reporter mouse (left) to the MOGi-cre strain. (b) EYFP + cells were sorted and stained with CC1-, GFP-, Iba1-, and PLP-specific antibodies following cytospin. Microglia/macrophage cells were excluded by positive CD45 and CD11b staining. Dead cells were excluded by Aqua Live/Dead staining reagent (Life Technologies). (c) Kinetics of photon emission acquired with an IVIS camera in anesthetized oLucR animals following intraperitoneal injection of 150 ng/kg of D-luciferin (mean ± SEM, n = 8). (d) In vivo bioluminescence recorded in a representative oLucR mouse (left) and a control LucR animal where the STOP codon impedes luciferase expression (right). Shown in red are the specific regions of interest (ROIs) for signal acquisition. (e) The CNS from oLucR mice of the indicated ages were homogenized and analyzed in a luminometer assay. Photon emission of the lysates is shown (mean ± SEM, n = 3). (f) oLucR mice were injected with luciferin every 3 days and bioluminescence recorded from specific brain and spinal cord ROIs over the course of 36 days (mean ± SEM, n = 4).

Techniques Used: In Vivo, Expressing, Luciferase, Staining, Plasmid Purification, Injection, Mouse Assay

15) Product Images from "Salvianolic acid A targeting the transgelin-actin complex to enhance vasoconstriction"

Article Title: Salvianolic acid A targeting the transgelin-actin complex to enhance vasoconstriction

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2018.10.041

SAA-30 promoted the contractility of vascular smooth muscle cells more than SAA and better achieved myocardial protection than SAA. (A) Overall scheme of muscular tension agonistic effects of SAA and SAA-30 on WT and transgelin (−/−) mice. (B) SAA and SAA-30 enhanced contractile capacities of vascular smooth muscle in isolated vascular smooth muscles of WT mice. (C) Diagram illustrating the generation of the MI model and the treatment schedule. (D) ST changes and (E) heart rate improvements observed in WT and transgelin (−/−) mice after IR injury with or without SAA and SAA-30 treatment. (F) Histopathological changes and (G) infraction sizes observed in MI-injured hearts of WT or transgelin (−/−) mice with or without SAA and SAA-30 administration. (H) Measurement of CK, CK-MB, AST and LDH levels in WT and transgelin (−/−) mice after IR injury with or without SAA and SAA-30. (I) A schematic diagram of SAA and SAA-30 treatments of myocardial ischemia. ⁎ P
Figure Legend Snippet: SAA-30 promoted the contractility of vascular smooth muscle cells more than SAA and better achieved myocardial protection than SAA. (A) Overall scheme of muscular tension agonistic effects of SAA and SAA-30 on WT and transgelin (−/−) mice. (B) SAA and SAA-30 enhanced contractile capacities of vascular smooth muscle in isolated vascular smooth muscles of WT mice. (C) Diagram illustrating the generation of the MI model and the treatment schedule. (D) ST changes and (E) heart rate improvements observed in WT and transgelin (−/−) mice after IR injury with or without SAA and SAA-30 treatment. (F) Histopathological changes and (G) infraction sizes observed in MI-injured hearts of WT or transgelin (−/−) mice with or without SAA and SAA-30 administration. (H) Measurement of CK, CK-MB, AST and LDH levels in WT and transgelin (−/−) mice after IR injury with or without SAA and SAA-30. (I) A schematic diagram of SAA and SAA-30 treatments of myocardial ischemia. ⁎ P

Techniques Used: Mouse Assay, Isolation, AST Assay

SAA and SAA-30 increased cell contractions in vitro. (A) Multi-angle light scattering results show that the aggregated state of actin was enhanced when transgelin was added. SAA and SAA-30 further promoted actin aggregation. (B) Negative staining electron microscopy analysis of actin, actin/transgelin, actin/transgelin/SAA and actin/transgelin/SAA-30 groups. The results show that transgelin enhanced actin polymerization, which was further markedly enhanced by SAA and SAA-30. SAA-30 was the most active. (C) Quantitative analysis of F-actin. The F-actin area, F-actin percentage area and total F-actin length were increased when transgelin was added. SAA and SAA-30 further promoted actin aggregation. (D) Accurate multidirectional co-localization analysis of SAA probes and of SAA-30 probe interactions with transgelin and actin via confocal and N-STROM. Green, SAA probe; Cyan, SAA-30 probe; Red, Actin; Blue, Transgelin. The results show the structural characteristics of SAA and SAA-30 bound to transgelin and actin. SAA and SAA-30 were distributed along with the transgelin-actin complex and promoted the uniform distribution of actin. (E F) SAA and SAA-30 promoted the co-localization of transgelin and actin and cell contractions. Actin is shown in red and transgelin is shown in green. (G) The SEM results show that SAA and SAA-30 enhanced HVSMC contractility levels. Cytoskeletons were clearly aggregated, and their adhesion capacities were enhanced. Cells were assayed after treatment with 20 μM SAA and SAA-30 for 24 h. (H) SAA and SAA-30 combatted inhibition by an actin inhibitor. The depolymerization capacities of actin inhibitor were antagonized by SAA and SAA-30. (I) SAA and SAA-30 limited hypoxia-induced actin degradation. Results were obtained from three independent experiments with each experiment performed in triplicate; N.S. denotes no significance, and error bars represent standard deviations ( ⁎ P
Figure Legend Snippet: SAA and SAA-30 increased cell contractions in vitro. (A) Multi-angle light scattering results show that the aggregated state of actin was enhanced when transgelin was added. SAA and SAA-30 further promoted actin aggregation. (B) Negative staining electron microscopy analysis of actin, actin/transgelin, actin/transgelin/SAA and actin/transgelin/SAA-30 groups. The results show that transgelin enhanced actin polymerization, which was further markedly enhanced by SAA and SAA-30. SAA-30 was the most active. (C) Quantitative analysis of F-actin. The F-actin area, F-actin percentage area and total F-actin length were increased when transgelin was added. SAA and SAA-30 further promoted actin aggregation. (D) Accurate multidirectional co-localization analysis of SAA probes and of SAA-30 probe interactions with transgelin and actin via confocal and N-STROM. Green, SAA probe; Cyan, SAA-30 probe; Red, Actin; Blue, Transgelin. The results show the structural characteristics of SAA and SAA-30 bound to transgelin and actin. SAA and SAA-30 were distributed along with the transgelin-actin complex and promoted the uniform distribution of actin. (E F) SAA and SAA-30 promoted the co-localization of transgelin and actin and cell contractions. Actin is shown in red and transgelin is shown in green. (G) The SEM results show that SAA and SAA-30 enhanced HVSMC contractility levels. Cytoskeletons were clearly aggregated, and their adhesion capacities were enhanced. Cells were assayed after treatment with 20 μM SAA and SAA-30 for 24 h. (H) SAA and SAA-30 combatted inhibition by an actin inhibitor. The depolymerization capacities of actin inhibitor were antagonized by SAA and SAA-30. (I) SAA and SAA-30 limited hypoxia-induced actin degradation. Results were obtained from three independent experiments with each experiment performed in triplicate; N.S. denotes no significance, and error bars represent standard deviations ( ⁎ P

Techniques Used: In Vitro, Negative Staining, Electron Microscopy, Inhibition

Chemical probe synthesis, target identification and validation of SAA. (A) Structure of the SAA probe. The alkyne reporter group is shown in red. (B) Evaluation of the capacity of the SAA probe to enhance contraction effects in HVSMCs. (C) Overall scheme of the SAA probe target-captured experiments for identifying SAA targets. (D) Molecular docking scores of SAA bonds to six potential targets. (E) The cotreatment of transgelin siRNA counteracted the effect of SAA in enhancing contraction effects in HVSMCs. (F) Biacore analysis of SAA bonds to transgelin. Concentrations of SAA were set to 0 μM, 1.5625 μM, 3.125 μM, 4.6875 μM, 6.25 μM, 12.5 μM, 18.75 μM and 25 μM. The equilibrium dissociation constant (KD) was set to 7.11 μM. (G) Microscale thermophoresis analysis of SAA bonds to transgelin. (H) Biacore analysis of SAA bonds to actin. (I) A diagram of the Biacore analysis on how SAA promotes the interaction of transgelin according to actin experiments. (J) Binding levels and stability analysis results show that protein binding signal values of transgelin and actin were increased when SAA was added. (K) N-STORM images of SAA bound to transgelin and actin. These results show that single molecules of SAA bound to transgelin and actin, and structural information for the image shows that SAA and transgelin were distributed along with F-actin. (L) F-actin features for the control and SAA-treated groups. N.S., not significant. For all data, N.S. denotes no significance, * P
Figure Legend Snippet: Chemical probe synthesis, target identification and validation of SAA. (A) Structure of the SAA probe. The alkyne reporter group is shown in red. (B) Evaluation of the capacity of the SAA probe to enhance contraction effects in HVSMCs. (C) Overall scheme of the SAA probe target-captured experiments for identifying SAA targets. (D) Molecular docking scores of SAA bonds to six potential targets. (E) The cotreatment of transgelin siRNA counteracted the effect of SAA in enhancing contraction effects in HVSMCs. (F) Biacore analysis of SAA bonds to transgelin. Concentrations of SAA were set to 0 μM, 1.5625 μM, 3.125 μM, 4.6875 μM, 6.25 μM, 12.5 μM, 18.75 μM and 25 μM. The equilibrium dissociation constant (KD) was set to 7.11 μM. (G) Microscale thermophoresis analysis of SAA bonds to transgelin. (H) Biacore analysis of SAA bonds to actin. (I) A diagram of the Biacore analysis on how SAA promotes the interaction of transgelin according to actin experiments. (J) Binding levels and stability analysis results show that protein binding signal values of transgelin and actin were increased when SAA was added. (K) N-STORM images of SAA bound to transgelin and actin. These results show that single molecules of SAA bound to transgelin and actin, and structural information for the image shows that SAA and transgelin were distributed along with F-actin. (L) F-actin features for the control and SAA-treated groups. N.S., not significant. For all data, N.S. denotes no significance, * P

Techniques Used: Microscale Thermophoresis, Binding Assay, Protein Binding

Transgelin truncation and Biacore assay verification of key amino acid residues that interacted with SAA. (A) The binding mode of SAA at the protein interaction interface of the transgelin and actin complex. The results show that SAA directly targets bonds and “ropes” the transgelin-actin complex. (B) Three constructs of transgelin truncations were prepared. Transgelin truncation domains were modeled using Pymol software. (C) The Biacore binding assays show that SAA binds T1 and T2 at comparable levels while SAA cannot bind T3.
Figure Legend Snippet: Transgelin truncation and Biacore assay verification of key amino acid residues that interacted with SAA. (A) The binding mode of SAA at the protein interaction interface of the transgelin and actin complex. The results show that SAA directly targets bonds and “ropes” the transgelin-actin complex. (B) Three constructs of transgelin truncations were prepared. Transgelin truncation domains were modeled using Pymol software. (C) The Biacore binding assays show that SAA binds T1 and T2 at comparable levels while SAA cannot bind T3.

Techniques Used: Binding Assay, Construct, Software

Comparison of SAA and SAA-30 structural and binding modes. (A) The structural energy levels of SAA-30 was lower than that of SAA. (B) Biacore results for SAA-30 bound to transgelin. SAA-30 was three times more active than SAA. (C) The binding mode of SAA and SAA-30 in the active site of the transgelin and actin complex. SAA-30 generated a higher docking score of −7.105 relative to that of the transgelin-actin complex. The green dotted line denotes the cation-π interaction. The black dotted line denotes hydrogen bond interactions, and the green solid line denotes hydrophobic interactions. (D) Table showing docking scores, binding energy levels, hydrogen bond interactions, cation-π interactions and hydrophobic interactions between SAA and SAA-30 and the transgelin-actin complex. SAA-30 shows lower binding energy levels in the transgelin-actin complex than in the SAA.
Figure Legend Snippet: Comparison of SAA and SAA-30 structural and binding modes. (A) The structural energy levels of SAA-30 was lower than that of SAA. (B) Biacore results for SAA-30 bound to transgelin. SAA-30 was three times more active than SAA. (C) The binding mode of SAA and SAA-30 in the active site of the transgelin and actin complex. SAA-30 generated a higher docking score of −7.105 relative to that of the transgelin-actin complex. The green dotted line denotes the cation-π interaction. The black dotted line denotes hydrogen bond interactions, and the green solid line denotes hydrophobic interactions. (D) Table showing docking scores, binding energy levels, hydrogen bond interactions, cation-π interactions and hydrophobic interactions between SAA and SAA-30 and the transgelin-actin complex. SAA-30 shows lower binding energy levels in the transgelin-actin complex than in the SAA.

Techniques Used: Binding Assay, Generated

16) Product Images from "A SNX10/V-ATPase pathway regulates ciliogenesis in vitro and in vivo"

Article Title: A SNX10/V-ATPase pathway regulates ciliogenesis in vitro and in vivo

Journal: Cell Research

doi: 10.1038/cr.2011.134

SNX10 regulates ciliogenesis in cultured cells. (A) Subcellular distribution of SNX10. RCC10/VHL cells are transfected with plasmid-encoding SNX10-GFP or SNX10-Flag plus Rab11-GFP. Cells are fixed 24 h after transfection and stained with the indicated endogenous markers: PCM-1 (pericentriolar material), Ninein or Pericentrin (centrosome). A fraction of SNX10 is detected around Rab11 (recycling endosome), PCM-1, Ninein or Pericentrin. In ciliated cells, SNX10 localizes to the base of cilia. (B) SNX10 is required for ciliogenesis in RCC10/VHL cells. Cells are transfected with a scramble control siRNA (siCTL), a positive control siRNA (siRab8a) or siRNAs to SNX10. Cilia are induced by serum starvation and visualized by immunofluorescence staining with the anti-acetylated α-tubulin antibody (red). Nuclei are counterstained with DAPI (blue). The percentages of cells with cilia are reduced upon treatment with siRNAs to SNX10. (C) Real-time RT-PCR analysis for the efficiency of siRNAs to Rab8a and SNX10. β-actin is used as the internal control. The expression level of Rab8a or SNX10 in the siCTL-treated cells is arbitrary set as 100%. More than 80% inhibition of the target gene at the mRNA level is achieved in each case. (D) Statistical analysis for B . Assays are repeated at least three times and at least 400 cells are counted for each treatment. Data represent mean ± SD from three independent experiments ( P
Figure Legend Snippet: SNX10 regulates ciliogenesis in cultured cells. (A) Subcellular distribution of SNX10. RCC10/VHL cells are transfected with plasmid-encoding SNX10-GFP or SNX10-Flag plus Rab11-GFP. Cells are fixed 24 h after transfection and stained with the indicated endogenous markers: PCM-1 (pericentriolar material), Ninein or Pericentrin (centrosome). A fraction of SNX10 is detected around Rab11 (recycling endosome), PCM-1, Ninein or Pericentrin. In ciliated cells, SNX10 localizes to the base of cilia. (B) SNX10 is required for ciliogenesis in RCC10/VHL cells. Cells are transfected with a scramble control siRNA (siCTL), a positive control siRNA (siRab8a) or siRNAs to SNX10. Cilia are induced by serum starvation and visualized by immunofluorescence staining with the anti-acetylated α-tubulin antibody (red). Nuclei are counterstained with DAPI (blue). The percentages of cells with cilia are reduced upon treatment with siRNAs to SNX10. (C) Real-time RT-PCR analysis for the efficiency of siRNAs to Rab8a and SNX10. β-actin is used as the internal control. The expression level of Rab8a or SNX10 in the siCTL-treated cells is arbitrary set as 100%. More than 80% inhibition of the target gene at the mRNA level is achieved in each case. (D) Statistical analysis for B . Assays are repeated at least three times and at least 400 cells are counted for each treatment. Data represent mean ± SD from three independent experiments ( P

Techniques Used: Cell Culture, Transfection, Plasmid Preparation, Staining, Positive Control, Immunofluorescence, Quantitative RT-PCR, Expressing, Inhibition

17) Product Images from "Propofol produces preventive analgesia via GluN2B-containing NMDA receptor/ERK1/2 signaling pathway in a rat model of inflammatory pain"

Article Title: Propofol produces preventive analgesia via GluN2B-containing NMDA receptor/ERK1/2 signaling pathway in a rat model of inflammatory pain

Journal: Molecular Pain

doi: 10.1177/1744806917737462

Distribution and co-localization of c-Fos-positive and p-ERK-positive-labeled neurons in the spinal cord dorsal horn following different treatment groups, including Naive group without any treatment (Naïve), formalin treatment group with formalin injection only (Formalin), and formalin injection with pretreatment of propofol followed by a recovery time of 30 min (P-30 min) or 2 h (P-2 h). (a) Confocal fluorescence images illustrate the co-localization of c-Fos-positive cells (red), NeuN (green), and DAPI (blue) in the different treatment groups. (b) Confocal fluorescence images illustrate the co-localization of p-ERK-positive cells (red), NeuN (green), and DAPI (blue) in the different treatment groups. Dotted lines show the estimated edges of laminae I. Scale bar: 50µm. The column figures on the right panel show the summary data following different treatments. * P
Figure Legend Snippet: Distribution and co-localization of c-Fos-positive and p-ERK-positive-labeled neurons in the spinal cord dorsal horn following different treatment groups, including Naive group without any treatment (Naïve), formalin treatment group with formalin injection only (Formalin), and formalin injection with pretreatment of propofol followed by a recovery time of 30 min (P-30 min) or 2 h (P-2 h). (a) Confocal fluorescence images illustrate the co-localization of c-Fos-positive cells (red), NeuN (green), and DAPI (blue) in the different treatment groups. (b) Confocal fluorescence images illustrate the co-localization of p-ERK-positive cells (red), NeuN (green), and DAPI (blue) in the different treatment groups. Dotted lines show the estimated edges of laminae I. Scale bar: 50µm. The column figures on the right panel show the summary data following different treatments. * P

Techniques Used: Labeling, Injection, Fluorescence

18) Product Images from "Lateral cortical Cdca7 expression levels are regulated by Pax6 and influence the production of intermediate progenitors"

Article Title: Lateral cortical Cdca7 expression levels are regulated by Pax6 and influence the production of intermediate progenitors

Journal: BMC Neuroscience

doi: 10.1186/s12868-017-0365-0

Effects of forced expression of Cdca7 in vivo. A Schematic showing the expression patterns of Pax6 (in radial glial cells), Tbr2 (in intermediate progenitor cells) and Tbr1 (in postmitotic neurons). Area in box is shown in E – G . B C dca7 expression vector pCAGGS_Cdca7 containing Cdca7 open reading frame. C Cdca7 expression plasmid was transiently transfected into HEK293 cells. Cell lysates were collected 48 h post transfection and Cdca7 expression was verified using an antibody against the HA epitope. The molecular weight of Cdca7 was approximately 43 kDa. GAPDH was used as a loading control. D Control (pCAGGS_GFP) or Cdca7 expression vector was used for in utero electroporation at E12.5 or E14.5. BrdU was injected 30 min before termination. E – J Immunofluorescence images show the expression of GFP and Cdca7 in the electroporated lateral cortex. H – J Almost all GFP+ cells co-express Cdca7 at levels obviously higher than endogenous. Outlines of GFP+ cells are overlaid on Cdca7 cells in J . White arrows indicate a cell with strong GFP but Cdca7 levels that are not obviously raised above endogenous. Red arrows indicate a cell with a low GFP level but a high Cdca7 level. Scale bars E – G 200 µm; H – J 50 µm
Figure Legend Snippet: Effects of forced expression of Cdca7 in vivo. A Schematic showing the expression patterns of Pax6 (in radial glial cells), Tbr2 (in intermediate progenitor cells) and Tbr1 (in postmitotic neurons). Area in box is shown in E – G . B C dca7 expression vector pCAGGS_Cdca7 containing Cdca7 open reading frame. C Cdca7 expression plasmid was transiently transfected into HEK293 cells. Cell lysates were collected 48 h post transfection and Cdca7 expression was verified using an antibody against the HA epitope. The molecular weight of Cdca7 was approximately 43 kDa. GAPDH was used as a loading control. D Control (pCAGGS_GFP) or Cdca7 expression vector was used for in utero electroporation at E12.5 or E14.5. BrdU was injected 30 min before termination. E – J Immunofluorescence images show the expression of GFP and Cdca7 in the electroporated lateral cortex. H – J Almost all GFP+ cells co-express Cdca7 at levels obviously higher than endogenous. Outlines of GFP+ cells are overlaid on Cdca7 cells in J . White arrows indicate a cell with strong GFP but Cdca7 levels that are not obviously raised above endogenous. Red arrows indicate a cell with a low GFP level but a high Cdca7 level. Scale bars E – G 200 µm; H – J 50 µm

Techniques Used: Expressing, In Vivo, Plasmid Preparation, Transfection, Molecular Weight, In Utero, Electroporation, Injection, Immunofluorescence

The distribution of electroporated cells with raised Cdca7 levels in lateral cortex is not different from control. A GFP+ ( green ) and Tbr2+ ( red ) cells were counted in ten 250 µm-wide bins of equal depth from ventricular zone to pia (counts were from three E12.5+16h embryos electroporated with control and three E12.5+16h embryos electroporated with Cdca7-expressing constructs). B , C Average proportions of GFP+ and Tbr2+ cells in each bin are plotted. The average depth of GFP+ cells on the scale of 1–10 was 4.95 ± 0.42 (SEM) in control and 4.78 ± 0.31 in experimental animals (ns, p = 0.76, Student’s t-test). The average depth of Tbr2+ cells was 5.48 ± 0.39 in control and 5.23 ± 0.22 in experimental animals (ns, p = 0.60, Student’s t-test)
Figure Legend Snippet: The distribution of electroporated cells with raised Cdca7 levels in lateral cortex is not different from control. A GFP+ ( green ) and Tbr2+ ( red ) cells were counted in ten 250 µm-wide bins of equal depth from ventricular zone to pia (counts were from three E12.5+16h embryos electroporated with control and three E12.5+16h embryos electroporated with Cdca7-expressing constructs). B , C Average proportions of GFP+ and Tbr2+ cells in each bin are plotted. The average depth of GFP+ cells on the scale of 1–10 was 4.95 ± 0.42 (SEM) in control and 4.78 ± 0.31 in experimental animals (ns, p = 0.76, Student’s t-test). The average depth of Tbr2+ cells was 5.48 ± 0.39 in control and 5.23 ± 0.22 in experimental animals (ns, p = 0.60, Student’s t-test)

Techniques Used: Expressing, Construct

Raising Cdca7 levels in E12.5 lateral cortex affects the production of Tbr2- and Tbr1-expressing cells. Brains electroporated with either the control or Cdca7 plasmid at A – D , K – N E12.5 and F – I E14.5 reacted for A – D , F – I Tbr2 or K – N Tbr1. B ′, D ′, G ′, I ′, L ′, N ′ High magnification of the areas outlined in B , D , G , I , L , N . Arrows point to cells co-labelled with Tbr2/Tbr1 and GFP and B ″, G ″ and L ″ show magnified examples. E The frequencies of Tbr2+ cells in the population of GFP+ cells after E12.5 electroporation (mean ± SEM; control, n = 4: Cdca7, n = 3): the proportion in the Cdca7 group is significantly lower than that in the control group (p
Figure Legend Snippet: Raising Cdca7 levels in E12.5 lateral cortex affects the production of Tbr2- and Tbr1-expressing cells. Brains electroporated with either the control or Cdca7 plasmid at A – D , K – N E12.5 and F – I E14.5 reacted for A – D , F – I Tbr2 or K – N Tbr1. B ′, D ′, G ′, I ′, L ′, N ′ High magnification of the areas outlined in B , D , G , I , L , N . Arrows point to cells co-labelled with Tbr2/Tbr1 and GFP and B ″, G ″ and L ″ show magnified examples. E The frequencies of Tbr2+ cells in the population of GFP+ cells after E12.5 electroporation (mean ± SEM; control, n = 4: Cdca7, n = 3): the proportion in the Cdca7 group is significantly lower than that in the control group (p

Techniques Used: Expressing, Plasmid Preparation, Electroporation

19) Product Images from "The Polarity Protein Scribble Regulates Myelination and Remyelination in the Central Nervous System"

Article Title: The Polarity Protein Scribble Regulates Myelination and Remyelination in the Central Nervous System

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1002107

Conditional elimination of Scribble expression in oligodendrocytes disrupts paranodal axo-glial junctions and axonal domain organisation at the node of Ranvier. A–F; In P40 Scribble cKO mice (C), paranodal axo-glial junctions in spinal cord or optic nerve (not shown) displayed an increased proportion of loops that had disengaged from the axonal surface (white arrows) relative to those in the CNS of wild-type littermates (A). This is quantified in B; WT ON: 0.8% ± 0.4%, cKO ON: 10.6% ± 1.8%, WT SC: 12.7% ± 2.4%, cKO SC: 24.1% ± 4.3%. Of the paranodal loops that faced the axolemma, a significantly increased proportion found in Scribble cKO mice were either linked to the axonal membrane by disordered electron density (F, arrow) instead of the ordered transverse bands seen in WT animals (E), or lacked transverse bands entirely (F, arrowhead). This is quantified in D; WT ON: 14.4% ± 1.7%, cKO ON: 53.4% ± 3.9%, WT SC: 19.9% ± 1.6%, cKO SC: 40.7% ± 3.5%. Results were presented as mean ± SEM. Student's t test was used. At least 20 nodes of Ranvier were analysed from each of three animals per genotype. G–I; Teased fibre preparations from P40 ventral spinal cord were used to assess myelin domain organisation at nodes of Ranvier in Scribble cKO mice and wild-type littermates. In Scribble cKO nerves, voltage-gated potassium channels normally localised to the juxtaparanode (Kv1.1, green) invade the paranode (J, arrowheads), encroaching on voltage-gated sodium channels at the node of Ranvier (Nav, red), while normal spacing is maintained in wild-type nerves (H), resulting in significantly reduced distances between sodium and potassium channel labelled regions (G; WT: 2.47 ± 0.09 μm, cKO: 1.13 ± 0.15 μm). This is due at least in part to widening of the Kv1.1-immunolabelled domain (I; WT: 18.0 ± 0.6 μm, cKO: 22.4 ± 1.3 μm). Results were presented as mean ± SEM. Student's t test was used. At least 30 nodes of Ranvier were analysed from each of four animals per genotype. * p
Figure Legend Snippet: Conditional elimination of Scribble expression in oligodendrocytes disrupts paranodal axo-glial junctions and axonal domain organisation at the node of Ranvier. A–F; In P40 Scribble cKO mice (C), paranodal axo-glial junctions in spinal cord or optic nerve (not shown) displayed an increased proportion of loops that had disengaged from the axonal surface (white arrows) relative to those in the CNS of wild-type littermates (A). This is quantified in B; WT ON: 0.8% ± 0.4%, cKO ON: 10.6% ± 1.8%, WT SC: 12.7% ± 2.4%, cKO SC: 24.1% ± 4.3%. Of the paranodal loops that faced the axolemma, a significantly increased proportion found in Scribble cKO mice were either linked to the axonal membrane by disordered electron density (F, arrow) instead of the ordered transverse bands seen in WT animals (E), or lacked transverse bands entirely (F, arrowhead). This is quantified in D; WT ON: 14.4% ± 1.7%, cKO ON: 53.4% ± 3.9%, WT SC: 19.9% ± 1.6%, cKO SC: 40.7% ± 3.5%. Results were presented as mean ± SEM. Student's t test was used. At least 20 nodes of Ranvier were analysed from each of three animals per genotype. G–I; Teased fibre preparations from P40 ventral spinal cord were used to assess myelin domain organisation at nodes of Ranvier in Scribble cKO mice and wild-type littermates. In Scribble cKO nerves, voltage-gated potassium channels normally localised to the juxtaparanode (Kv1.1, green) invade the paranode (J, arrowheads), encroaching on voltage-gated sodium channels at the node of Ranvier (Nav, red), while normal spacing is maintained in wild-type nerves (H), resulting in significantly reduced distances between sodium and potassium channel labelled regions (G; WT: 2.47 ± 0.09 μm, cKO: 1.13 ± 0.15 μm). This is due at least in part to widening of the Kv1.1-immunolabelled domain (I; WT: 18.0 ± 0.6 μm, cKO: 22.4 ± 1.3 μm). Results were presented as mean ± SEM. Student's t test was used. At least 30 nodes of Ranvier were analysed from each of four animals per genotype. * p

Techniques Used: Expressing, Mouse Assay

20) Product Images from "Therapeutic effect and mechanism of electroacupuncture at Zusanli on plasticity of interstitial cells of Cajal: a study of rat ileum"

Article Title: Therapeutic effect and mechanism of electroacupuncture at Zusanli on plasticity of interstitial cells of Cajal: a study of rat ileum

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/1472-6882-14-186

Confocal images of ICC oral to the clip labeled with DOG1 on frozen sections (A, C, E, G, I) and on whole-mount preparations (B, D, F, H, J) showing the alterations of distribution and morphology in control (A, B), sham-operated (C, D), operated without EA treatment group (E, F), operated and EA at Zusanli (G, H), operated and EA treatment at Yinglingquan (I, J). In the N and S group, DOG1-like immunoreactivity (DOG1-LI) was localized in two distinct populations of ICC (ICC-MY and ICC-DMP) within the tunica muscularis and DOG1-LI ICCs were normally present with famose processes and intact cellular networks (A-D) . In the O-N group, the density of DOG1-LI ICCs were significantly reduced, the distribution was not continuous, and ICC networks were partly damaged (E, F) . In the O-EZ group, an almost intact cellular networks could be observed and DOG1+ cell numbers had recovered to control values (G, H) . In the O-EY group, the DOG1 + cell density was still with the same to the O-N group (I, J) . Comparison between groups in positive cell counts (K) . N group: Normal group; S group: Sham-operated group; O-N group: operated without EA treatment group; O-EZ group: operated and EA treatment at ST36 Zusanli point group; O-EY group: operated and EA treatment at SP9 Yinglingquan point group. *represents significant difference from N group, † represents significant difference from S group. ‡ represents significant difference from O-N group. § represents significant difference from O-EZ group.
Figure Legend Snippet: Confocal images of ICC oral to the clip labeled with DOG1 on frozen sections (A, C, E, G, I) and on whole-mount preparations (B, D, F, H, J) showing the alterations of distribution and morphology in control (A, B), sham-operated (C, D), operated without EA treatment group (E, F), operated and EA at Zusanli (G, H), operated and EA treatment at Yinglingquan (I, J). In the N and S group, DOG1-like immunoreactivity (DOG1-LI) was localized in two distinct populations of ICC (ICC-MY and ICC-DMP) within the tunica muscularis and DOG1-LI ICCs were normally present with famose processes and intact cellular networks (A-D) . In the O-N group, the density of DOG1-LI ICCs were significantly reduced, the distribution was not continuous, and ICC networks were partly damaged (E, F) . In the O-EZ group, an almost intact cellular networks could be observed and DOG1+ cell numbers had recovered to control values (G, H) . In the O-EY group, the DOG1 + cell density was still with the same to the O-N group (I, J) . Comparison between groups in positive cell counts (K) . N group: Normal group; S group: Sham-operated group; O-N group: operated without EA treatment group; O-EZ group: operated and EA treatment at ST36 Zusanli point group; O-EY group: operated and EA treatment at SP9 Yinglingquan point group. *represents significant difference from N group, † represents significant difference from S group. ‡ represents significant difference from O-N group. § represents significant difference from O-EZ group.

Techniques Used: Immunocytochemistry, Cross-linking Immunoprecipitation, Labeling

Confocal images showing the ICCs in the rat colon immunolabeled for DOG1. (A-D) on frozen sections, (E-H) on whole-mount preparations, (A, E) : normal small intestine, (B, F) : oral to the occlusion clip, (C, G) : beneath the occlusion clip, (D, H) : aboral to the occlusion clip. In the normal small intestine, DOG1-like immunoreactivity (DOG1-LI) was localized in two distinct populations of ICC (ICC-MY and ICC-DMP) within the tunica muscularis and DOG1-LI ICCs were normally present with famose processes and intact cellular networks (A, E) . Beneath and oral to the clip, the density of DOG1-LI ICCs were significantly reduced, the distribution was not continuous, and ICC networks were partly damaged (B, C, F, G) . Aboral to the clip, ICC networks and function were essentially normal (D, H) .
Figure Legend Snippet: Confocal images showing the ICCs in the rat colon immunolabeled for DOG1. (A-D) on frozen sections, (E-H) on whole-mount preparations, (A, E) : normal small intestine, (B, F) : oral to the occlusion clip, (C, G) : beneath the occlusion clip, (D, H) : aboral to the occlusion clip. In the normal small intestine, DOG1-like immunoreactivity (DOG1-LI) was localized in two distinct populations of ICC (ICC-MY and ICC-DMP) within the tunica muscularis and DOG1-LI ICCs were normally present with famose processes and intact cellular networks (A, E) . Beneath and oral to the clip, the density of DOG1-LI ICCs were significantly reduced, the distribution was not continuous, and ICC networks were partly damaged (B, C, F, G) . Aboral to the clip, ICC networks and function were essentially normal (D, H) .

Techniques Used: Immunolabeling, Cross-linking Immunoprecipitation, Immunocytochemistry

21) Product Images from "Functional characterization of the interactions between endosomal adaptor protein APPL1 and the NuRD co-repressor complex"

Article Title: Functional characterization of the interactions between endosomal adaptor protein APPL1 and the NuRD co-repressor complex

Journal: Biochemical Journal

doi: 10.1042/BJ20090086

APPL1 interacts with the NuRD subunits in both cytoplasmic and nuclear fractions independently of HDAC enzymatic activity ( A ) Cytoplasmic (C) and nuclear (N) fractions along with total extracts (T) of three different cell lines, HeLa, HEK-293 and A431, were analysed for the presence of several NuRD subunits by Western blotting with different antibodies as indicated. For detection with a given antibody, equal amounts of proteins from all fractions and three cell lines were loaded (20 μg of protein for blotting with anti-p66α/β, -MBD2/3 and -EEA1 antibodies; 15 μg of protein for anti-APPL1, -HDAC2, -RbAp46 and -GAPDH; 10 μg of protein for anti-MTA2, -HDAC1, -RbAp48 and -Histone H3; the different amounts of protein loaded were chosen to match different sensitivities of the antibodies used). Cytoplasmic (GAPDH and EEA1) and nuclear (histone H3) markers were used to demonstrate the purity of fractions. ( B ) HeLa cells were transfected for 72 h with two oligonucleotides (a, b) against HDAC2 or with non-specific control oligonucleotide (Φ). Cytoplasmic and nuclear fractions were prepared and subjected to immunoprecipitation (IP) using anti-APPL1 antibody or non-specific immunoglobulins (IgG). Immunoprecipitates were tested for the presence of several NuRD subunits by immunoblotting with various antibodies as indicated. Right panel: 10% of the input material (cytoplasmic and nuclear fractions) were analysed for the knockdown efficiency using anti-HDAC1 and anti-HDAC2 antibodies, as well as for the fraction purity with anti-GAPDH and anti-histone H3 antibodies. ( C ) Immunoprecipitation from cytoplasmic (C) or nuclear (N) fractions of HeLa cells treated for 20 h with 100 ng/ml of TSA (left panel) or 25 mM sodium butyrate (BUT; right panel) was performed using anti-APPL1, anti-MTA2 or non-specific rabbit IgG. Precipitates along with 10% of the input material were analysed by Western blotting using different antibodies, as indicated. Ctr, control.
Figure Legend Snippet: APPL1 interacts with the NuRD subunits in both cytoplasmic and nuclear fractions independently of HDAC enzymatic activity ( A ) Cytoplasmic (C) and nuclear (N) fractions along with total extracts (T) of three different cell lines, HeLa, HEK-293 and A431, were analysed for the presence of several NuRD subunits by Western blotting with different antibodies as indicated. For detection with a given antibody, equal amounts of proteins from all fractions and three cell lines were loaded (20 μg of protein for blotting with anti-p66α/β, -MBD2/3 and -EEA1 antibodies; 15 μg of protein for anti-APPL1, -HDAC2, -RbAp46 and -GAPDH; 10 μg of protein for anti-MTA2, -HDAC1, -RbAp48 and -Histone H3; the different amounts of protein loaded were chosen to match different sensitivities of the antibodies used). Cytoplasmic (GAPDH and EEA1) and nuclear (histone H3) markers were used to demonstrate the purity of fractions. ( B ) HeLa cells were transfected for 72 h with two oligonucleotides (a, b) against HDAC2 or with non-specific control oligonucleotide (Φ). Cytoplasmic and nuclear fractions were prepared and subjected to immunoprecipitation (IP) using anti-APPL1 antibody or non-specific immunoglobulins (IgG). Immunoprecipitates were tested for the presence of several NuRD subunits by immunoblotting with various antibodies as indicated. Right panel: 10% of the input material (cytoplasmic and nuclear fractions) were analysed for the knockdown efficiency using anti-HDAC1 and anti-HDAC2 antibodies, as well as for the fraction purity with anti-GAPDH and anti-histone H3 antibodies. ( C ) Immunoprecipitation from cytoplasmic (C) or nuclear (N) fractions of HeLa cells treated for 20 h with 100 ng/ml of TSA (left panel) or 25 mM sodium butyrate (BUT; right panel) was performed using anti-APPL1, anti-MTA2 or non-specific rabbit IgG. Precipitates along with 10% of the input material were analysed by Western blotting using different antibodies, as indicated. Ctr, control.

Techniques Used: Activity Assay, Western Blot, Transfection, Immunoprecipitation

APPL1 overexpression affects the composition of HDAC1-containing NuRD complex and the expression of HDAC1 target p21 WAF1/CIP ( A ) APPL1 overexpression impairs the interactions of HDAC1 with other NuRD subunits. HEK-293 cells overexpressing untagged APPL1 at moderate or high levels (m.o., moderate overexpression of pAPPL1; h.o., high overexpression of pAPPL1) were subjected to immunoprecipitation (IP) with anti-HDAC1 or non-specific rabbit (IgG) antibodies. Immunoprecipitates along with 10% of the extracts used (input, right panel) were tested by immunoblotting for the presence of several NuRD subunits, as indicated. Some non-specific binding of APPL1 to IgG-covered Protein G beads is marked with an asterisk. ( B ) APPL1 overexpression reduces the association of HDAC1 with other NuRD components in the nuclear fraction. HDAC1 was immunoprecipitated from the nuclear extracts of HEK-293 cells with endogenous (vector) or overexpressed APPL1. Immunoprecipitates and 5% of the starting material (input, right panel) were blotted for the presence of the indicated NuRD components. ( C ) APPL1 influences the level of HDAC1 target gene product p21 WAF1/CIP1 . The level of p21 WAF1/CIP1 expression was analysed by Western blotting using anti-p21 antibody in extracts of cells with overexpression or silenced expression of APPL1. Left panel: Extracts of HEK-293 cells overexpressing APPL1 (either untagged, pAPPL1, or MYC-tagged, pAPPL1–MYC) or transfected with a control vector for 48 h were immunoblotted as indicated. No efficient overexpression of APPL1 could be achieved in HeLa cells. Right panel: APPL1 expression was reduced by esiRNA against APPL1 in HEK-293 or HeLa cells, using esiRNA against luciferase (luc) as a specificity control. Transfections with esiRNA were performed for 72 h (HEK-293) or 48 h (HeLa). The resulting extracts were immunoblotted against APPL1, Myc and p21. GAPDH was included as a loading control.
Figure Legend Snippet: APPL1 overexpression affects the composition of HDAC1-containing NuRD complex and the expression of HDAC1 target p21 WAF1/CIP ( A ) APPL1 overexpression impairs the interactions of HDAC1 with other NuRD subunits. HEK-293 cells overexpressing untagged APPL1 at moderate or high levels (m.o., moderate overexpression of pAPPL1; h.o., high overexpression of pAPPL1) were subjected to immunoprecipitation (IP) with anti-HDAC1 or non-specific rabbit (IgG) antibodies. Immunoprecipitates along with 10% of the extracts used (input, right panel) were tested by immunoblotting for the presence of several NuRD subunits, as indicated. Some non-specific binding of APPL1 to IgG-covered Protein G beads is marked with an asterisk. ( B ) APPL1 overexpression reduces the association of HDAC1 with other NuRD components in the nuclear fraction. HDAC1 was immunoprecipitated from the nuclear extracts of HEK-293 cells with endogenous (vector) or overexpressed APPL1. Immunoprecipitates and 5% of the starting material (input, right panel) were blotted for the presence of the indicated NuRD components. ( C ) APPL1 influences the level of HDAC1 target gene product p21 WAF1/CIP1 . The level of p21 WAF1/CIP1 expression was analysed by Western blotting using anti-p21 antibody in extracts of cells with overexpression or silenced expression of APPL1. Left panel: Extracts of HEK-293 cells overexpressing APPL1 (either untagged, pAPPL1, or MYC-tagged, pAPPL1–MYC) or transfected with a control vector for 48 h were immunoblotted as indicated. No efficient overexpression of APPL1 could be achieved in HeLa cells. Right panel: APPL1 expression was reduced by esiRNA against APPL1 in HEK-293 or HeLa cells, using esiRNA against luciferase (luc) as a specificity control. Transfections with esiRNA were performed for 72 h (HEK-293) or 48 h (HeLa). The resulting extracts were immunoblotted against APPL1, Myc and p21. GAPDH was included as a loading control.

Techniques Used: Over Expression, Expressing, Immunoprecipitation, Binding Assay, Plasmid Preparation, Western Blot, Transfection, esiRNA, Luciferase

HDAC2 is critical for binding of APPL1 to the NuRD complex ( A and B ) Extracts from HeLa cells transfected for 72 h with two (a, b) or three (a, b, c) different siRNA oligonucleotides per gene against: HDAC1, HDAC2, MTA2, RbAp48 and RbAp46 or non-specific siRNA (Φ) were subjected to immunoprecipitation (IP) using: ( A ) anti-APPL1 antibody; ( B ) anti-HDAC1 antibody (left panel) or anti-MTA2 antibody (right panel). Non-specific antibodies (IgG) were used as controls. Input indicates 10% of total cell extracts used for immunoprecipitation. Immunoprecipitates and input extracts were analysed by Western blotting using different antibodies as indicated. ( C ) To verify the direct interactions between APPL1 and HDAC1 or HDAC2, in vitro translated HDAC1–FLAG and untagged HDAC2 were subjected to GST pull-down assay using GST alone (Φ) or GST fused to the N- or C-terminal parts of APPL1 (APPL1-N or APPL1-C, respectively). Input indicates 10% of in vitro translated material used for the pull-down assay. Bound proteins were analysed by Western blotting using anti-HDAC1 and anti-HDAC2 antibodies. ND, not determined.
Figure Legend Snippet: HDAC2 is critical for binding of APPL1 to the NuRD complex ( A and B ) Extracts from HeLa cells transfected for 72 h with two (a, b) or three (a, b, c) different siRNA oligonucleotides per gene against: HDAC1, HDAC2, MTA2, RbAp48 and RbAp46 or non-specific siRNA (Φ) were subjected to immunoprecipitation (IP) using: ( A ) anti-APPL1 antibody; ( B ) anti-HDAC1 antibody (left panel) or anti-MTA2 antibody (right panel). Non-specific antibodies (IgG) were used as controls. Input indicates 10% of total cell extracts used for immunoprecipitation. Immunoprecipitates and input extracts were analysed by Western blotting using different antibodies as indicated. ( C ) To verify the direct interactions between APPL1 and HDAC1 or HDAC2, in vitro translated HDAC1–FLAG and untagged HDAC2 were subjected to GST pull-down assay using GST alone (Φ) or GST fused to the N- or C-terminal parts of APPL1 (APPL1-N or APPL1-C, respectively). Input indicates 10% of in vitro translated material used for the pull-down assay. Bound proteins were analysed by Western blotting using anti-HDAC1 and anti-HDAC2 antibodies. ND, not determined.

Techniques Used: Binding Assay, Transfection, Immunoprecipitation, Western Blot, In Vitro, Pull Down Assay

The interactions with NuRD affect cellular distribution of APPL1 ( A ) APPL1 protein levels do not depend on HDAC1 or HDAC2. Extracts of HeLa cells with reduced HDAC1 or HDAC2 levels by siRNA [two different oligonucleotides (a, b) per gene or non-specific oligo (Φ), transfected for 72 h; top panel] and HEK-293 cells transfected for 48 h with plasmids encoding FLAG-tagged HDAC1 (pHDAC1–FLAG), HDAC2 (pHDAC2–FLAG) or with a control vector (bottom panel), were analysed by Western blotting using anti-APPL1 antibodies. To demonstrate the efficiency of silencing or overexpression, extracts were probed using anti-HDAC1 and anti-HDAC2 antibodies. GAPDH was included as a loading control. ( B ) Microscopy-based analysis of APPL1 nuclear localization upon silencing of HDAC1 or HDAC2. HeLa cells were transfected with siRNA oligonucleotides: one non-specific (Φ) and two specific per gene (a and b) against HDAC1 and HDAC2 for 72 h, followed by fixation and immunostaining for APPL1. Acquired microscopic images were analysed by Metamorph software and the average pixel intensities corresponding to APPL1 in the nuclei (as visualized by Hoechst staining, not shown) were calculated. The results of a representative experiment are shown in the graph. The values are normalized with respect to the average pixel intensity of nuclear APPL1 in cells transfected with non-specific siRNA, assigned one arbitrary unit. Error bars indicate standard error (minimum 100 cells from each transfection were used for the analysis). The results were statistically analysed by GraphPad Prism4 software, and the values obtained for each knockdown experiment were significantly different from the control at P
Figure Legend Snippet: The interactions with NuRD affect cellular distribution of APPL1 ( A ) APPL1 protein levels do not depend on HDAC1 or HDAC2. Extracts of HeLa cells with reduced HDAC1 or HDAC2 levels by siRNA [two different oligonucleotides (a, b) per gene or non-specific oligo (Φ), transfected for 72 h; top panel] and HEK-293 cells transfected for 48 h with plasmids encoding FLAG-tagged HDAC1 (pHDAC1–FLAG), HDAC2 (pHDAC2–FLAG) or with a control vector (bottom panel), were analysed by Western blotting using anti-APPL1 antibodies. To demonstrate the efficiency of silencing or overexpression, extracts were probed using anti-HDAC1 and anti-HDAC2 antibodies. GAPDH was included as a loading control. ( B ) Microscopy-based analysis of APPL1 nuclear localization upon silencing of HDAC1 or HDAC2. HeLa cells were transfected with siRNA oligonucleotides: one non-specific (Φ) and two specific per gene (a and b) against HDAC1 and HDAC2 for 72 h, followed by fixation and immunostaining for APPL1. Acquired microscopic images were analysed by Metamorph software and the average pixel intensities corresponding to APPL1 in the nuclei (as visualized by Hoechst staining, not shown) were calculated. The results of a representative experiment are shown in the graph. The values are normalized with respect to the average pixel intensity of nuclear APPL1 in cells transfected with non-specific siRNA, assigned one arbitrary unit. Error bars indicate standard error (minimum 100 cells from each transfection were used for the analysis). The results were statistically analysed by GraphPad Prism4 software, and the values obtained for each knockdown experiment were significantly different from the control at P

Techniques Used: Transfection, Plasmid Preparation, Western Blot, Over Expression, Microscopy, Immunostaining, Software, Staining

Deacetylase activity detected in APPL1 complexes derives mainly from HDAC2 HDAC enzymatic activity was measured using a fluorimetric method (as described in the Experimental section) in immunoprecipitates from HeLa cells. ( A ) APPL1 binds active HDACs from class I or II. The APPL1 immunoprecipitate (IP) was divided into three equal parts: one left untreated and the other two treated with HDAC inhibitors: 1 mM nicotinamide and 1 μM TSA. Non-specific rabbit IgG was used as a control. ( B ) HeLa cells were silenced for HDAC1, HDAC2 and MTA2 [using two (a, b) different siRNA oligonucleotides per gene or non-specific siRNA Φ] prior to immunoprecipitation using APPL1 antibodies or non-specific rabbit IgG. The same extracts as presented in Figure 1 (A) were used (one third of the immunoprecipitates was measured in the HDAC activity assay, two thirds blotted as shown in Figure 1 A). The intensity of fluorescence emitted by the deacetylated substrate is expressed in arbitrary units in ( A ) and ( B ).
Figure Legend Snippet: Deacetylase activity detected in APPL1 complexes derives mainly from HDAC2 HDAC enzymatic activity was measured using a fluorimetric method (as described in the Experimental section) in immunoprecipitates from HeLa cells. ( A ) APPL1 binds active HDACs from class I or II. The APPL1 immunoprecipitate (IP) was divided into three equal parts: one left untreated and the other two treated with HDAC inhibitors: 1 mM nicotinamide and 1 μM TSA. Non-specific rabbit IgG was used as a control. ( B ) HeLa cells were silenced for HDAC1, HDAC2 and MTA2 [using two (a, b) different siRNA oligonucleotides per gene or non-specific siRNA Φ] prior to immunoprecipitation using APPL1 antibodies or non-specific rabbit IgG. The same extracts as presented in Figure 1 (A) were used (one third of the immunoprecipitates was measured in the HDAC activity assay, two thirds blotted as shown in Figure 1 A). The intensity of fluorescence emitted by the deacetylated substrate is expressed in arbitrary units in ( A ) and ( B ).

Techniques Used: Histone Deacetylase Assay, Activity Assay, Immunoprecipitation, HDAC Activity Assay, Fluorescence

22) Product Images from "PKC-Theta is a Novel SC35 Splicing Factor Regulator in Response to T Cell Activation"

Article Title: PKC-Theta is a Novel SC35 Splicing Factor Regulator in Response to T Cell Activation

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2015.00562

Identification of phosphorylated residues on the SC35 splicing protein. (A) Mean peptide signal and all SC35 peptide constructs used to examine SC35 for phosphorylation by PKC-θ. The red bar indicates the cut-off for positive signal for phosphorylation. The distribution curve for SC35 peptide phosphorylation is also displayed: the y -axis is the kernel density estimate of the normalized phosphorylation signal intensities on the x -axis, with anything equal to or greater than the red bar (2 × SD above the mean) considered a positive phosphorylation event. (B) The amino acid sequence of SC35 indicating the location of the top nine peptides in descending order and their location; the other five peptides correspond to overlaps with the first nine peptide locations. Green highlight denotes the RRM domain, the yellow highlight denotes RS dipeptide repeats. Peptides are numbered in order of mean signal intensity: red bar is the highest signal, blue bars are the other positive SC35 peptides, and possible serine or threonine phosphorylation residues are indicated by red or blue diamonds. (C) The top three peptide sequences and their overlap with the SC35 amino acid sequence. Hutti et al. ( 46 ) reported a serine/threonine specific motif for PKC-θ-mediated phosphorylation; using this information, the section of SC35 that scored the top three hits for peptide phosphorylation signals was analyzed for these motifs. Seven separate phosphorylation motifs were identified.
Figure Legend Snippet: Identification of phosphorylated residues on the SC35 splicing protein. (A) Mean peptide signal and all SC35 peptide constructs used to examine SC35 for phosphorylation by PKC-θ. The red bar indicates the cut-off for positive signal for phosphorylation. The distribution curve for SC35 peptide phosphorylation is also displayed: the y -axis is the kernel density estimate of the normalized phosphorylation signal intensities on the x -axis, with anything equal to or greater than the red bar (2 × SD above the mean) considered a positive phosphorylation event. (B) The amino acid sequence of SC35 indicating the location of the top nine peptides in descending order and their location; the other five peptides correspond to overlaps with the first nine peptide locations. Green highlight denotes the RRM domain, the yellow highlight denotes RS dipeptide repeats. Peptides are numbered in order of mean signal intensity: red bar is the highest signal, blue bars are the other positive SC35 peptides, and possible serine or threonine phosphorylation residues are indicated by red or blue diamonds. (C) The top three peptide sequences and their overlap with the SC35 amino acid sequence. Hutti et al. ( 46 ) reported a serine/threonine specific motif for PKC-θ-mediated phosphorylation; using this information, the section of SC35 that scored the top three hits for peptide phosphorylation signals was analyzed for these motifs. Seven separate phosphorylation motifs were identified.

Techniques Used: Construct, Sequencing

Interplay between SC35p, histone PTMs, and RNA-Pol-II ser2 in the Jurkat T cell model . Jurkat T cells stimulated as described in the section “ Materials and Methods ” (NS, no stimulation; ST, stimulation; SW, stimulus withdrawal; RST, re-stimulation) were fixed and probed with a primary mouse antibody to a phospho-epitope of human SC35 and primary rabbit antibody to H3K27ac (A) , H3k4me3 (B) , or RNA-Pol-II ser-2 (C) followed by visualization with a secondary goat antibody to mouse immunoglobulins conjugated to Alexa-Fluor 568 and secondary antibodies to rabbit immunoglobulins conjugated to Alexa-Fluor 488, respectively. Confocal laser scanning microscopy was used to measure expression of SC35p and H3K27ac, H3k4me3, or RNA-Pol-II-ser-2. Representative images for each stimulation point are shown with a 10-μm scale bar. Channels were overlaid to examine colocalization of the antibody targets. Pearson’s co-localizaton coefficient (PCC) and mean fluorescent intensity line scans were calculated with Fiji-ImageJ as described in the section “ Materials and Methods .” Data represent the mean ± SEM, n = 20 for each dataset with significant differences between datasets indicated. Red = SC35; green = H3K27ac, H3k4me3, or RNA-Pol-II-ser2; and yellow = visual overlap between the fluorescence signals.
Figure Legend Snippet: Interplay between SC35p, histone PTMs, and RNA-Pol-II ser2 in the Jurkat T cell model . Jurkat T cells stimulated as described in the section “ Materials and Methods ” (NS, no stimulation; ST, stimulation; SW, stimulus withdrawal; RST, re-stimulation) were fixed and probed with a primary mouse antibody to a phospho-epitope of human SC35 and primary rabbit antibody to H3K27ac (A) , H3k4me3 (B) , or RNA-Pol-II ser-2 (C) followed by visualization with a secondary goat antibody to mouse immunoglobulins conjugated to Alexa-Fluor 568 and secondary antibodies to rabbit immunoglobulins conjugated to Alexa-Fluor 488, respectively. Confocal laser scanning microscopy was used to measure expression of SC35p and H3K27ac, H3k4me3, or RNA-Pol-II-ser-2. Representative images for each stimulation point are shown with a 10-μm scale bar. Channels were overlaid to examine colocalization of the antibody targets. Pearson’s co-localizaton coefficient (PCC) and mean fluorescent intensity line scans were calculated with Fiji-ImageJ as described in the section “ Materials and Methods .” Data represent the mean ± SEM, n = 20 for each dataset with significant differences between datasets indicated. Red = SC35; green = H3K27ac, H3k4me3, or RNA-Pol-II-ser2; and yellow = visual overlap between the fluorescence signals.

Techniques Used: Confocal Laser Scanning Microscopy, Expressing, Periodic Counter-current Chromatography, Fluorescence

Interplay between PKC- θ and SC35 phosphorylation . (A) Naive OT-1 CD8 + CD44 lo/intermediate T-cells or influenza-specific effector OT-1 CD8 + T-cells were fixed and probed with a primary mouse antibody to a phospho-epitope of SC35 and primary rabbit antibody to PKC-θ followed by visualization with a secondary goat antibody to mouse immunoglobulins conjugated to Alexa-Fluor 568 and secondary antibodies to rabbit immunoglobulins conjugated to Alexa-Fluor 488, respectively. Confocal laser scanning microscopy was used to measure expression of SC35 and PKC-θ. Representative images for each stimulation point are shown with a 5-μm scale bar. Channels were overlaid to examine colocalization of the antibody targets. Pearson’s colocalization coefficient (PCC) and mean fluorescent intensity line scans were calculated with Fiji-ImageJ as described in the section “ Materials and Methods .” Data represent the mean ± SEM, n = 20 for each dataset with significant differences between datasets indicated. Red = SC35; green = PKC-θ; and yellow = visual overlap between the fluorescence signals. (B) Cell lysates of primary human CD4 + cells were untreated (mock) or treated with PKC-θ siRNA1 (Life Technologies) or siRNA2 (Santa Cruz). Effect on SC35 was analyzed by immunoblotting with a mouse raised primary antibody to a phospho-epitope of SC35, measuring band intensity with Fiji-ImageJ for each sample. A representative image of SC35 labeling for three separate experiments ( n = 3) is displayed (labeled SC35), with the mean intensity plotted with significant differences displayed for each treatment along with a representative loading control (LC) as described in the section “ Materials and Methods .” The effect of rottlerin treatment (C) , a PKC-θ-specific kinase inhibitor, on SC35 phosphorylation was also examined by immunoblotting as described above. A representative loading control (LC) is shown along with a representative SC35p-probed blot for three separate experiments ( n = 3). The mean intensity is plotted with significant differences displayed for each treatment.
Figure Legend Snippet: Interplay between PKC- θ and SC35 phosphorylation . (A) Naive OT-1 CD8 + CD44 lo/intermediate T-cells or influenza-specific effector OT-1 CD8 + T-cells were fixed and probed with a primary mouse antibody to a phospho-epitope of SC35 and primary rabbit antibody to PKC-θ followed by visualization with a secondary goat antibody to mouse immunoglobulins conjugated to Alexa-Fluor 568 and secondary antibodies to rabbit immunoglobulins conjugated to Alexa-Fluor 488, respectively. Confocal laser scanning microscopy was used to measure expression of SC35 and PKC-θ. Representative images for each stimulation point are shown with a 5-μm scale bar. Channels were overlaid to examine colocalization of the antibody targets. Pearson’s colocalization coefficient (PCC) and mean fluorescent intensity line scans were calculated with Fiji-ImageJ as described in the section “ Materials and Methods .” Data represent the mean ± SEM, n = 20 for each dataset with significant differences between datasets indicated. Red = SC35; green = PKC-θ; and yellow = visual overlap between the fluorescence signals. (B) Cell lysates of primary human CD4 + cells were untreated (mock) or treated with PKC-θ siRNA1 (Life Technologies) or siRNA2 (Santa Cruz). Effect on SC35 was analyzed by immunoblotting with a mouse raised primary antibody to a phospho-epitope of SC35, measuring band intensity with Fiji-ImageJ for each sample. A representative image of SC35 labeling for three separate experiments ( n = 3) is displayed (labeled SC35), with the mean intensity plotted with significant differences displayed for each treatment along with a representative loading control (LC) as described in the section “ Materials and Methods .” The effect of rottlerin treatment (C) , a PKC-θ-specific kinase inhibitor, on SC35 phosphorylation was also examined by immunoblotting as described above. A representative loading control (LC) is shown along with a representative SC35p-probed blot for three separate experiments ( n = 3). The mean intensity is plotted with significant differences displayed for each treatment.

Techniques Used: Confocal Laser Scanning Microscopy, Expressing, Periodic Counter-current Chromatography, Fluorescence, Labeling

Localization of PKC- θ in T cells and effect on SC35p . The full-length PKC-θ wild-type gene sequence ( HA PKC-θ WT) and the NLS mutation ( HA PKC-θ NLS) were cloned into the pTracer-CMV vector in frame with a C-terminal HA tag. Transfected Hut-78 T cells (A) and Jurkat T cells (B) and were fixed and probed with a rabbit antibody to the HA-tag followed by visualization with a secondary goat antibody to rabbit immunoglobulins conjugated to Alexa-Fluor 568. Confocal laser scanning microscopy was also used to assess PKC-θ localization as described in the section “ Materials and Methods .” Representative images for each construct are shown. Fn/c values for each construct are shown with significant differences between datasets indicated. Data represent the mean ± SEM, n = 20 for each dataset. (C) Jurkat T cells were transfected, stimulated, and nuclear extracts created to examine nuclear localization of the PKC-θ wild-type and PKCθNLS mutant. A representative immunoblot image of nuclear extracts showing HA PKC-θ localization in the nucleus is shown. Jurkat T cells were transfected as described in the section “ Materials and Methods ” and subsequently stimulated (NS, non-stimulated; ST, stimulated; RST, re-stimulated). Cells were fixed and probed with a mouse antibody to a phospho-epitope of human SC35 with secondary antibodies to mouse immunoglobulins conjugated to Alexa-Fluor 568. Confocal laser scanning microscopy was used to study SC35p expression in HA tag-positive cells. Representative images for each construct are shown with a 10-μm scale bar: (D) vector only (VO), (E) HA PKC-θ WT, and (F) HA PKC-θ NLS. Total nuclear fluorescence values for each construct are shown with significant differences between datasets indicated (G) . Data represent the mean ± SEM, n = 20 for each dataset. (H) Nuclear extracts were made from Jurkat T cells stimulated as previously described (NS, no stimulation; ST, stimulation; SW, stimulus withdrawal; RST, re-stimulation) and subjected to half-way CHIP using PKC-θ pull down or a no antibody control. Samples were probed with a primary mouse antibody to a phospho-epitope of human SC35 as described in the section “ Materials and Methods ”; representative bands are shown. SC35 band intensity was plotted using Fiji-ImageJ software minus background for n = 3 with mean ± SEM.
Figure Legend Snippet: Localization of PKC- θ in T cells and effect on SC35p . The full-length PKC-θ wild-type gene sequence ( HA PKC-θ WT) and the NLS mutation ( HA PKC-θ NLS) were cloned into the pTracer-CMV vector in frame with a C-terminal HA tag. Transfected Hut-78 T cells (A) and Jurkat T cells (B) and were fixed and probed with a rabbit antibody to the HA-tag followed by visualization with a secondary goat antibody to rabbit immunoglobulins conjugated to Alexa-Fluor 568. Confocal laser scanning microscopy was also used to assess PKC-θ localization as described in the section “ Materials and Methods .” Representative images for each construct are shown. Fn/c values for each construct are shown with significant differences between datasets indicated. Data represent the mean ± SEM, n = 20 for each dataset. (C) Jurkat T cells were transfected, stimulated, and nuclear extracts created to examine nuclear localization of the PKC-θ wild-type and PKCθNLS mutant. A representative immunoblot image of nuclear extracts showing HA PKC-θ localization in the nucleus is shown. Jurkat T cells were transfected as described in the section “ Materials and Methods ” and subsequently stimulated (NS, non-stimulated; ST, stimulated; RST, re-stimulated). Cells were fixed and probed with a mouse antibody to a phospho-epitope of human SC35 with secondary antibodies to mouse immunoglobulins conjugated to Alexa-Fluor 568. Confocal laser scanning microscopy was used to study SC35p expression in HA tag-positive cells. Representative images for each construct are shown with a 10-μm scale bar: (D) vector only (VO), (E) HA PKC-θ WT, and (F) HA PKC-θ NLS. Total nuclear fluorescence values for each construct are shown with significant differences between datasets indicated (G) . Data represent the mean ± SEM, n = 20 for each dataset. (H) Nuclear extracts were made from Jurkat T cells stimulated as previously described (NS, no stimulation; ST, stimulation; SW, stimulus withdrawal; RST, re-stimulation) and subjected to half-way CHIP using PKC-θ pull down or a no antibody control. Samples were probed with a primary mouse antibody to a phospho-epitope of human SC35 as described in the section “ Materials and Methods ”; representative bands are shown. SC35 band intensity was plotted using Fiji-ImageJ software minus background for n = 3 with mean ± SEM.

Techniques Used: Sequencing, Mutagenesis, Clone Assay, Plasmid Preparation, Transfection, Confocal Laser Scanning Microscopy, Construct, Expressing, Fluorescence, Chromatin Immunoprecipitation, Software

Nuclear PKC- θ as a novel regulator of SC35 in T-cells . Our model of the interaction between PKC-θ and SC35 in the context of TM in Jurkat T cells. Blue cylinders represent histones, and the colored ovals represent histone PTMs: red = repressive, purple (H3k27ac), or green (H3k4me3). Light green oval is PKC-θ, red oval is RNA-Pol-II, the darker green oval represents various TFs, and the orange oval is SC35p speckles. Upon stimulation, PKC-θ enters the nucleus and binds to a chromatin platform incorporating RNA-Pol-II and various transcription factors. At the same time, PKC-θ phosphorylates SC35, relocating the speckle to the site of transcription to potentially form a linked complex between the chromatin platform, the splicing speckle, and active histone marks. After stimulus withdrawal, this association between PKC-θ/TFs, histone PTMs, and SC35 speckles is maintained and expands even further upon re-stimulation to effect massive recruitment of phosphorylated SC35 splicing speckles.
Figure Legend Snippet: Nuclear PKC- θ as a novel regulator of SC35 in T-cells . Our model of the interaction between PKC-θ and SC35 in the context of TM in Jurkat T cells. Blue cylinders represent histones, and the colored ovals represent histone PTMs: red = repressive, purple (H3k27ac), or green (H3k4me3). Light green oval is PKC-θ, red oval is RNA-Pol-II, the darker green oval represents various TFs, and the orange oval is SC35p speckles. Upon stimulation, PKC-θ enters the nucleus and binds to a chromatin platform incorporating RNA-Pol-II and various transcription factors. At the same time, PKC-θ phosphorylates SC35, relocating the speckle to the site of transcription to potentially form a linked complex between the chromatin platform, the splicing speckle, and active histone marks. After stimulus withdrawal, this association between PKC-θ/TFs, histone PTMs, and SC35 speckles is maintained and expands even further upon re-stimulation to effect massive recruitment of phosphorylated SC35 splicing speckles.

Techniques Used:

Expression of phosphorylated SC35 in T cells and in primary mouse OT-1 T cells . (A) Jurkat T cells were either unstimulated (NS) or PMA/I-activated (ST) for 2 h or (B) naive OT-1 CD8 + CD44 lo/intermediate T-cells or influenza-specific effector OT-1 CD8 + T cells were fixed and probed with a mouse antibody to a phospho-epitope of SC35 followed by visualization with a secondary goat antibody to mouse immunoglobulins conjugated to Alexa-Fluor 568. Confocal laser scanning microscopy was used to measure SC35 expression as detailed in the section “ Materials and Methods .” Representative images for each treatment are shown, with a 10-μm scale bar for (A) and a 5-μm scale bar for (B) . Total nuclear fluorescence intensity (NFI) was measured with Fiji-ImageJ. Data represent the mean ± SEM, n = 20 for each dataset with significant differences between datasets indicated. Naive OT-1 CD8 + CD44 lo/intermediate T-cells or influenza-specific effector OT-1 CD8 + T cells were fixed and probed with a primary mouse antibody to a phospho-epitope of human SC35 and primary rabbit antibody to H3K27ac (C) , H3k4me3 (D) , or RNA-Pol-II ser-2 phosphorylation (E) followed by visualization with a secondary goat antibody to mouse immunoglobulins conjugated to Alexa-Fluor 568 and secondary antibodies to rabbit immunoglobulins conjugated to Alexa-Fluor 488, respectively. Confocal laser scanning microscopy was used to measure expression of SC35 and H3K27ac, H3k4me3, or RNA-Pol-II ser-2 as described in the section “ Materials and Methods .” Representative images for each treatment are shown with a 5-μm scale bar. Channels were overlaid to examine colocalization of the antibody targets. Pearson’s colocalization coefficient (PCC) and mean fluorescent intensity line scans were calculated with Fiji-ImageJ as described in the section “ Materials and Methods .” Data represent the mean ± SEM, n = 20 for each dataset with significant differences between datasets indicated. Red = SC35p; green = H3K27ac, H3k4me3, or RNA-Pol-II-ser2; and yellow = visual overlap between the fluorescence signals.
Figure Legend Snippet: Expression of phosphorylated SC35 in T cells and in primary mouse OT-1 T cells . (A) Jurkat T cells were either unstimulated (NS) or PMA/I-activated (ST) for 2 h or (B) naive OT-1 CD8 + CD44 lo/intermediate T-cells or influenza-specific effector OT-1 CD8 + T cells were fixed and probed with a mouse antibody to a phospho-epitope of SC35 followed by visualization with a secondary goat antibody to mouse immunoglobulins conjugated to Alexa-Fluor 568. Confocal laser scanning microscopy was used to measure SC35 expression as detailed in the section “ Materials and Methods .” Representative images for each treatment are shown, with a 10-μm scale bar for (A) and a 5-μm scale bar for (B) . Total nuclear fluorescence intensity (NFI) was measured with Fiji-ImageJ. Data represent the mean ± SEM, n = 20 for each dataset with significant differences between datasets indicated. Naive OT-1 CD8 + CD44 lo/intermediate T-cells or influenza-specific effector OT-1 CD8 + T cells were fixed and probed with a primary mouse antibody to a phospho-epitope of human SC35 and primary rabbit antibody to H3K27ac (C) , H3k4me3 (D) , or RNA-Pol-II ser-2 phosphorylation (E) followed by visualization with a secondary goat antibody to mouse immunoglobulins conjugated to Alexa-Fluor 568 and secondary antibodies to rabbit immunoglobulins conjugated to Alexa-Fluor 488, respectively. Confocal laser scanning microscopy was used to measure expression of SC35 and H3K27ac, H3k4me3, or RNA-Pol-II ser-2 as described in the section “ Materials and Methods .” Representative images for each treatment are shown with a 5-μm scale bar. Channels were overlaid to examine colocalization of the antibody targets. Pearson’s colocalization coefficient (PCC) and mean fluorescent intensity line scans were calculated with Fiji-ImageJ as described in the section “ Materials and Methods .” Data represent the mean ± SEM, n = 20 for each dataset with significant differences between datasets indicated. Red = SC35p; green = H3K27ac, H3k4me3, or RNA-Pol-II-ser2; and yellow = visual overlap between the fluorescence signals.

Techniques Used: Expressing, Confocal Laser Scanning Microscopy, Fluorescence, Periodic Counter-current Chromatography

23) Product Images from "To unite or divide: mitochondrial dynamics in the murine outer retina that preceded age related photoreceptor loss"

Article Title: To unite or divide: mitochondrial dynamics in the murine outer retina that preceded age related photoreceptor loss

Journal: Oncotarget

doi:

Changes in levels and distribution of mitochondrial fission proteins in the ageing retina of C57BL/6 mice A. Retinal sections of both young and old mice were immunostained with a mitochondrial fission antibody, Fis1 (red) and the nuclei counterstained with 4′, 6-diamino-2-phenylindole (DAPI) (Blue). Expression of Fis1 is stronger in the ganglion cell layer of the young retina compared to the old, while the old retina express more Fis1 in the photoreceptor layer than the young. B. Graph showing the quantification of Fis1 expression in the ganglion cell layer. There is a significant decrease in Fis1 expression in the ganglion cell layer of the old retina when compared to the young ( P = 0.0048). C. Graph showing that Fis1 is significantly increased in the inner segments of the photoreceptor layer ( P = 0.0333). D. Western blot results for the Fis1 protein showed that Fis1 in the old retina is reduced by 60% when compared to young retinae ( P = 0.0910). Mean ± SEM. Scale bar = 25μm. GCL; ganglion cell layer, IPL; Inner plexiform layer, INL; inner nuclear layer, OPL; outer plexiform layer, ONL; outer nuclear layer, PR; photoreceptor layer and RPE; retinal pigment epithelial.
Figure Legend Snippet: Changes in levels and distribution of mitochondrial fission proteins in the ageing retina of C57BL/6 mice A. Retinal sections of both young and old mice were immunostained with a mitochondrial fission antibody, Fis1 (red) and the nuclei counterstained with 4′, 6-diamino-2-phenylindole (DAPI) (Blue). Expression of Fis1 is stronger in the ganglion cell layer of the young retina compared to the old, while the old retina express more Fis1 in the photoreceptor layer than the young. B. Graph showing the quantification of Fis1 expression in the ganglion cell layer. There is a significant decrease in Fis1 expression in the ganglion cell layer of the old retina when compared to the young ( P = 0.0048). C. Graph showing that Fis1 is significantly increased in the inner segments of the photoreceptor layer ( P = 0.0333). D. Western blot results for the Fis1 protein showed that Fis1 in the old retina is reduced by 60% when compared to young retinae ( P = 0.0910). Mean ± SEM. Scale bar = 25μm. GCL; ganglion cell layer, IPL; Inner plexiform layer, INL; inner nuclear layer, OPL; outer plexiform layer, ONL; outer nuclear layer, PR; photoreceptor layer and RPE; retinal pigment epithelial.

Techniques Used: Mouse Assay, Expressing, Western Blot

24) Product Images from "Activation of Wnt/?-Catenin Signalling Affects Differentiation of Cells Arising from the Cerebellar Ventricular Zone"

Article Title: Activation of Wnt/?-Catenin Signalling Affects Differentiation of Cells Arising from the Cerebellar Ventricular Zone

Journal: PLoS ONE

doi: 10.1371/journal.pone.0042572

Effects of in vivo activation of Wnt/β-catenin signalling on development of VZ-derived cells. Apc lox/+ and Apc lox/lox embryos were electroporated with a Cre-GFP plasmid at E13.5 and analysed at E18.5. Electroporated cells and their progeny, marked by expression of GFP, were examined for expression of Sox9 (A–C), Pax2 (D–F) and PCNA (G–I). Immunohistochemical analyses were quantitated by counting the number of GFP+ cells expressing each marker (white arrows) as a proportion of total GFP+ cell numbers per section and comparing by Student's T-test between the two genotypes for each marker. For each test, n = 4, error bars = SEM, * = p
Figure Legend Snippet: Effects of in vivo activation of Wnt/β-catenin signalling on development of VZ-derived cells. Apc lox/+ and Apc lox/lox embryos were electroporated with a Cre-GFP plasmid at E13.5 and analysed at E18.5. Electroporated cells and their progeny, marked by expression of GFP, were examined for expression of Sox9 (A–C), Pax2 (D–F) and PCNA (G–I). Immunohistochemical analyses were quantitated by counting the number of GFP+ cells expressing each marker (white arrows) as a proportion of total GFP+ cell numbers per section and comparing by Student's T-test between the two genotypes for each marker. For each test, n = 4, error bars = SEM, * = p

Techniques Used: In Vivo, Activation Assay, Derivative Assay, Plasmid Preparation, Expressing, Immunohistochemistry, Marker

25) Product Images from "Signalling through AMPA receptors on oligodendrocyte precursors promotes myelination by enhancing oligodendrocyte survival"

Article Title: Signalling through AMPA receptors on oligodendrocyte precursors promotes myelination by enhancing oligodendrocyte survival

Journal: eLife

doi: 10.7554/eLife.28080

Gria3 null 2 –/– 4 –/– mice generate fewer OLs in the corpus callosum. ( A ) CC1 immunolabelling marks differentiated OLs in Gria3 null and Gria3 null 2 –/– 4 –/– mice. ( B ) The density of CC1 + OLs is significantly less (by ∼22% at P14 and ~26% at P53) in Gria3 null 2 –/– 4 –/– mice relative to Gria3 null controls (p=0.026 at P14, p=0.029 at P53, Mann-Whitney test; > 900 cells were counted per mouse at all ages). ( C ) Pdgfra + OPs in Gria3 null and Gria3 null 2 –/– 4 –/– mice. ( D ) There were no significant differences in the density of Pdgfra + OPs at P14 or P53 in Gria3 null 2 –/– 4 –/– versus Gria3 null mice (p=0.11 at P14; p=0.7 at P53, Mann-Whitney test; > 400 cells were counted per mouse at all ages). ( E ) Cleaved Caspase-3 + , Olig2 + OL lineage cells in Gria3 null 2 –/– 4 –/– mice. Cells in the rectangle (dotted line) are shown on the right at higher magnification. ( F ) There was a ~24% increase in the fraction of Olig2 + cells that expressed cleaved Caspase-3 in Gria3 null 2 –/– 4 –/– compared to Gria3 null mice (p=0.004, Mann-Whitney test; > 1500 Olig2 + cells were counted in each mouse). ( G ) Dye-filled OLs in P14 corpus callosum of Gria3 null and Gria3 null 2 –/– 4 –/– mice. ( H ) There was no change in the length of the internodes (p=0.93, Student’s t-test) or the number of internodes per OL (p=0.41, Student’s t-test) in Gria3 null 2 –/– 4 –/– compared to Gria3 null mice. Numbers of mice analyzed are indicated in ( B ), ( D ) and ( F ) and numbers of cells in ( H ). Scale bars: 50 μm. DOI: http://dx.doi.org/10.7554/eLife.28080.017
Figure Legend Snippet: Gria3 null 2 –/– 4 –/– mice generate fewer OLs in the corpus callosum. ( A ) CC1 immunolabelling marks differentiated OLs in Gria3 null and Gria3 null 2 –/– 4 –/– mice. ( B ) The density of CC1 + OLs is significantly less (by ∼22% at P14 and ~26% at P53) in Gria3 null 2 –/– 4 –/– mice relative to Gria3 null controls (p=0.026 at P14, p=0.029 at P53, Mann-Whitney test; > 900 cells were counted per mouse at all ages). ( C ) Pdgfra + OPs in Gria3 null and Gria3 null 2 –/– 4 –/– mice. ( D ) There were no significant differences in the density of Pdgfra + OPs at P14 or P53 in Gria3 null 2 –/– 4 –/– versus Gria3 null mice (p=0.11 at P14; p=0.7 at P53, Mann-Whitney test; > 400 cells were counted per mouse at all ages). ( E ) Cleaved Caspase-3 + , Olig2 + OL lineage cells in Gria3 null 2 –/– 4 –/– mice. Cells in the rectangle (dotted line) are shown on the right at higher magnification. ( F ) There was a ~24% increase in the fraction of Olig2 + cells that expressed cleaved Caspase-3 in Gria3 null 2 –/– 4 –/– compared to Gria3 null mice (p=0.004, Mann-Whitney test; > 1500 Olig2 + cells were counted in each mouse). ( G ) Dye-filled OLs in P14 corpus callosum of Gria3 null and Gria3 null 2 –/– 4 –/– mice. ( H ) There was no change in the length of the internodes (p=0.93, Student’s t-test) or the number of internodes per OL (p=0.41, Student’s t-test) in Gria3 null 2 –/– 4 –/– compared to Gria3 null mice. Numbers of mice analyzed are indicated in ( B ), ( D ) and ( F ) and numbers of cells in ( H ). Scale bars: 50 μm. DOI: http://dx.doi.org/10.7554/eLife.28080.017

Techniques Used: Mouse Assay, MANN-WHITNEY

26) Product Images from "Regulation of the subcellular distribution of key cellular RNA-processing factors during permissive human cytomegalovirus infection"

Article Title: Regulation of the subcellular distribution of key cellular RNA-processing factors during permissive human cytomegalovirus infection

Journal: The Journal of General Virology

doi: 10.1099/vir.0.020313-0

(a) HCMV infection induces the formation of fibrillarin-containing caps. G 0 -HFFs were mock infected or HCMV infected and harvested at 96 h p.i. as in Fig. 3 . Cells were stained with anti-fibrillarin (green, 1 : 250), anti-PSF (red, 1 : 250) and anti-ppUL57 (blue, 1 : 250) antibodies and corresponding secondary antibodies. The left and middle panels are greyscale and the rightmost panels show all three channels merged. The bottom panel shows an enlarged HCMV-infected cell. (b) Co-sedimentation of cellular RNA-processing factors and nucleolar components with HCMV ppUL57, a VRC component. G 0 -HFFs were mock infected (M) or HCMV infected (H, m.o.i.=1) as in Fig. 2 and harvested at 96 h p.i. Cells were fractionated into cytoplasmic fraction, subnuclear pellet and supernatant (Sup) by modification of the nucleolar isolation procedure of Andersen et al. (2002) . The cytoplasmic (Cyto) fraction was concentrated 5-fold and proteins (15 μg per well) were resolved by SDS-PAGE and blotted as described in Fig. 2 . The blots were reacted with antibodies against CstF-64 (1 : 2000), SRPK1 (1 : 1000), fibrillarin (1 : 1000), PSF (1 : 2000), ppUL57 (1 : 500) or LDH (1 : 1000). Probed blots were stripped of bound primary and secondary antibodies after exposure and reprobed as required. The fold inductions in CstF-64, SRPK1, fibrillarin and PSF abundance were determined by comparison of the densities of the bands from HCMV-infected cells divided by the densities of the corresponding mock-infected cells as in Fig. 2 except where the mock- or HCMV-infected band was not detectable ( nd ).
Figure Legend Snippet: (a) HCMV infection induces the formation of fibrillarin-containing caps. G 0 -HFFs were mock infected or HCMV infected and harvested at 96 h p.i. as in Fig. 3 . Cells were stained with anti-fibrillarin (green, 1 : 250), anti-PSF (red, 1 : 250) and anti-ppUL57 (blue, 1 : 250) antibodies and corresponding secondary antibodies. The left and middle panels are greyscale and the rightmost panels show all three channels merged. The bottom panel shows an enlarged HCMV-infected cell. (b) Co-sedimentation of cellular RNA-processing factors and nucleolar components with HCMV ppUL57, a VRC component. G 0 -HFFs were mock infected (M) or HCMV infected (H, m.o.i.=1) as in Fig. 2 and harvested at 96 h p.i. Cells were fractionated into cytoplasmic fraction, subnuclear pellet and supernatant (Sup) by modification of the nucleolar isolation procedure of Andersen et al. (2002) . The cytoplasmic (Cyto) fraction was concentrated 5-fold and proteins (15 μg per well) were resolved by SDS-PAGE and blotted as described in Fig. 2 . The blots were reacted with antibodies against CstF-64 (1 : 2000), SRPK1 (1 : 1000), fibrillarin (1 : 1000), PSF (1 : 2000), ppUL57 (1 : 500) or LDH (1 : 1000). Probed blots were stripped of bound primary and secondary antibodies after exposure and reprobed as required. The fold inductions in CstF-64, SRPK1, fibrillarin and PSF abundance were determined by comparison of the densities of the bands from HCMV-infected cells divided by the densities of the corresponding mock-infected cells as in Fig. 2 except where the mock- or HCMV-infected band was not detectable ( nd ).

Techniques Used: Infection, Staining, Sedimentation, Modification, Isolation, SDS Page

27) Product Images from "Interaction of neurotrophin signaling with Bcl-2 localized to the mitochondria and endoplasmic reticulum on spiral ganglion neuron survival and neurite growth"

Article Title: Interaction of neurotrophin signaling with Bcl-2 localized to the mitochondria and endoplasmic reticulum on spiral ganglion neuron survival and neurite growth

Journal: Journal of Neuroscience Research

doi: 10.1002/jnr.22381

Mitochondrially targeted Bcl-2 decreases SGN survival in the presence of NT-3. Mean SGN survival in cultures transfected with GFP and maintained in neurotrophin-3 (NT-3, 50 ng/ml) is defined as 100%. Expression of GFP-Bcl-2-Maob in the presence of NT-3 decreased mean SGN survival by 81% relative to cultures expressing GFP-Bcl-2-Maob in the absence of NT-3. Each condition was performed in duplicate or triplicate and repeated at least three times. Error bars present standard deviation from each repetition. Significance of difference among means was determined by one-way ANOVA followed by a post-hoc Tukey analysis.
Figure Legend Snippet: Mitochondrially targeted Bcl-2 decreases SGN survival in the presence of NT-3. Mean SGN survival in cultures transfected with GFP and maintained in neurotrophin-3 (NT-3, 50 ng/ml) is defined as 100%. Expression of GFP-Bcl-2-Maob in the presence of NT-3 decreased mean SGN survival by 81% relative to cultures expressing GFP-Bcl-2-Maob in the absence of NT-3. Each condition was performed in duplicate or triplicate and repeated at least three times. Error bars present standard deviation from each repetition. Significance of difference among means was determined by one-way ANOVA followed by a post-hoc Tukey analysis.

Techniques Used: Transfection, Expressing, Standard Deviation

Mitochondrially targeted Bcl-2 decreases SGN survival specifically in the presence of neurotrophins. Mean SGN survival in cultures transfected with GFP and maintained in neurotrophin-3 (NT-3, 50 ng/ml) is defined as 100%. Maintenance of cultures expressing GFP-Bcl-2-Maob in brain derived neurotrophic factor (BDNF, 50 ng/ml) decreased mean SGN survival by 64% relative to cultures expressing GFP-Bcl-2-Maob in the absence of neurotrophic factors. Conversely, maintenance of cultures expressing GFP-Bcl-2-Maob in 30 mM extracellular K + (30K) or pro-nerve growth factor (proNGF, 3 nM) did not reduce SGN survival. Error bars present standard deviation from each repetition. Each condition was performed in duplicate or triplicate and repeated at least three times. Significance of difference among means was determined by one-way ANOVA followed by a post-hoc Tukey analysis.
Figure Legend Snippet: Mitochondrially targeted Bcl-2 decreases SGN survival specifically in the presence of neurotrophins. Mean SGN survival in cultures transfected with GFP and maintained in neurotrophin-3 (NT-3, 50 ng/ml) is defined as 100%. Maintenance of cultures expressing GFP-Bcl-2-Maob in brain derived neurotrophic factor (BDNF, 50 ng/ml) decreased mean SGN survival by 64% relative to cultures expressing GFP-Bcl-2-Maob in the absence of neurotrophic factors. Conversely, maintenance of cultures expressing GFP-Bcl-2-Maob in 30 mM extracellular K + (30K) or pro-nerve growth factor (proNGF, 3 nM) did not reduce SGN survival. Error bars present standard deviation from each repetition. Each condition was performed in duplicate or triplicate and repeated at least three times. Significance of difference among means was determined by one-way ANOVA followed by a post-hoc Tukey analysis.

Techniques Used: Transfection, Expressing, Derivative Assay, Standard Deviation

Bcl-2 targeted to the mitochondria disrupts the mitochondrial transmembrane potential. Spiral ganglion cultures were transfected with GFP-Maob or GFP-Bcl-2-Maob and loaded for 15 min with MitoTracker ® Red CMXRos 24 h after transgene expression. Cultures were subsequently immunostained with anti-NF200 antibodies (blue) and imaged with laser confocal microscopy. SGNs expressing GFP-Bcl-2-Maob, but not GFP-Maob, fail to label with MitoTracker Red indicating disruption of the mitochondrial transmembrane potential.
Figure Legend Snippet: Bcl-2 targeted to the mitochondria disrupts the mitochondrial transmembrane potential. Spiral ganglion cultures were transfected with GFP-Maob or GFP-Bcl-2-Maob and loaded for 15 min with MitoTracker ® Red CMXRos 24 h after transgene expression. Cultures were subsequently immunostained with anti-NF200 antibodies (blue) and imaged with laser confocal microscopy. SGNs expressing GFP-Bcl-2-Maob, but not GFP-Maob, fail to label with MitoTracker Red indicating disruption of the mitochondrial transmembrane potential.

Techniques Used: Transfection, Expressing, Confocal Microscopy

Treatment with NT-3 leads to activation of caspase 3 in SGNs expressing mitochondrially targeted Bcl-2. Spiral ganglion cultures were transfected with GFP-Bcl-2-Maob and maintained in the absence (A) or presence (B) of NT-3 for 24 h after transgene expression. Cultures were immunostained with anti-NF200 (blue) and anti-activated caspase-3 (red) antibodies and imaged with laser confocal microscopy. SGNs transfected with GFP-Bcl-2-Maob and treated with NT-3 (B) demonstrate activated caspase-3 immunoreactivity. Scale bar=10 μm.
Figure Legend Snippet: Treatment with NT-3 leads to activation of caspase 3 in SGNs expressing mitochondrially targeted Bcl-2. Spiral ganglion cultures were transfected with GFP-Bcl-2-Maob and maintained in the absence (A) or presence (B) of NT-3 for 24 h after transgene expression. Cultures were immunostained with anti-NF200 (blue) and anti-activated caspase-3 (red) antibodies and imaged with laser confocal microscopy. SGNs transfected with GFP-Bcl-2-Maob and treated with NT-3 (B) demonstrate activated caspase-3 immunoreactivity. Scale bar=10 μm.

Techniques Used: Activation Assay, Expressing, Transfection, Confocal Microscopy

Transfection of GFP-Bcl-2-WT, GFP-Bcl-2-Maob or GFP-Bcl-2-Cb5 results in increased Bcl-2 expression. Spiral ganglion cultures were transfected with GFP-Bcl-2-WT, GFP-Bcl-2-Maob or GFP-Bcl-2-Cb5, or GFP as indicated. Twenty four hrs after transfection, the cultures were immunostained with anti-NF200 (red) and anti-Bcl-2 (scaled, right panels) antibodies and imaged with laser confocal microscopy. The intensity of Bcl-2 immunostaining is scaled (0-255) in the right panels. Images were obtained a lower magnification to allow demonstration of a transfected (GFP-positive, green, arrow) and untransfected neuron (GFP-negative, arrowhead) in each condition. SGNs expressing GFP-Bcl-2-WT, GFP-Bcl-2-Maob or GFP-Bcl-2-Cb5 demonstrate increased Bcl-2 immunoreactivity compared with SGNs expressing GFP, GFP-Maob (not shown), or GFP-Cb5 (not shown). Scale bar=20 μm.
Figure Legend Snippet: Transfection of GFP-Bcl-2-WT, GFP-Bcl-2-Maob or GFP-Bcl-2-Cb5 results in increased Bcl-2 expression. Spiral ganglion cultures were transfected with GFP-Bcl-2-WT, GFP-Bcl-2-Maob or GFP-Bcl-2-Cb5, or GFP as indicated. Twenty four hrs after transfection, the cultures were immunostained with anti-NF200 (red) and anti-Bcl-2 (scaled, right panels) antibodies and imaged with laser confocal microscopy. The intensity of Bcl-2 immunostaining is scaled (0-255) in the right panels. Images were obtained a lower magnification to allow demonstration of a transfected (GFP-positive, green, arrow) and untransfected neuron (GFP-negative, arrowhead) in each condition. SGNs expressing GFP-Bcl-2-WT, GFP-Bcl-2-Maob or GFP-Bcl-2-Cb5 demonstrate increased Bcl-2 immunoreactivity compared with SGNs expressing GFP, GFP-Maob (not shown), or GFP-Cb5 (not shown). Scale bar=20 μm.

Techniques Used: Transfection, Expressing, Confocal Microscopy, Immunostaining

Expression of Bcl-2 targeted to the mitochondrial or endoplasmic reticulum promotes spiral ganglion neuron (SGN) survival. Mean SGN survival in cultures transfected with GFP and maintained in neurotrophin-3 (NT-3, 50 ng/ml) is defined as 100%. The number of surviving SGNs in control conditions was 127±24 (mean±standard deviation) across all repetitions. Transfection of GFP-Bcl-2-Cb5 or GFP-Bcl-2-Maob increases mean SGN survival 5 and 1.5 times, respectively, compared with transfection of GFP and maintenance of cultures in NT-3 (control). Each condition was performed in duplicate or triplicate and repeated at least three times. Error bars present standard deviation from each repetition. Significance of difference among means was determined by one-way ANOVA followed by a post-hoc Tukey analysis.
Figure Legend Snippet: Expression of Bcl-2 targeted to the mitochondrial or endoplasmic reticulum promotes spiral ganglion neuron (SGN) survival. Mean SGN survival in cultures transfected with GFP and maintained in neurotrophin-3 (NT-3, 50 ng/ml) is defined as 100%. The number of surviving SGNs in control conditions was 127±24 (mean±standard deviation) across all repetitions. Transfection of GFP-Bcl-2-Cb5 or GFP-Bcl-2-Maob increases mean SGN survival 5 and 1.5 times, respectively, compared with transfection of GFP and maintenance of cultures in NT-3 (control). Each condition was performed in duplicate or triplicate and repeated at least three times. Error bars present standard deviation from each repetition. Significance of difference among means was determined by one-way ANOVA followed by a post-hoc Tukey analysis.

Techniques Used: Expressing, Transfection, Standard Deviation

Expression of Bcl-2 to the mitochondrial or endoplasmic reticulum inhibits SGN neurite growth. A, B, and C . Representative epifluorescence images of spiral ganglion cultures transfected with GFP, GFP-Bcl-2-Cb5, or GFP-Bcl-2-Maob and maintained in the presence of NT-3 (50 ng/ml) for 48 h following transgene expression. Cultures were immunolabeled with anti-NF200 antibodies (red). SGNs transfected with GFP-Bcl-2-Cb5 ( B ) or GFP-Bcl-2-Maob ( C ) extend short neurites relative to SGNs transfected with GFP ( A ). D . Neurite length was determined by measuring the longest process extending from each transfected SGN. Results are presented as cumulative percent histograms where conditions with shorter neurites are shifted to the left compared with conditions with longer neurites. Each condition was performed in duplicate or triplicate and repeated at least three times. n=total cumulative number of SGNs scored for each condition for all repetitions. Neurite length in cultures transfected with wild-type GFP-Bcl-2, GFP-Bcl-2-Cb5, or GFP-Bcl-2-Maob was significantly different from cultures transfected with GFP (p
Figure Legend Snippet: Expression of Bcl-2 to the mitochondrial or endoplasmic reticulum inhibits SGN neurite growth. A, B, and C . Representative epifluorescence images of spiral ganglion cultures transfected with GFP, GFP-Bcl-2-Cb5, or GFP-Bcl-2-Maob and maintained in the presence of NT-3 (50 ng/ml) for 48 h following transgene expression. Cultures were immunolabeled with anti-NF200 antibodies (red). SGNs transfected with GFP-Bcl-2-Cb5 ( B ) or GFP-Bcl-2-Maob ( C ) extend short neurites relative to SGNs transfected with GFP ( A ). D . Neurite length was determined by measuring the longest process extending from each transfected SGN. Results are presented as cumulative percent histograms where conditions with shorter neurites are shifted to the left compared with conditions with longer neurites. Each condition was performed in duplicate or triplicate and repeated at least three times. n=total cumulative number of SGNs scored for each condition for all repetitions. Neurite length in cultures transfected with wild-type GFP-Bcl-2, GFP-Bcl-2-Cb5, or GFP-Bcl-2-Maob was significantly different from cultures transfected with GFP (p

Techniques Used: Expressing, Transfection, Immunolabeling

Treatment with NT-3 leads to apoptosis of SGNs expressing mitochondrially targeted Bcl-2. Spiral ganglion cultures were transfected with GFP-Bcl-2-Maob and maintained in the absence (A) or presence (B) of NT-3 for 24 h after transgene expression. Cultures were immunostained with anti-NF200 (red) antibodies and labeled with TUNEL (green, right panels) and imaged with laser confocal microscopy. SGNs transfected with GFP-Bcl-2-Maob and treated with NT-3 (B), but not control SGNs (A) demonstrate TUNEL-positive condensed and fragmented nuclei.
Figure Legend Snippet: Treatment with NT-3 leads to apoptosis of SGNs expressing mitochondrially targeted Bcl-2. Spiral ganglion cultures were transfected with GFP-Bcl-2-Maob and maintained in the absence (A) or presence (B) of NT-3 for 24 h after transgene expression. Cultures were immunostained with anti-NF200 (red) antibodies and labeled with TUNEL (green, right panels) and imaged with laser confocal microscopy. SGNs transfected with GFP-Bcl-2-Maob and treated with NT-3 (B), but not control SGNs (A) demonstrate TUNEL-positive condensed and fragmented nuclei.

Techniques Used: Expressing, Transfection, Labeling, TUNEL Assay, Confocal Microscopy

Targeting of Bcl-2 to the mitochondria and endoplasmic reticulum in spiral ganglion neurons. Spiral ganglion neurons were transfected with GFP-Bcl-2-Maob (A) or GFP-Bcl-2-Cb5 (B). Cultures were immunostained with anti-heat shock protein (Hsp) 60 (A, red) or anti-calnexin (B, red) and anti-neurofilament 200 (NF200, blue) antibodies and imaged with laser confocal microscopy. GFP-Bcl-2-GFP-Maob colocalizes with Hsp60 immunoreactivity and GFP-Bcl-2-GFP-Cb5 colocalizes with calnexin immunoreactivity.
Figure Legend Snippet: Targeting of Bcl-2 to the mitochondria and endoplasmic reticulum in spiral ganglion neurons. Spiral ganglion neurons were transfected with GFP-Bcl-2-Maob (A) or GFP-Bcl-2-Cb5 (B). Cultures were immunostained with anti-heat shock protein (Hsp) 60 (A, red) or anti-calnexin (B, red) and anti-neurofilament 200 (NF200, blue) antibodies and imaged with laser confocal microscopy. GFP-Bcl-2-GFP-Maob colocalizes with Hsp60 immunoreactivity and GFP-Bcl-2-GFP-Cb5 colocalizes with calnexin immunoreactivity.

Techniques Used: Transfection, Confocal Microscopy

Activation of PI3-K promotes SGN neurite growth but fails to rescue neurites in SGNs expressing Bcl-2. A and B . Representative epifluorescence images of spiral ganglion cultures cotransfected with GFP and MEKΔEE (A) or P110 (B). Cultures were immunolabeled with anti-NF200 antibodies (red). C . Neurite length was determined as above for spiral ganglion cultures cotransfected with empty vector, P110, or MEKΔEE and GFP in the presence (empty vector) or absence (P110 and MEKΔEE) of NT-3. n=total cumulative number of SGNs scored for each condition for all repetitions. Neurite length in cultures cotransfected with P110 and GFP was significantly different from cultures cotransfected with MEKΔEE and GFP or transfected with GFP (p
Figure Legend Snippet: Activation of PI3-K promotes SGN neurite growth but fails to rescue neurites in SGNs expressing Bcl-2. A and B . Representative epifluorescence images of spiral ganglion cultures cotransfected with GFP and MEKΔEE (A) or P110 (B). Cultures were immunolabeled with anti-NF200 antibodies (red). C . Neurite length was determined as above for spiral ganglion cultures cotransfected with empty vector, P110, or MEKΔEE and GFP in the presence (empty vector) or absence (P110 and MEKΔEE) of NT-3. n=total cumulative number of SGNs scored for each condition for all repetitions. Neurite length in cultures cotransfected with P110 and GFP was significantly different from cultures cotransfected with MEKΔEE and GFP or transfected with GFP (p

Techniques Used: Activation Assay, Expressing, Immunolabeling, Plasmid Preparation, Transfection

28) Product Images from "Cholera toxin inhibits SNX27-retromer-mediated delivery of cargo proteins to the plasma membrane"

Article Title: Cholera toxin inhibits SNX27-retromer-mediated delivery of cargo proteins to the plasma membrane

Journal: Journal of Cell Science

doi: 10.1242/jcs.218610

CT decreases retromer protein expression. (A) Schematic diagram of an EE illustrating the SNX27–retromer and the WASH complex. Branched tubules (arrow) represents discrete domains into which specific proteins are sorted and targeted to their respective destinations. (B) Expression of retromer proteins VPS35 and VPS26 in untreated and CT-treated (8 h and 4 times over 48 h) 14-day post confluent Caco-2/bbe cells. Western blot analysis of a representative blot is shown above. Quantitative analysis of the blots showing levels of VPS35 and VPS26 normalized to total levels of actin is shown below. Results are means±s.e.m., n =3. * P
Figure Legend Snippet: CT decreases retromer protein expression. (A) Schematic diagram of an EE illustrating the SNX27–retromer and the WASH complex. Branched tubules (arrow) represents discrete domains into which specific proteins are sorted and targeted to their respective destinations. (B) Expression of retromer proteins VPS35 and VPS26 in untreated and CT-treated (8 h and 4 times over 48 h) 14-day post confluent Caco-2/bbe cells. Western blot analysis of a representative blot is shown above. Quantitative analysis of the blots showing levels of VPS35 and VPS26 normalized to total levels of actin is shown below. Results are means±s.e.m., n =3. * P

Techniques Used: Expressing, Western Blot

A pharmacological chaperone rescues CT-dependent retromer destabilization and stimulates fluid absorption by intestinal epithelial cells. (A) Caco-2/bbe cells were treated with either CT (8 h or 48 h) or R55 (5 μM for 48 h) or both, in pre- (CT treatment before R55 treatment) or post-combinations (CT treatment after R55 treatment) for indicated periods of time, and levels of retromer proteins VPS26 and VPS35 were analyzed using western blot analysis. A representative blot is shown. (B) Quantitative analysis of the immunoblots in A. Levels of VPS26 and VPS35 were normalized to total levels of actin ( n =4 per group). * P
Figure Legend Snippet: A pharmacological chaperone rescues CT-dependent retromer destabilization and stimulates fluid absorption by intestinal epithelial cells. (A) Caco-2/bbe cells were treated with either CT (8 h or 48 h) or R55 (5 μM for 48 h) or both, in pre- (CT treatment before R55 treatment) or post-combinations (CT treatment after R55 treatment) for indicated periods of time, and levels of retromer proteins VPS26 and VPS35 were analyzed using western blot analysis. A representative blot is shown. (B) Quantitative analysis of the immunoblots in A. Levels of VPS26 and VPS35 were normalized to total levels of actin ( n =4 per group). * P

Techniques Used: Western Blot

29) Product Images from "Cholera toxin inhibits SNX27-retromer-mediated delivery of cargo proteins to the plasma membrane"

Article Title: Cholera toxin inhibits SNX27-retromer-mediated delivery of cargo proteins to the plasma membrane

Journal: Journal of Cell Science

doi: 10.1242/jcs.218610

CT decreases retromer protein expression. (A) Schematic diagram of an EE illustrating the SNX27–retromer and the WASH complex. Branched tubules (arrow) represents discrete domains into which specific proteins are sorted and targeted to their respective destinations. (B) Expression of retromer proteins VPS35 and VPS26 in untreated and CT-treated (8 h and 4 times over 48 h) 14-day post confluent Caco-2/bbe cells. Western blot analysis of a representative blot is shown above. Quantitative analysis of the blots showing levels of VPS35 and VPS26 normalized to total levels of actin is shown below. Results are means±s.e.m., n =3. * P
Figure Legend Snippet: CT decreases retromer protein expression. (A) Schematic diagram of an EE illustrating the SNX27–retromer and the WASH complex. Branched tubules (arrow) represents discrete domains into which specific proteins are sorted and targeted to their respective destinations. (B) Expression of retromer proteins VPS35 and VPS26 in untreated and CT-treated (8 h and 4 times over 48 h) 14-day post confluent Caco-2/bbe cells. Western blot analysis of a representative blot is shown above. Quantitative analysis of the blots showing levels of VPS35 and VPS26 normalized to total levels of actin is shown below. Results are means±s.e.m., n =3. * P

Techniques Used: Expressing, Western Blot

A pharmacological chaperone rescues CT-dependent retromer destabilization and stimulates fluid absorption by intestinal epithelial cells. (A) Caco-2/bbe cells were treated with either CT (8 h or 48 h) or R55 (5 μM for 48 h) or both, in pre- (CT treatment before R55 treatment) or post-combinations (CT treatment after R55 treatment) for indicated periods of time, and levels of retromer proteins VPS26 and VPS35 were analyzed using western blot analysis. A representative blot is shown. (B) Quantitative analysis of the immunoblots in A. Levels of VPS26 and VPS35 were normalized to total levels of actin ( n =4 per group). * P
Figure Legend Snippet: A pharmacological chaperone rescues CT-dependent retromer destabilization and stimulates fluid absorption by intestinal epithelial cells. (A) Caco-2/bbe cells were treated with either CT (8 h or 48 h) or R55 (5 μM for 48 h) or both, in pre- (CT treatment before R55 treatment) or post-combinations (CT treatment after R55 treatment) for indicated periods of time, and levels of retromer proteins VPS26 and VPS35 were analyzed using western blot analysis. A representative blot is shown. (B) Quantitative analysis of the immunoblots in A. Levels of VPS26 and VPS35 were normalized to total levels of actin ( n =4 per group). * P

Techniques Used: Western Blot

30) Product Images from "Naturally Occurring Variants in LRP1 (Low-Density Lipoprotein Receptor–Related Protein 1) Affect HDL (High-Density Lipoprotein) Metabolism Through ABCA1 (ATP-Binding Cassette A1) and SR-B1 (Scavenger Receptor Class B Type 1) in Humans"

Article Title: Naturally Occurring Variants in LRP1 (Low-Density Lipoprotein Receptor–Related Protein 1) Affect HDL (High-Density Lipoprotein) Metabolism Through ABCA1 (ATP-Binding Cassette A1) and SR-B1 (Scavenger Receptor Class B Type 1) in Humans

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/ATVBAHA.117.310309

ABCA1 (ATP-binding cassette A1) and SR-B1 (scavenger receptor class B type 1) protein levels in the LRP1 (low-density lipoprotein receptor–related protein 1) variant carrier and control fibroblasts. A , Real-time polymerase chain reaction quantification of LRP1 expression in total cell lysates. β-actin and GAPDH were used as housekeeping genes. B , Representative Western blot showing LRP1 expression in 3 control individuals and the variant carrier. Quantification of LRP1 expression in controls (black bar) and variant carrier (white bar). C , Recovery of LRP1 mutant expression by culturing fibroblasts at both 37°C and 30°C for 48 hours. Bars represent fold induction. Quantification of LRP1 expression. D , Wild-type and E3983D-mutant LRP1 quantification using mass spectrometry. Endogenous signal is shown in light gray, standard in dark gray. Carrier ( left ) and 3 controls ( right ) are shown. E , PSAP (prosaposin; upper ) and CTSD (cathepsin D; lower ) expression levels in controls and variant carrier. Quantification of PSAP and CTSD expression. F , ABCA1 protein expression in total lysates ( left ) and on the cell surface ( right ). G , ABCA1 and SR-B1 mRNA expression levels. H , SR-B1 protein expression in total lysates ( left ) and on the cell surface ( right ). I , Immunofluorescence studies. Control ( left ) and carrier ( right ) fibroblasts were assessed for LRP1 (green), ABCA1 (red), and SR-B1 (purple) expression using confocal microscopy. In the above experiments, unchanged levels of β-actin or α-tubulin are shown as loading controls. Fibroblasts from controls were averaged and used as reference. Groups were compared using unpaired t tests. Data are expressed as mean±SEM of 3 independent experiments. * P
Figure Legend Snippet: ABCA1 (ATP-binding cassette A1) and SR-B1 (scavenger receptor class B type 1) protein levels in the LRP1 (low-density lipoprotein receptor–related protein 1) variant carrier and control fibroblasts. A , Real-time polymerase chain reaction quantification of LRP1 expression in total cell lysates. β-actin and GAPDH were used as housekeeping genes. B , Representative Western blot showing LRP1 expression in 3 control individuals and the variant carrier. Quantification of LRP1 expression in controls (black bar) and variant carrier (white bar). C , Recovery of LRP1 mutant expression by culturing fibroblasts at both 37°C and 30°C for 48 hours. Bars represent fold induction. Quantification of LRP1 expression. D , Wild-type and E3983D-mutant LRP1 quantification using mass spectrometry. Endogenous signal is shown in light gray, standard in dark gray. Carrier ( left ) and 3 controls ( right ) are shown. E , PSAP (prosaposin; upper ) and CTSD (cathepsin D; lower ) expression levels in controls and variant carrier. Quantification of PSAP and CTSD expression. F , ABCA1 protein expression in total lysates ( left ) and on the cell surface ( right ). G , ABCA1 and SR-B1 mRNA expression levels. H , SR-B1 protein expression in total lysates ( left ) and on the cell surface ( right ). I , Immunofluorescence studies. Control ( left ) and carrier ( right ) fibroblasts were assessed for LRP1 (green), ABCA1 (red), and SR-B1 (purple) expression using confocal microscopy. In the above experiments, unchanged levels of β-actin or α-tubulin are shown as loading controls. Fibroblasts from controls were averaged and used as reference. Groups were compared using unpaired t tests. Data are expressed as mean±SEM of 3 independent experiments. * P

Techniques Used: Binding Assay, Variant Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Mutagenesis, Mass Spectrometry, Immunofluorescence, Confocal Microscopy

ABCA1 (ATP-binding cassette A1) and SR-B1 (scavenger receptor class B type 1) levels in LRP1 (low-density lipoprotein receptor–related protein 1) CRISPR/Cas9 knockout cells. A , Representative Western blot showing LRP1 expression in 3 wild-type (WT) and knockout (KO) cell lines, respectively. Quantification of LRP1 expression in WT (black bar) and KO (white bar). B , Quantification of CTSD (cathepsin D) and PSAP (prosaposin) expression in controls and KO cells. C , ABCA1 and SR-B1 protein expression in total lysates and on the cell surface and quantification of ABCA1 and SR-B1 expression. Unchanged levels of β-actin, α-tubulin are shown as loading controls. WT cell lines were used as reference. Groups were compared using unpaired t tests. Data are expressed as mean±SEM of 3 independent experiments. * P
Figure Legend Snippet: ABCA1 (ATP-binding cassette A1) and SR-B1 (scavenger receptor class B type 1) levels in LRP1 (low-density lipoprotein receptor–related protein 1) CRISPR/Cas9 knockout cells. A , Representative Western blot showing LRP1 expression in 3 wild-type (WT) and knockout (KO) cell lines, respectively. Quantification of LRP1 expression in WT (black bar) and KO (white bar). B , Quantification of CTSD (cathepsin D) and PSAP (prosaposin) expression in controls and KO cells. C , ABCA1 and SR-B1 protein expression in total lysates and on the cell surface and quantification of ABCA1 and SR-B1 expression. Unchanged levels of β-actin, α-tubulin are shown as loading controls. WT cell lines were used as reference. Groups were compared using unpaired t tests. Data are expressed as mean±SEM of 3 independent experiments. * P

Techniques Used: Binding Assay, CRISPR, Knock-Out, Western Blot, Expressing

31) Product Images from "Dual extra-retinal origins of microglia in the model of retinal microglia repopulation"

Article Title: Dual extra-retinal origins of microglia in the model of retinal microglia repopulation

Journal: Cell Discovery

doi: 10.1038/s41421-018-0011-8

Repopulated microglia replenish the entire retina after removal of CSF1R inhibition . a Scheme of microglial repopulation and time points for examination. b Spatial distributions of retinal microglia show that microglia repopulate the whole retina after removal of PLX5622. Each green dot represents a microglial cell. c Zoom-in images of microglia in the OPL. Green: GFP; magenta: BrdU. d Quantification of microglial density in the OPL during microglial repopulation. The red line and pink area indicate the mean and SD of microglial density in normal retinas, respectively. NS not significant; * p
Figure Legend Snippet: Repopulated microglia replenish the entire retina after removal of CSF1R inhibition . a Scheme of microglial repopulation and time points for examination. b Spatial distributions of retinal microglia show that microglia repopulate the whole retina after removal of PLX5622. Each green dot represents a microglial cell. c Zoom-in images of microglia in the OPL. Green: GFP; magenta: BrdU. d Quantification of microglial density in the OPL during microglial repopulation. The red line and pink area indicate the mean and SD of microglial density in normal retinas, respectively. NS not significant; * p

Techniques Used: Inhibition

32) Product Images from "Naturally Occurring Variants in LRP1 (Low-Density Lipoprotein Receptor–Related Protein 1) Affect HDL (High-Density Lipoprotein) Metabolism Through ABCA1 (ATP-Binding Cassette A1) and SR-B1 (Scavenger Receptor Class B Type 1) in Humans"

Article Title: Naturally Occurring Variants in LRP1 (Low-Density Lipoprotein Receptor–Related Protein 1) Affect HDL (High-Density Lipoprotein) Metabolism Through ABCA1 (ATP-Binding Cassette A1) and SR-B1 (Scavenger Receptor Class B Type 1) in Humans

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/ATVBAHA.117.310309

ABCA1 (ATP-binding cassette A1) and SR-B1 (scavenger receptor class B type 1) protein levels in the LRP1 (low-density lipoprotein receptor–related protein 1) variant carrier and control fibroblasts. A , Real-time polymerase chain reaction quantification of LRP1 expression in total cell lysates. β-actin and GAPDH were used as housekeeping genes. B , Representative Western blot showing LRP1 expression in 3 control individuals and the variant carrier. Quantification of LRP1 expression in controls (black bar) and variant carrier (white bar). C , Recovery of LRP1 mutant expression by culturing fibroblasts at both 37°C and 30°C for 48 hours. Bars represent fold induction. Quantification of LRP1 expression. D , Wild-type and E3983D-mutant LRP1 quantification using mass spectrometry. Endogenous signal is shown in light gray, standard in dark gray. Carrier ( left ) and 3 controls ( right ) are shown. E , PSAP (prosaposin; upper ) and CTSD (cathepsin D; lower ) expression levels in controls and variant carrier. Quantification of PSAP and CTSD expression. F , ABCA1 protein expression in total lysates ( left ) and on the cell surface ( right ). G , ABCA1 and SR-B1 mRNA expression levels. H , SR-B1 protein expression in total lysates ( left ) and on the cell surface ( right ). I , Immunofluorescence studies. Control ( left ) and carrier ( right ) fibroblasts were assessed for LRP1 (green), ABCA1 (red), and SR-B1 (purple) expression using confocal microscopy. In the above experiments, unchanged levels of β-actin or α-tubulin are shown as loading controls. Fibroblasts from controls were averaged and used as reference. Groups were compared using unpaired t tests. Data are expressed as mean±SEM of 3 independent experiments. * P
Figure Legend Snippet: ABCA1 (ATP-binding cassette A1) and SR-B1 (scavenger receptor class B type 1) protein levels in the LRP1 (low-density lipoprotein receptor–related protein 1) variant carrier and control fibroblasts. A , Real-time polymerase chain reaction quantification of LRP1 expression in total cell lysates. β-actin and GAPDH were used as housekeeping genes. B , Representative Western blot showing LRP1 expression in 3 control individuals and the variant carrier. Quantification of LRP1 expression in controls (black bar) and variant carrier (white bar). C , Recovery of LRP1 mutant expression by culturing fibroblasts at both 37°C and 30°C for 48 hours. Bars represent fold induction. Quantification of LRP1 expression. D , Wild-type and E3983D-mutant LRP1 quantification using mass spectrometry. Endogenous signal is shown in light gray, standard in dark gray. Carrier ( left ) and 3 controls ( right ) are shown. E , PSAP (prosaposin; upper ) and CTSD (cathepsin D; lower ) expression levels in controls and variant carrier. Quantification of PSAP and CTSD expression. F , ABCA1 protein expression in total lysates ( left ) and on the cell surface ( right ). G , ABCA1 and SR-B1 mRNA expression levels. H , SR-B1 protein expression in total lysates ( left ) and on the cell surface ( right ). I , Immunofluorescence studies. Control ( left ) and carrier ( right ) fibroblasts were assessed for LRP1 (green), ABCA1 (red), and SR-B1 (purple) expression using confocal microscopy. In the above experiments, unchanged levels of β-actin or α-tubulin are shown as loading controls. Fibroblasts from controls were averaged and used as reference. Groups were compared using unpaired t tests. Data are expressed as mean±SEM of 3 independent experiments. * P

Techniques Used: Binding Assay, Variant Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Mutagenesis, Mass Spectrometry, Immunofluorescence, Confocal Microscopy

ABCA1 (ATP-binding cassette A1) and SR-B1 (scavenger receptor class B type 1) levels in LRP1 (low-density lipoprotein receptor–related protein 1) CRISPR/Cas9 knockout cells. A , Representative Western blot showing LRP1 expression in 3 wild-type (WT) and knockout (KO) cell lines, respectively. Quantification of LRP1 expression in WT (black bar) and KO (white bar). B , Quantification of CTSD (cathepsin D) and PSAP (prosaposin) expression in controls and KO cells. C , ABCA1 and SR-B1 protein expression in total lysates and on the cell surface and quantification of ABCA1 and SR-B1 expression. Unchanged levels of β-actin, α-tubulin are shown as loading controls. WT cell lines were used as reference. Groups were compared using unpaired t tests. Data are expressed as mean±SEM of 3 independent experiments. * P
Figure Legend Snippet: ABCA1 (ATP-binding cassette A1) and SR-B1 (scavenger receptor class B type 1) levels in LRP1 (low-density lipoprotein receptor–related protein 1) CRISPR/Cas9 knockout cells. A , Representative Western blot showing LRP1 expression in 3 wild-type (WT) and knockout (KO) cell lines, respectively. Quantification of LRP1 expression in WT (black bar) and KO (white bar). B , Quantification of CTSD (cathepsin D) and PSAP (prosaposin) expression in controls and KO cells. C , ABCA1 and SR-B1 protein expression in total lysates and on the cell surface and quantification of ABCA1 and SR-B1 expression. Unchanged levels of β-actin, α-tubulin are shown as loading controls. WT cell lines were used as reference. Groups were compared using unpaired t tests. Data are expressed as mean±SEM of 3 independent experiments. * P

Techniques Used: Binding Assay, CRISPR, Knock-Out, Western Blot, Expressing

33) Product Images from "Hippocampal interleukin-1 mediates stress-enhanced fear learning: A potential role for astrocyte-derived interleukin-1β"

Article Title: Hippocampal interleukin-1 mediates stress-enhanced fear learning: A potential role for astrocyte-derived interleukin-1β

Journal: Brain, behavior, and immunity

doi: 10.1016/j.bbi.2017.09.016

Severe stress increases hippocampal IL-1β immunoreactivity The stress-induced increase in hippocampal IL-1β that we previously reported is replicated here. Representative images of IL-1β immunoreactivity in the dentate gyrus of the DH acquired at 10X are shown from stressed (Foot Shock in context A) and non-stressed (No Foot Shock in Context A) rats. Top panel shows a tiled 10X image, while bottom panel shows a single 10X image. For the bottom panel, Bitplane Imaris was used for background subtraction to better visualize individual cells presented. Paxinos and Watson (2007) schematic shows the approximate region of the DH where images were acquired, AP −3.36 from bregma. Quantification of IL-1β immunoreactivity revealed that exposure to severe stress (15 foot shocks) significantly increased IL-1β immunoreactivity in the DH 48 hours post-stress. * p
Figure Legend Snippet: Severe stress increases hippocampal IL-1β immunoreactivity The stress-induced increase in hippocampal IL-1β that we previously reported is replicated here. Representative images of IL-1β immunoreactivity in the dentate gyrus of the DH acquired at 10X are shown from stressed (Foot Shock in context A) and non-stressed (No Foot Shock in Context A) rats. Top panel shows a tiled 10X image, while bottom panel shows a single 10X image. For the bottom panel, Bitplane Imaris was used for background subtraction to better visualize individual cells presented. Paxinos and Watson (2007) schematic shows the approximate region of the DH where images were acquired, AP −3.36 from bregma. Quantification of IL-1β immunoreactivity revealed that exposure to severe stress (15 foot shocks) significantly increased IL-1β immunoreactivity in the DH 48 hours post-stress. * p

Techniques Used:

IL-1β signal is colocalized with GFAP, and not with Iba-1 or NeuN, in the dorsal hippocampus in stressed and non-stressed animals A. Representative images of IL-1β, NeuN, Iba-1, and GFAP immunoreactivity in the dentate gyrus of the DH (AP −3.36 mm from bregma) acquired at 20X are shown. Because we did not detect any differences in colocalization between stressed and non-stressed rats, all images here are taken from animals that received stress exposure. Bitplane Imaris was used for background subtraction to better visualize individual cells presented. B. Bitplane Imaris software was used to calculate the colocalization of the IL-1β signal with GFAP, Iba-1, and NeuN. Colocalization analyses revealed that the percent of the IL-1β signal colocalized with GFAP was significantly greater than the percent of the IL-1β signal colocalized with either Iba-1 or NeuN. * p
Figure Legend Snippet: IL-1β signal is colocalized with GFAP, and not with Iba-1 or NeuN, in the dorsal hippocampus in stressed and non-stressed animals A. Representative images of IL-1β, NeuN, Iba-1, and GFAP immunoreactivity in the dentate gyrus of the DH (AP −3.36 mm from bregma) acquired at 20X are shown. Because we did not detect any differences in colocalization between stressed and non-stressed rats, all images here are taken from animals that received stress exposure. Bitplane Imaris was used for background subtraction to better visualize individual cells presented. B. Bitplane Imaris software was used to calculate the colocalization of the IL-1β signal with GFAP, Iba-1, and NeuN. Colocalization analyses revealed that the percent of the IL-1β signal colocalized with GFAP was significantly greater than the percent of the IL-1β signal colocalized with either Iba-1 or NeuN. * p

Techniques Used: Software

34) Product Images from "Acute inflammation sensitizes knee-innervating sensory neurons and decreases mouse digging behavior in a TRPV1-dependent manner"

Article Title: Acute inflammation sensitizes knee-innervating sensory neurons and decreases mouse digging behavior in a TRPV1-dependent manner

Journal: Neuropharmacology

doi: 10.1016/j.neuropharm.2018.09.014

Digging behavior after injection of the TRPV1 antagonist A-425619. A) Experimental timeline highlighting the days where behavioral testing and injection was performed. Distribution of time spent digging and the number of burrows in the CFA (B, C, n = 10) and saline group (D, E, n = 7). * indicates p
Figure Legend Snippet: Digging behavior after injection of the TRPV1 antagonist A-425619. A) Experimental timeline highlighting the days where behavioral testing and injection was performed. Distribution of time spent digging and the number of burrows in the CFA (B, C, n = 10) and saline group (D, E, n = 7). * indicates p

Techniques Used: Injection

TRP agonist response profile of knee neurons following acute knee inflammation. Representative traces of 10 μM capsaicin (pink, Ai), 100 μM cinnamaldehyde (orange, Bi) and 100 μM menthol (blue, Ci) response from a CFA neuron and their respective percentage frequency (Aii, Bii, Cii). The numbers above the bars indicate the number of responsive neurons. D) Heat map of Cntrl (i, n = 27) and CFA (ii, n = 25) neurons responding to capsaicin, cinnamaldehyde and menthol. E (i) Representative images of a whole DRG section from L4 showing fast blue (FB) labeling from the knee (blue), TRPV1 expression (pink), TrkA expression (green) and a merged image; sections from an animal injected with CFA. White arrowhead shows a knee neuron that only expresses TrkA, white arrowhead with asterisk shows a knee neuron that expresses only TRPV1 and white arrow shows a knee neuron that co-expresses TRPV1 and TrkA. (ii) Proportion of knee neurons (L2-L5) that express TRPV1 (pink), TrkA (green) and both TRPV1 and TrkA (green and pink stripes) from Cntrl (n = 1334) and CFA (n = 1089) injected side. Numbers above the bars represent neurons stained positive with respective antibodies. * indicates p
Figure Legend Snippet: TRP agonist response profile of knee neurons following acute knee inflammation. Representative traces of 10 μM capsaicin (pink, Ai), 100 μM cinnamaldehyde (orange, Bi) and 100 μM menthol (blue, Ci) response from a CFA neuron and their respective percentage frequency (Aii, Bii, Cii). The numbers above the bars indicate the number of responsive neurons. D) Heat map of Cntrl (i, n = 27) and CFA (ii, n = 25) neurons responding to capsaicin, cinnamaldehyde and menthol. E (i) Representative images of a whole DRG section from L4 showing fast blue (FB) labeling from the knee (blue), TRPV1 expression (pink), TrkA expression (green) and a merged image; sections from an animal injected with CFA. White arrowhead shows a knee neuron that only expresses TrkA, white arrowhead with asterisk shows a knee neuron that expresses only TRPV1 and white arrow shows a knee neuron that co-expresses TRPV1 and TrkA. (ii) Proportion of knee neurons (L2-L5) that express TRPV1 (pink), TrkA (green) and both TRPV1 and TrkA (green and pink stripes) from Cntrl (n = 1334) and CFA (n = 1089) injected side. Numbers above the bars represent neurons stained positive with respective antibodies. * indicates p

Techniques Used: Labeling, Expressing, Injection, Staining

35) Product Images from "Acute inflammation sensitizes knee-innervating sensory neurons and decreases mouse digging behavior in a TRPV1-dependent manner"

Article Title: Acute inflammation sensitizes knee-innervating sensory neurons and decreases mouse digging behavior in a TRPV1-dependent manner

Journal: Neuropharmacology

doi: 10.1016/j.neuropharm.2018.09.014

Digging behavior after injection of the TRPV1 antagonist A-425619. A) Experimental timeline highlighting the days where behavioral testing and injection was performed. Distribution of time spent digging and the number of burrows in the CFA (B, C, n = 10) and saline group (D, E, n = 7). * indicates p
Figure Legend Snippet: Digging behavior after injection of the TRPV1 antagonist A-425619. A) Experimental timeline highlighting the days where behavioral testing and injection was performed. Distribution of time spent digging and the number of burrows in the CFA (B, C, n = 10) and saline group (D, E, n = 7). * indicates p

Techniques Used: Injection

TRP agonist response profile of knee neurons following acute knee inflammation. Representative traces of 10 μM capsaicin (pink, Ai), 100 μM cinnamaldehyde (orange, Bi) and 100 μM menthol (blue, Ci) response from a CFA neuron and their respective percentage frequency (Aii, Bii, Cii). The numbers above the bars indicate the number of responsive neurons. D) Heat map of Cntrl (i, n = 27) and CFA (ii, n = 25) neurons responding to capsaicin, cinnamaldehyde and menthol. E (i) Representative images of a whole DRG section from L4 showing fast blue (FB) labeling from the knee (blue), TRPV1 expression (pink), TrkA expression (green) and a merged image; sections from an animal injected with CFA. White arrowhead shows a knee neuron that only expresses TrkA, white arrowhead with asterisk shows a knee neuron that expresses only TRPV1 and white arrow shows a knee neuron that co-expresses TRPV1 and TrkA. (ii) Proportion of knee neurons (L2-L5) that express TRPV1 (pink), TrkA (green) and both TRPV1 and TrkA (green and pink stripes) from Cntrl (n = 1334) and CFA (n = 1089) injected side. Numbers above the bars represent neurons stained positive with respective antibodies. * indicates p
Figure Legend Snippet: TRP agonist response profile of knee neurons following acute knee inflammation. Representative traces of 10 μM capsaicin (pink, Ai), 100 μM cinnamaldehyde (orange, Bi) and 100 μM menthol (blue, Ci) response from a CFA neuron and their respective percentage frequency (Aii, Bii, Cii). The numbers above the bars indicate the number of responsive neurons. D) Heat map of Cntrl (i, n = 27) and CFA (ii, n = 25) neurons responding to capsaicin, cinnamaldehyde and menthol. E (i) Representative images of a whole DRG section from L4 showing fast blue (FB) labeling from the knee (blue), TRPV1 expression (pink), TrkA expression (green) and a merged image; sections from an animal injected with CFA. White arrowhead shows a knee neuron that only expresses TrkA, white arrowhead with asterisk shows a knee neuron that expresses only TRPV1 and white arrow shows a knee neuron that co-expresses TRPV1 and TrkA. (ii) Proportion of knee neurons (L2-L5) that express TRPV1 (pink), TrkA (green) and both TRPV1 and TrkA (green and pink stripes) from Cntrl (n = 1334) and CFA (n = 1089) injected side. Numbers above the bars represent neurons stained positive with respective antibodies. * indicates p

Techniques Used: Labeling, Expressing, Injection, Staining

36) Product Images from "Dynamics of Salmonella infection of macrophages at the single cell level"

Article Title: Dynamics of Salmonella infection of macrophages at the single cell level

Journal: Journal of the Royal Society Interface

doi: 10.1098/rsif.2012.0163

Salmonella Typhimurium are contained in Salmonella -containing vacuoles (SCVs) at different stages of maturation, indicating that reinfection events have occurred. Overnight cultures of S. Typhimurium SL1344 expressing GFP (G) were added to BMDM at an MOI of 100. Samples were fixed at 10 min post-infection and immunolocalization for extracellular bacteria was performed using anti-O5 antisera. The panels show the localization of extracellular G bacteria, the early phagosomal protein EEA1, the late phagosomal protein LAMP-1, the overlaid image of G, EEA1 and LAMP-1 and the merged picture of all the images in phase contrast. The arrows indicate SCVs of differing maturity containing S. Typhimurium. A total of 100 BMDM were assessed per experiment and this was repeated on three separate occasions.
Figure Legend Snippet: Salmonella Typhimurium are contained in Salmonella -containing vacuoles (SCVs) at different stages of maturation, indicating that reinfection events have occurred. Overnight cultures of S. Typhimurium SL1344 expressing GFP (G) were added to BMDM at an MOI of 100. Samples were fixed at 10 min post-infection and immunolocalization for extracellular bacteria was performed using anti-O5 antisera. The panels show the localization of extracellular G bacteria, the early phagosomal protein EEA1, the late phagosomal protein LAMP-1, the overlaid image of G, EEA1 and LAMP-1 and the merged picture of all the images in phase contrast. The arrows indicate SCVs of differing maturity containing S. Typhimurium. A total of 100 BMDM were assessed per experiment and this was repeated on three separate occasions.

Techniques Used: Expressing, Infection

37) Product Images from "Apolipoprotein E4 impairs spontaneous blood brain barrier repair following traumatic brain injury"

Article Title: Apolipoprotein E4 impairs spontaneous blood brain barrier repair following traumatic brain injury

Journal: Molecular Neurodegeneration

doi: 10.1186/s13024-018-0249-5

APOE4 mice display prolonged BBB dysfunction at the NVU following TBI. ( a ) Immunohistochemistry analysis of lectin (inset: arrowheads = vessel, arrows = glial) and CD31 staining in the ipsilateral pericontusional cortex following TBI ( n = 3–4). ( b-c ) Quantitative immunohistochemistry showing the expression of Claudin-5 and Zonula Occludens-1 (ZO-1) in the ipsilateral pericontusional cortex of APOE3 and APOE4 sham and injured mice. ( n = 4, * p
Figure Legend Snippet: APOE4 mice display prolonged BBB dysfunction at the NVU following TBI. ( a ) Immunohistochemistry analysis of lectin (inset: arrowheads = vessel, arrows = glial) and CD31 staining in the ipsilateral pericontusional cortex following TBI ( n = 3–4). ( b-c ) Quantitative immunohistochemistry showing the expression of Claudin-5 and Zonula Occludens-1 (ZO-1) in the ipsilateral pericontusional cortex of APOE3 and APOE4 sham and injured mice. ( n = 4, * p

Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Expressing

38) Product Images from "Covalent modification of pericardial patches for sustained rapamycin delivery inhibits venous neointimal hyperplasia"

Article Title: Covalent modification of pericardial patches for sustained rapamycin delivery inhibits venous neointimal hyperplasia

Journal: Scientific Reports

doi: 10.1038/srep40142

NP-rapamycin inhibits SMC and macrophage infiltration and proliferation. ( a ) Representative Western blot showing expression of α-actin, CD68 and GAPDH in NP-control or NP-rapamycin-eluting patch neointimas at day 7 or day 30; n = 3. ( b ) Bar graph showing SMC density. *p
Figure Legend Snippet: NP-rapamycin inhibits SMC and macrophage infiltration and proliferation. ( a ) Representative Western blot showing expression of α-actin, CD68 and GAPDH in NP-control or NP-rapamycin-eluting patch neointimas at day 7 or day 30; n = 3. ( b ) Bar graph showing SMC density. *p

Techniques Used: Western Blot, Expressing

39) Product Images from "Induction of Pluripotent Stem Cells from Human Third Molar Mesenchymal Stromal Cells *"

Article Title: Induction of Pluripotent Stem Cells from Human Third Molar Mesenchymal Stromal Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.055889

Characterization of iPS cells from clonally expanded MSCs and HDF. A , immunocytochemistry of SSEA-3, SSEA-4, TRA-1–60, TRA-1–81, OCT3/4, and NANOG for iPS cells. Scale bars = 100 μm. B , DNA methylation states of the OCT3/4 and
Figure Legend Snippet: Characterization of iPS cells from clonally expanded MSCs and HDF. A , immunocytochemistry of SSEA-3, SSEA-4, TRA-1–60, TRA-1–81, OCT3/4, and NANOG for iPS cells. Scale bars = 100 μm. B , DNA methylation states of the OCT3/4 and

Techniques Used: Immunocytochemistry, DNA Methylation Assay

40) Product Images from "Poly(ethylene glycol)-modified silk fibroin membrane as a carrier for limbal epithelial stem cell transplantation in a rabbit LSCD model"

Article Title: Poly(ethylene glycol)-modified silk fibroin membrane as a carrier for limbal epithelial stem cell transplantation in a rabbit LSCD model

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-017-0707-y

Rabbit LESCs cultured on PEG-modified SF membranes maintain characteristics of stem cells. a , b LESCs from the tissue explant cultures expressed p63α and ABCB5 on PEG-modified SF membranes. c Flow cytometric analysis for p63α and ABCB5 of LESCs cultured on culture plates and PEG-modified SF membranes. d LESCs cultured on PEG-modified SF membranes maintained high proliferative capacity (Ki67 staining). e Schematic summary of the experimental design for BrdU chase experiments. LESCs labeled by BrdU for 1 day, and chased in BrdU-free DMEM/F12 medium for 10 days. f Specific staining of BrdU-retaining LESCs cultured on PEG-modified SF membranes at 0, 4, and 10 days. Percentage of BrdU-retaining LESCs was quantified. Data was shown as mean ± SD from three independent experiments. One-way ANOVA analysis: * P
Figure Legend Snippet: Rabbit LESCs cultured on PEG-modified SF membranes maintain characteristics of stem cells. a , b LESCs from the tissue explant cultures expressed p63α and ABCB5 on PEG-modified SF membranes. c Flow cytometric analysis for p63α and ABCB5 of LESCs cultured on culture plates and PEG-modified SF membranes. d LESCs cultured on PEG-modified SF membranes maintained high proliferative capacity (Ki67 staining). e Schematic summary of the experimental design for BrdU chase experiments. LESCs labeled by BrdU for 1 day, and chased in BrdU-free DMEM/F12 medium for 10 days. f Specific staining of BrdU-retaining LESCs cultured on PEG-modified SF membranes at 0, 4, and 10 days. Percentage of BrdU-retaining LESCs was quantified. Data was shown as mean ± SD from three independent experiments. One-way ANOVA analysis: * P

Techniques Used: Cell Culture, Modification, Flow Cytometry, Staining, Labeling

Tissue explant and single cell-suspension cultures produced more LESCs and fewer stromal cells. a , b ABCB5 and p63α positivity in rabbit limbus, with negativity in central cornea. Arrows point to the ABCB5 + and p63α + LESCs in the limbus. c , d Expressions of proposed LESC markers (p63α and ABCB5) and stromal cell marker (α-SMA) in P1 and P2 LESCs from the three cultures. Data were given as mean ± SD from three independent experiments. One-way ANOVA analysis: * P
Figure Legend Snippet: Tissue explant and single cell-suspension cultures produced more LESCs and fewer stromal cells. a , b ABCB5 and p63α positivity in rabbit limbus, with negativity in central cornea. Arrows point to the ABCB5 + and p63α + LESCs in the limbus. c , d Expressions of proposed LESC markers (p63α and ABCB5) and stromal cell marker (α-SMA) in P1 and P2 LESCs from the three cultures. Data were given as mean ± SD from three independent experiments. One-way ANOVA analysis: * P

Techniques Used: Produced, Marker

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Western Blot:

Article Title: A Bivalent Typhoid Live Vector Vaccine Expressing both Chromosome- and Plasmid-Encoded Yersinia pestis Antigens Fully Protects against Murine Lethal Pulmonary Plague Infection
Article Snippet: .. After separation, proteins were transferred to polyvinylidene difluoride (PVDF) membranes, and the production of F1 and LcrV proteins was examined by Western immunoblot analysis using mouse monoclonal anti-F1 IgG (clone Va13; Abcam, Cambridge, MA) or mouse monoclonal anti-LcrV IgG (clone YPF19; Abcam) antibody followed by goat anti-mouse IgG conjugated to horseradish peroxidase (KPL, Gaithersburg, MD). .. After applying enhanced chemiluminescence (ECL) substrate (PerkinElmer, Waltham, MA) to the PVDF membranes, the chemiluminescence signal was detected using an LAS-4000 imaging analyzer system (Fujifilm Life Science USA, Stamford, CT).

Article Title: Cancer cell secretion of the DAMP protein HMGB1 supports progression in malignant mesothelioma
Article Snippet: .. A total of 50 μg of protein lysates were used for Western blotting performed as previously described , using mouse monoclonal anti-HMGB1, rabbit polyclonal anti-RAGE, mouse monoclonal anti-TLR2 and goat polyclonal anti-TLR4 (Abcam). .. Anti-α-Tubulin (Calbiochem) and anti-Lamin B (Abcam) were used as loading controls for the cytoplasmic and nuclear fractions, respectively.

Incubation:

Article Title: Early activated hepatic stellate cell-derived molecules reverse acute hepatic injury
Article Snippet: .. After blocking with 5% BSA (Sigma Aldrich, St. Louis, MO, United States) in PBS, cells were incubated with mouse monoclonal anti-α-SMA IgG (Abcam, Cambridge, MA; 1:100) or rabbit anti-desmin (Abcam, Cambridge, MA; 1:100) for 2 h at room temperature, followed by development with donkey anti-mouse Alexa Fluor 488 (Invitrogen, Carlsbad, California, United States; 1:500) or donkey anti-rabbit Alexa Fluor 594 (Invitrogen, Carlsbad, California, United States; 1:500) for 30 min at room temperature, respectively. .. All plates were examined under an Axiovert 200 (Carl-Zeiss, Jena, Germany) using a computer-assisted image analysis program (AxioVision Ver.

Article Title: The expression profile of Dopamine D2 receptor, MGMT and VEGF in different histological subtypes of pituitary adenomas: a study of 197 cases and indications for the medical therapy
Article Snippet: .. Slides were first incubated with rabbit polyclonal anti-D2R (Abcam, Shanghai, China; 1:50), mouse monoclonal anti-MGMT (Abcam, Shanghai, China; 1:50) or mouse monoclonal anti-VEGF (Abcam, Shanghai, China; 1:50) overnight at 4°C, and then rinsed twice with PBS. .. Slides were then incubated with a secondary antibody for 15 min at 37°C followed by treatment with streptavidin–peroxidase reagent for 15 min, and rinsed twice with PBS.

Blocking Assay:

Article Title: Early activated hepatic stellate cell-derived molecules reverse acute hepatic injury
Article Snippet: .. After blocking with 5% BSA (Sigma Aldrich, St. Louis, MO, United States) in PBS, cells were incubated with mouse monoclonal anti-α-SMA IgG (Abcam, Cambridge, MA; 1:100) or rabbit anti-desmin (Abcam, Cambridge, MA; 1:100) for 2 h at room temperature, followed by development with donkey anti-mouse Alexa Fluor 488 (Invitrogen, Carlsbad, California, United States; 1:500) or donkey anti-rabbit Alexa Fluor 594 (Invitrogen, Carlsbad, California, United States; 1:500) for 30 min at room temperature, respectively. .. All plates were examined under an Axiovert 200 (Carl-Zeiss, Jena, Germany) using a computer-assisted image analysis program (AxioVision Ver.

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