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    Thermo Fisher gfp tag polyclonal antibody
    BRN2 rescues neurogenesis, but not survival, in Scz organoids. a Schematic of self-regulating BRN2 lentiviral vector and supplementation strategy for BRN2 rescue experiments. Briefly, we modified a previously validated lentiviral construct that transiently induces exogenous BRN2 expression but “switches-off” upon completion of neuronal differentiation and assumption of a post-mitotic neuronal fate. After supplementing neuronal transcriptional programs, exogenous BRN2 -Virus transcripts are decayed via binding of the neuron-specific noncoding RNA, miRNA-124, to recognition units embedded within viral transcripts (see Fig. S7 ). This BRN2 -Virus construct thus allows transient supplementation of BRN2 levels in Scz progenitors undergoing differentiation without sustained overexpression of this target in mature neurons. b – e , BRN2 supplementation increases neuron numbers in Scz organoids. Ctrl and Scz organoids were infected with CTRL-Virus and BRN2 -Virus during organoid neural induction. Images show <t>GFP+</t> cells within ventricular zones. Viral infection rates appeared similar between Ctrl and Scz organoids, and viral <t>GFP</t> expression was detected within progenitor pools as expected ( b ). Scz organoids infected with CTRL -Virus exhibited fewer neurons than Ctrl organoids. However, when comparing Scz organoids infected with CTRL- Virus versus BRN2- Virus, we observed a significant rescue of BRN2+ neuron number. Scz organoids infected with BRN2 -Virus exhibited substantially increased BRN2+ neuron numbers, which were comparable to Ctrl organoids. Enlarged whole-organoid images are provided in Supplementary Material (see Fig. S8 ), and graphs reflect raw data for complete data transparency of rescue effects ( d , CTRL -Virus Ctrl organoids n = 43 fields, n = 16 organoids, and n = 3 independent Ctrl lines; BRN2 -Virus Ctrl organoids n = 40 fields, n = 18 organoids, and n = 3 Ctrl independent lines; CTRL -Virus Scz organoids n = 65 fields, n = 25 organoids, and n = 5 independent Scz lines; BRN2 -Virus Scz organoids n = 42 fields, n = 14 organoids, and n = 4 independent Scz lines). To determine if this effect was reflected in pan neuronal numbers, we also examined MAP2+ neurons in infected organoids. Consistent with data in Fig. 2 , CTRL -Virus+ Scz organoids exhibited a decreased number of MAP2+ neurons compared to Ctrl organoids. However, MAP2+ neuron numbers were significantly increased in the BRN2 -Virus+ Scz organoids ( e , CTRL -Virus Ctrl organoids n = 73 fields, n = 35 organoids, and n = 5 independent Ctrl lines; BRN2 -Virus Ctrl organoids n = 55 fields, n = 26 organoids, and n = 3 Ctrl independent lines; CTRL -Virus Scz organoids n = 52 fields, n = 25 organoids, and n = 5 independent Scz lines; BRN2 -Virus Scz organoids n = 64 fields, n = 28 organoids, and n = 5 independent Scz lines). Thus, transient BRN2 supplementation resulted in a significant recovery of neurons in 3D Scz patient-derived organoids, which confirms a mechanistic role for BRN2 within Scz organoids. Each data point on graphs reflects raw data (an independent, non-overlapping, cortical field) for complete data transparency, with the average of individual iPSC lines provided in Supplementary Material (see Fig. S7d ). f No effect of BRN2 supplementation on progenitor cell death in Scz organoids. Scz organoids are associated with increased rates of cell death of ventricular zone neural progenitors (Fig. 1 ). To determine if BRN2 regulates the survival of progenitors, we assessed the number of CAS3+ in infected organoids. In both CTRL- and BRN2 -Virus infected Scz organoids, there was an increase in progenitor death relative to Ctrl samples ( e , CTRL -Virus Ctrl organoids n = 34 fields, n = 19 organoids, and n = 3 independent Ctrl lines; BRN2 -Virus Ctrl organoids n = 35 fields, n = 19 organoids, and n = 3 independent Ctrl lines; CTRL -Virus Scz organoids n = 35 fields, n = 20 organoids, and n = 4 independent Scz lines; BRN2 -Virus Scz organoids n = 45 fields, n = 23 organoids, and n = 4 independent Scz lines). These data indicate that decreased levels of BRN2 do not contribute to the increased apoptosis of progenitors in Scz organoids. Each data point on graphs reflects raw data (comprising an independent ventricular zone) for complete data transparency (for the average of groups, see Fig. S7d ). In sum, we found that BRN2 has a mechanistic role in promoting neuron production in Scz organoids, but not the survival of neuronal progenitors. This selective rescuing effect of BRN2 highlights that multiple factors and pathways likely combine to produce progenitor and neuronal pathology in developing cortical assemblies of Scz organoids. **** p
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    Images

    1) Product Images from "Schizophrenia is defined by cell-specific neuropathology and multiple neurodevelopmental mechanisms in patient-derived cerebral organoids"

    Article Title: Schizophrenia is defined by cell-specific neuropathology and multiple neurodevelopmental mechanisms in patient-derived cerebral organoids

    Journal: Molecular Psychiatry

    doi: 10.1038/s41380-021-01316-6

    BRN2 rescues neurogenesis, but not survival, in Scz organoids. a Schematic of self-regulating BRN2 lentiviral vector and supplementation strategy for BRN2 rescue experiments. Briefly, we modified a previously validated lentiviral construct that transiently induces exogenous BRN2 expression but “switches-off” upon completion of neuronal differentiation and assumption of a post-mitotic neuronal fate. After supplementing neuronal transcriptional programs, exogenous BRN2 -Virus transcripts are decayed via binding of the neuron-specific noncoding RNA, miRNA-124, to recognition units embedded within viral transcripts (see Fig. S7 ). This BRN2 -Virus construct thus allows transient supplementation of BRN2 levels in Scz progenitors undergoing differentiation without sustained overexpression of this target in mature neurons. b – e , BRN2 supplementation increases neuron numbers in Scz organoids. Ctrl and Scz organoids were infected with CTRL-Virus and BRN2 -Virus during organoid neural induction. Images show GFP+ cells within ventricular zones. Viral infection rates appeared similar between Ctrl and Scz organoids, and viral GFP expression was detected within progenitor pools as expected ( b ). Scz organoids infected with CTRL -Virus exhibited fewer neurons than Ctrl organoids. However, when comparing Scz organoids infected with CTRL- Virus versus BRN2- Virus, we observed a significant rescue of BRN2+ neuron number. Scz organoids infected with BRN2 -Virus exhibited substantially increased BRN2+ neuron numbers, which were comparable to Ctrl organoids. Enlarged whole-organoid images are provided in Supplementary Material (see Fig. S8 ), and graphs reflect raw data for complete data transparency of rescue effects ( d , CTRL -Virus Ctrl organoids n = 43 fields, n = 16 organoids, and n = 3 independent Ctrl lines; BRN2 -Virus Ctrl organoids n = 40 fields, n = 18 organoids, and n = 3 Ctrl independent lines; CTRL -Virus Scz organoids n = 65 fields, n = 25 organoids, and n = 5 independent Scz lines; BRN2 -Virus Scz organoids n = 42 fields, n = 14 organoids, and n = 4 independent Scz lines). To determine if this effect was reflected in pan neuronal numbers, we also examined MAP2+ neurons in infected organoids. Consistent with data in Fig. 2 , CTRL -Virus+ Scz organoids exhibited a decreased number of MAP2+ neurons compared to Ctrl organoids. However, MAP2+ neuron numbers were significantly increased in the BRN2 -Virus+ Scz organoids ( e , CTRL -Virus Ctrl organoids n = 73 fields, n = 35 organoids, and n = 5 independent Ctrl lines; BRN2 -Virus Ctrl organoids n = 55 fields, n = 26 organoids, and n = 3 Ctrl independent lines; CTRL -Virus Scz organoids n = 52 fields, n = 25 organoids, and n = 5 independent Scz lines; BRN2 -Virus Scz organoids n = 64 fields, n = 28 organoids, and n = 5 independent Scz lines). Thus, transient BRN2 supplementation resulted in a significant recovery of neurons in 3D Scz patient-derived organoids, which confirms a mechanistic role for BRN2 within Scz organoids. Each data point on graphs reflects raw data (an independent, non-overlapping, cortical field) for complete data transparency, with the average of individual iPSC lines provided in Supplementary Material (see Fig. S7d ). f No effect of BRN2 supplementation on progenitor cell death in Scz organoids. Scz organoids are associated with increased rates of cell death of ventricular zone neural progenitors (Fig. 1 ). To determine if BRN2 regulates the survival of progenitors, we assessed the number of CAS3+ in infected organoids. In both CTRL- and BRN2 -Virus infected Scz organoids, there was an increase in progenitor death relative to Ctrl samples ( e , CTRL -Virus Ctrl organoids n = 34 fields, n = 19 organoids, and n = 3 independent Ctrl lines; BRN2 -Virus Ctrl organoids n = 35 fields, n = 19 organoids, and n = 3 independent Ctrl lines; CTRL -Virus Scz organoids n = 35 fields, n = 20 organoids, and n = 4 independent Scz lines; BRN2 -Virus Scz organoids n = 45 fields, n = 23 organoids, and n = 4 independent Scz lines). These data indicate that decreased levels of BRN2 do not contribute to the increased apoptosis of progenitors in Scz organoids. Each data point on graphs reflects raw data (comprising an independent ventricular zone) for complete data transparency (for the average of groups, see Fig. S7d ). In sum, we found that BRN2 has a mechanistic role in promoting neuron production in Scz organoids, but not the survival of neuronal progenitors. This selective rescuing effect of BRN2 highlights that multiple factors and pathways likely combine to produce progenitor and neuronal pathology in developing cortical assemblies of Scz organoids. **** p
    Figure Legend Snippet: BRN2 rescues neurogenesis, but not survival, in Scz organoids. a Schematic of self-regulating BRN2 lentiviral vector and supplementation strategy for BRN2 rescue experiments. Briefly, we modified a previously validated lentiviral construct that transiently induces exogenous BRN2 expression but “switches-off” upon completion of neuronal differentiation and assumption of a post-mitotic neuronal fate. After supplementing neuronal transcriptional programs, exogenous BRN2 -Virus transcripts are decayed via binding of the neuron-specific noncoding RNA, miRNA-124, to recognition units embedded within viral transcripts (see Fig. S7 ). This BRN2 -Virus construct thus allows transient supplementation of BRN2 levels in Scz progenitors undergoing differentiation without sustained overexpression of this target in mature neurons. b – e , BRN2 supplementation increases neuron numbers in Scz organoids. Ctrl and Scz organoids were infected with CTRL-Virus and BRN2 -Virus during organoid neural induction. Images show GFP+ cells within ventricular zones. Viral infection rates appeared similar between Ctrl and Scz organoids, and viral GFP expression was detected within progenitor pools as expected ( b ). Scz organoids infected with CTRL -Virus exhibited fewer neurons than Ctrl organoids. However, when comparing Scz organoids infected with CTRL- Virus versus BRN2- Virus, we observed a significant rescue of BRN2+ neuron number. Scz organoids infected with BRN2 -Virus exhibited substantially increased BRN2+ neuron numbers, which were comparable to Ctrl organoids. Enlarged whole-organoid images are provided in Supplementary Material (see Fig. S8 ), and graphs reflect raw data for complete data transparency of rescue effects ( d , CTRL -Virus Ctrl organoids n = 43 fields, n = 16 organoids, and n = 3 independent Ctrl lines; BRN2 -Virus Ctrl organoids n = 40 fields, n = 18 organoids, and n = 3 Ctrl independent lines; CTRL -Virus Scz organoids n = 65 fields, n = 25 organoids, and n = 5 independent Scz lines; BRN2 -Virus Scz organoids n = 42 fields, n = 14 organoids, and n = 4 independent Scz lines). To determine if this effect was reflected in pan neuronal numbers, we also examined MAP2+ neurons in infected organoids. Consistent with data in Fig. 2 , CTRL -Virus+ Scz organoids exhibited a decreased number of MAP2+ neurons compared to Ctrl organoids. However, MAP2+ neuron numbers were significantly increased in the BRN2 -Virus+ Scz organoids ( e , CTRL -Virus Ctrl organoids n = 73 fields, n = 35 organoids, and n = 5 independent Ctrl lines; BRN2 -Virus Ctrl organoids n = 55 fields, n = 26 organoids, and n = 3 Ctrl independent lines; CTRL -Virus Scz organoids n = 52 fields, n = 25 organoids, and n = 5 independent Scz lines; BRN2 -Virus Scz organoids n = 64 fields, n = 28 organoids, and n = 5 independent Scz lines). Thus, transient BRN2 supplementation resulted in a significant recovery of neurons in 3D Scz patient-derived organoids, which confirms a mechanistic role for BRN2 within Scz organoids. Each data point on graphs reflects raw data (an independent, non-overlapping, cortical field) for complete data transparency, with the average of individual iPSC lines provided in Supplementary Material (see Fig. S7d ). f No effect of BRN2 supplementation on progenitor cell death in Scz organoids. Scz organoids are associated with increased rates of cell death of ventricular zone neural progenitors (Fig. 1 ). To determine if BRN2 regulates the survival of progenitors, we assessed the number of CAS3+ in infected organoids. In both CTRL- and BRN2 -Virus infected Scz organoids, there was an increase in progenitor death relative to Ctrl samples ( e , CTRL -Virus Ctrl organoids n = 34 fields, n = 19 organoids, and n = 3 independent Ctrl lines; BRN2 -Virus Ctrl organoids n = 35 fields, n = 19 organoids, and n = 3 independent Ctrl lines; CTRL -Virus Scz organoids n = 35 fields, n = 20 organoids, and n = 4 independent Scz lines; BRN2 -Virus Scz organoids n = 45 fields, n = 23 organoids, and n = 4 independent Scz lines). These data indicate that decreased levels of BRN2 do not contribute to the increased apoptosis of progenitors in Scz organoids. Each data point on graphs reflects raw data (comprising an independent ventricular zone) for complete data transparency (for the average of groups, see Fig. S7d ). In sum, we found that BRN2 has a mechanistic role in promoting neuron production in Scz organoids, but not the survival of neuronal progenitors. This selective rescuing effect of BRN2 highlights that multiple factors and pathways likely combine to produce progenitor and neuronal pathology in developing cortical assemblies of Scz organoids. **** p

    Techniques Used: Plasmid Preparation, Modification, Construct, Expressing, Binding Assay, Over Expression, Infection, Derivative Assay

    2) Product Images from "Hypoxia potentiates the inflammatory fibroblast phenotype promoted by pancreatic cancer cell-derived cytokines"

    Article Title: Hypoxia potentiates the inflammatory fibroblast phenotype promoted by pancreatic cancer cell-derived cytokines

    Journal: bioRxiv

    doi: 10.1101/2022.07.26.501639

    Hypoxia potentiates the cytokine-induced inflammatory fibroblast phenotype. (A, B) Fluorescence intensity of IL6 -EGFP expressing PSCs cultured in normoxia or hypoxia and mock-treated or treated with cytokines (IL1/TNFα) for 48h. (A) Histogram of IL6 -EGFP fluorescence intensity. (B) Quantification of the relative MFI of IL6 -EGFP. N=3 biological replicates. Data represent mean+SD. P-values were calculated by two-way ANOVA. (C) Representative images of IL6 -EGFP and αSMA -DsRed expressing PSCs cultured in normoxia or hypoxia and mock-treated or treated with cytokines for 48h. Scale bar = 200 µm. (D-F) MEMIC experiment. (D) Schematic of the MEMIC, adapted from ( 31 , 32 ). PSCs expressing IL6 -EGFP were plated in the inner chamber and treated with cytokines the next day. Gradients were allowed to form for 48h. (E) Representative image. Cells were fixed and stained for GFP ( IL6 ). Nuclei are labeled with DAPI. Scale bar = 500 µM. Oxygen-rich (E’) and oxygen-poor (E’’) regions are highlighted. Scale bar = 100 µM. (F) Quantification of GFP ( IL6 ) fluorescence intensity per cell with increasing distance from the oxygen-rich opening. A.U. = arbitrary units. N=15,027 nuclei. Line represents median. P-value was calculated by Pearson’s Linear Correlation Coefficient. (G, H) PSC/Tumor organoid co-culture experiment. PSCs expressing IL6 -EGFP and αSMA -DsRed were cultured alone or together with KPC organoids in Matrigel for five days. In the last 48h, part of the cultures was incubated in hypoxia. (G) Histogram of IL6 -EGFP fluorescence intensity in PSCs. (H) Quantification of the relative MFI of IL6 -EGFP in PSCs. N=3 biological replicates. Data represent mean+SD. P-values were calculated by two-way ANOVA.
    Figure Legend Snippet: Hypoxia potentiates the cytokine-induced inflammatory fibroblast phenotype. (A, B) Fluorescence intensity of IL6 -EGFP expressing PSCs cultured in normoxia or hypoxia and mock-treated or treated with cytokines (IL1/TNFα) for 48h. (A) Histogram of IL6 -EGFP fluorescence intensity. (B) Quantification of the relative MFI of IL6 -EGFP. N=3 biological replicates. Data represent mean+SD. P-values were calculated by two-way ANOVA. (C) Representative images of IL6 -EGFP and αSMA -DsRed expressing PSCs cultured in normoxia or hypoxia and mock-treated or treated with cytokines for 48h. Scale bar = 200 µm. (D-F) MEMIC experiment. (D) Schematic of the MEMIC, adapted from ( 31 , 32 ). PSCs expressing IL6 -EGFP were plated in the inner chamber and treated with cytokines the next day. Gradients were allowed to form for 48h. (E) Representative image. Cells were fixed and stained for GFP ( IL6 ). Nuclei are labeled with DAPI. Scale bar = 500 µM. Oxygen-rich (E’) and oxygen-poor (E’’) regions are highlighted. Scale bar = 100 µM. (F) Quantification of GFP ( IL6 ) fluorescence intensity per cell with increasing distance from the oxygen-rich opening. A.U. = arbitrary units. N=15,027 nuclei. Line represents median. P-value was calculated by Pearson’s Linear Correlation Coefficient. (G, H) PSC/Tumor organoid co-culture experiment. PSCs expressing IL6 -EGFP and αSMA -DsRed were cultured alone or together with KPC organoids in Matrigel for five days. In the last 48h, part of the cultures was incubated in hypoxia. (G) Histogram of IL6 -EGFP fluorescence intensity in PSCs. (H) Quantification of the relative MFI of IL6 -EGFP in PSCs. N=3 biological replicates. Data represent mean+SD. P-values were calculated by two-way ANOVA.

    Techniques Used: Fluorescence, Expressing, Cell Culture, Staining, Labeling, Co-Culture Assay, Incubation

    3) Product Images from "Unraveling Complex Interplay between Heat Shock Factor 1 and 2 Splicing Isoforms"

    Article Title: Unraveling Complex Interplay between Heat Shock Factor 1 and 2 Splicing Isoforms

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056085

    Transcriptional activity of HSF1 and HSF2 isoforms. (A) Hsf1.2 −/− iMEFs were co-transfected with increasing quantity of pCR3.1-HSF1α (left), or pCR3.1-HSF1β (right), in addition to pHSE x2 -TATA-Luc used as a reporter gene. DNA quantities were adjusted with empty pCR3.1. Transfection efficiency was assessed using the pTK-Rluc reporter gene. Cells were treated with MG132 at 2.5 µM (white) or with DMSO (black) as control, for 8 h. Results correspond to the ratio between firefly luciferase (FLucif) and renilla luciferase (RLucif) activities. The data are from a representative experiment including three independent replicates (mean +/− SD). (B) Representative Western-blot showing the expression of HSF1 isoforms after transfection. Hsf1.2 −/− iMEFs were co-transfected with increasing quantity of pCR3.1-HSF1α (left) or pCR3.1-HSF1β (right) and pEGFP as control for transfection efficiency. Cells were treated with MG132 at 2.5 µM and immunoblots for HSF1 and GFP were performed. (C) Hsf1.2 −/− iMEFs were co-transfected with 12.5 ng of pCR3.1-HSF1α (full line), or pCR3.1-HSF1β (dotted line), with two reporter genes described previously. Cells were treated with MG132 at 2.5 µM or with DMSO, for 2 h, 4 h, 6 h or 8 h. Results correspond to the ratio between firefly luciferase (FLucif) and renilla luciferase (RLucif) activities and are the mean of three independent experiments +/− SD. (D) Hsf1.2 −/− iMEFs were co-transfected with 12.5 ng of pCR3.1-HSF2α, or pCR3.1-HSF2β, with the reporter genes as described in (A). Cells were treated with MG132 at 2.5 µM (black), or with DMSO (white), for 8 h. Results are expressed in percentage of empty vector and represent the mean of three independent experiments +/− SD (Student’s t test, ns: no significant). (E) Representative Western-blot showing the expression of HSF2 isoforms after transfection. Hsf1.2 −/− iMEFs were co-transfected with increasing quantity of pCR3.1-HSF2α (high panel) or pCR3.1-HSF2β (low panel) and pEGFP as control for efficiency. Cells were treated with MG132 at 2.5 µM and immunoblots for HSF2 and GFP were performed.
    Figure Legend Snippet: Transcriptional activity of HSF1 and HSF2 isoforms. (A) Hsf1.2 −/− iMEFs were co-transfected with increasing quantity of pCR3.1-HSF1α (left), or pCR3.1-HSF1β (right), in addition to pHSE x2 -TATA-Luc used as a reporter gene. DNA quantities were adjusted with empty pCR3.1. Transfection efficiency was assessed using the pTK-Rluc reporter gene. Cells were treated with MG132 at 2.5 µM (white) or with DMSO (black) as control, for 8 h. Results correspond to the ratio between firefly luciferase (FLucif) and renilla luciferase (RLucif) activities. The data are from a representative experiment including three independent replicates (mean +/− SD). (B) Representative Western-blot showing the expression of HSF1 isoforms after transfection. Hsf1.2 −/− iMEFs were co-transfected with increasing quantity of pCR3.1-HSF1α (left) or pCR3.1-HSF1β (right) and pEGFP as control for transfection efficiency. Cells were treated with MG132 at 2.5 µM and immunoblots for HSF1 and GFP were performed. (C) Hsf1.2 −/− iMEFs were co-transfected with 12.5 ng of pCR3.1-HSF1α (full line), or pCR3.1-HSF1β (dotted line), with two reporter genes described previously. Cells were treated with MG132 at 2.5 µM or with DMSO, for 2 h, 4 h, 6 h or 8 h. Results correspond to the ratio between firefly luciferase (FLucif) and renilla luciferase (RLucif) activities and are the mean of three independent experiments +/− SD. (D) Hsf1.2 −/− iMEFs were co-transfected with 12.5 ng of pCR3.1-HSF2α, or pCR3.1-HSF2β, with the reporter genes as described in (A). Cells were treated with MG132 at 2.5 µM (black), or with DMSO (white), for 8 h. Results are expressed in percentage of empty vector and represent the mean of three independent experiments +/− SD (Student’s t test, ns: no significant). (E) Representative Western-blot showing the expression of HSF2 isoforms after transfection. Hsf1.2 −/− iMEFs were co-transfected with increasing quantity of pCR3.1-HSF2α (high panel) or pCR3.1-HSF2β (low panel) and pEGFP as control for efficiency. Cells were treated with MG132 at 2.5 µM and immunoblots for HSF2 and GFP were performed.

    Techniques Used: Activity Assay, Transfection, Luciferase, Western Blot, Expressing, Plasmid Preparation

    4) Product Images from "Photoreceptors generate neuronal diversity in their target field through a Hedgehog morphogen gradient in Drosophila"

    Article Title: Photoreceptors generate neuronal diversity in their target field through a Hedgehog morphogen gradient in Drosophila

    Journal: eLife

    doi: 10.7554/eLife.78093

    Hh::GFP is distributed in a protein gradient in the lamina. ( A ) A maximum intensity projection of a hh-sfGFP/+ optic lobe and eye disc complex (fixed tissue). Hh::GFP (cyan) detected by immunohistochemistry was present in photoreceptor cell bodies in the eye disc (Embryonic lethal abnormal vision [Elav]; yellow), with higher levels in younger photoreceptors as reported previously ( Huang and Kunes, 1996 ) and photoreceptor axons in the optic stalk (Horseradish peroxidase [HRP]; magenta). The Hh::GFP expression decreased rapidly once photoreceptors entered the lamina (brackets). ( B ) A cross-sectional view of the lamina from a live explant of hh-sfGFP/+ . Hh::GFP puncta were visible more prominently in the distal lamina, with fewer and smaller puncta appearing in proximal regions. White dashed lines mark the lamina furrow. The yellow dashed line marks the youngest column. ( C ) Hh::GFP mean fluorescence intensity (MFI) plots from live explants normalised to the maximum MFI value for each plot (arbitrary units; a.u.) as a function of distance from distal to proximal cell position as indicated for the youngest lamina column (yellow dashed outline in B). The red line shows regression averaging of each of the MFI profiles (see Materials and methods section). Scale bar = 20 µm.
    Figure Legend Snippet: Hh::GFP is distributed in a protein gradient in the lamina. ( A ) A maximum intensity projection of a hh-sfGFP/+ optic lobe and eye disc complex (fixed tissue). Hh::GFP (cyan) detected by immunohistochemistry was present in photoreceptor cell bodies in the eye disc (Embryonic lethal abnormal vision [Elav]; yellow), with higher levels in younger photoreceptors as reported previously ( Huang and Kunes, 1996 ) and photoreceptor axons in the optic stalk (Horseradish peroxidase [HRP]; magenta). The Hh::GFP expression decreased rapidly once photoreceptors entered the lamina (brackets). ( B ) A cross-sectional view of the lamina from a live explant of hh-sfGFP/+ . Hh::GFP puncta were visible more prominently in the distal lamina, with fewer and smaller puncta appearing in proximal regions. White dashed lines mark the lamina furrow. The yellow dashed line marks the youngest column. ( C ) Hh::GFP mean fluorescence intensity (MFI) plots from live explants normalised to the maximum MFI value for each plot (arbitrary units; a.u.) as a function of distance from distal to proximal cell position as indicated for the youngest lamina column (yellow dashed outline in B). The red line shows regression averaging of each of the MFI profiles (see Materials and methods section). Scale bar = 20 µm.

    Techniques Used: Immunohistochemistry, Expressing, Fluorescence

    Lamina-specific misexpression of the repressive form of Cubitus interruptus ( Ci rep ) and Ci RNAi perturbs specification. ( A ) An optic lobe with a GFP-positive (cyan) smo 3 MARCM clone (dashed outline) labelled with Dachsund Dac; (magenta), Embryonic lethal abnormal vision Elav; (yellow) and Horseradish peroxidase (HRP ; white). Cells within the clone did not express Dac and did not incorporate into the lamina. ( B ) R27G05-Gal4, a lamina-specific Gal4 driving expression of CD8::GFP (GFP in cyan and Dac in magenta). GFP expression initiated in lamina precursor cells exiting the lamina furrow. ( C–F ) Quantifications of the number of each lamina neuron type per focal slice, L2-L3, L1, L4, or L5, for each condition. One-way ANOVA with Dunn’s multiple comparisons test. p **
    Figure Legend Snippet: Lamina-specific misexpression of the repressive form of Cubitus interruptus ( Ci rep ) and Ci RNAi perturbs specification. ( A ) An optic lobe with a GFP-positive (cyan) smo 3 MARCM clone (dashed outline) labelled with Dachsund Dac; (magenta), Embryonic lethal abnormal vision Elav; (yellow) and Horseradish peroxidase (HRP ; white). Cells within the clone did not express Dac and did not incorporate into the lamina. ( B ) R27G05-Gal4, a lamina-specific Gal4 driving expression of CD8::GFP (GFP in cyan and Dac in magenta). GFP expression initiated in lamina precursor cells exiting the lamina furrow. ( C–F ) Quantifications of the number of each lamina neuron type per focal slice, L2-L3, L1, L4, or L5, for each condition. One-way ANOVA with Dunn’s multiple comparisons test. p **

    Techniques Used: Expressing

    Intermediate levels of Hedgehog (Hh) signalling activity specify intermediate lamina neuron identities when evaluated by alternative neuron-type markers. ( A–C ) Lamina-specific misexpression of ( A ) CD8::GFP (control), ( B ) Cubitus interruptus (Ci rep ) shifted to 29°C, and ( C ) Ci rep shifted to 25°C, labelled with lamina neuron-type-specific markers sloppy paired 2 (Slp2; cyan), brain-specific homeobox (Bsh; yellow), and seven-up (Svp; magenta). ( D–E ) Quantifications of the proportion of each lamina neuron type per focal slice, normalised to the control. Error bars represent SD. One-way ANOVA with Dunn’s multiple comparison test. p *
    Figure Legend Snippet: Intermediate levels of Hedgehog (Hh) signalling activity specify intermediate lamina neuron identities when evaluated by alternative neuron-type markers. ( A–C ) Lamina-specific misexpression of ( A ) CD8::GFP (control), ( B ) Cubitus interruptus (Ci rep ) shifted to 29°C, and ( C ) Ci rep shifted to 25°C, labelled with lamina neuron-type-specific markers sloppy paired 2 (Slp2; cyan), brain-specific homeobox (Bsh; yellow), and seven-up (Svp; magenta). ( D–E ) Quantifications of the proportion of each lamina neuron type per focal slice, normalised to the control. Error bars represent SD. One-way ANOVA with Dunn’s multiple comparison test. p *

    Techniques Used: Activity Assay

    5) Product Images from "Parallel functional testing identifies enhancers active in early postnatal mouse brain"

    Article Title: Parallel functional testing identifies enhancers active in early postnatal mouse brain

    Journal: bioRxiv

    doi: 10.1101/2021.01.15.426772

    Validation of cell-type specific enhancer function in the STARR-seq orientation. ( A, B ) Representative confocal images of coronal sections of P7 mouse brain transduced by intracranial injection at P0 with scAAV9-Hsp68-EGFP-mDlx ( A ) or AAV9-mDlx-βGlobinMinP-EGFP ( B ) and a CAG-mRuby3 positive control. Sections were stained with an antibody for EGFP for signal amplification. Green, EGFP; red, mRuby3; grey, DAPI. ( C ) Quantification of the numbers of Pyramidal (light blue), Non-pyramidal (darker blue), and cells of ambiguous morphology (grey) observed in confocal imaging experiment in A and B above; N = 5 animals, 314 cells transduced with Hsp68-EGFP-mDlx (Hsp68-3’UTR) and 4 animals, 972 cells transduced with mDlx-βGlobinMinP-EGFP (βGlobin-5’UTR). ( D ) Quantification of images from experiment shown in Figure 4F , with mDlx-βGlobinMinP-EGFP included for comparison. Individual GFP+ and mRuby3+ cells were counted and scored for whether each cell contained a Ctip2-positive nucleus. Cell counts were summed across images for the same brain. Data is presented as mean proportion of Ctip2-positive cells (n = 5 animals co-injected with Hsp68-EGFP-#161 and CAG-mRuby3, 4 animals co-injected with Hsp68-EGFP-mDlx and CAG- mRuby3, and 2 animals co-injected with mDlx-βGlobinMinP-EGFP and CAG-mRuby3).
    Figure Legend Snippet: Validation of cell-type specific enhancer function in the STARR-seq orientation. ( A, B ) Representative confocal images of coronal sections of P7 mouse brain transduced by intracranial injection at P0 with scAAV9-Hsp68-EGFP-mDlx ( A ) or AAV9-mDlx-βGlobinMinP-EGFP ( B ) and a CAG-mRuby3 positive control. Sections were stained with an antibody for EGFP for signal amplification. Green, EGFP; red, mRuby3; grey, DAPI. ( C ) Quantification of the numbers of Pyramidal (light blue), Non-pyramidal (darker blue), and cells of ambiguous morphology (grey) observed in confocal imaging experiment in A and B above; N = 5 animals, 314 cells transduced with Hsp68-EGFP-mDlx (Hsp68-3’UTR) and 4 animals, 972 cells transduced with mDlx-βGlobinMinP-EGFP (βGlobin-5’UTR). ( D ) Quantification of images from experiment shown in Figure 4F , with mDlx-βGlobinMinP-EGFP included for comparison. Individual GFP+ and mRuby3+ cells were counted and scored for whether each cell contained a Ctip2-positive nucleus. Cell counts were summed across images for the same brain. Data is presented as mean proportion of Ctip2-positive cells (n = 5 animals co-injected with Hsp68-EGFP-#161 and CAG-mRuby3, 4 animals co-injected with Hsp68-EGFP-mDlx and CAG- mRuby3, and 2 animals co-injected with mDlx-βGlobinMinP-EGFP and CAG-mRuby3).

    Techniques Used: Injection, Positive Control, Staining, Amplification, Imaging, Transduction

    6) Product Images from "Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins"

    Article Title: Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0120751

    Plant-produced τGFP particles bind GFP. a) Ultracentrifuge tubes containing sucrose cushions photographed under UV light after ultracentrifugation. GFP-associated fluorescence remains in the supernatant when GFP-containing plant lysate is centrifuged alone or mixed with tEL-containing plant lysate; but migrates through the cushion when GFP-containing and τGFP-containing plant lysates are mixed. b) Detection of GFP by sandwich ELISA, after coating wells with τGFP (green), τglyc (orange) or an anti-GFP polyclonal IgG (blue) and adding GFP to the wells at four different concentrations after blocking. Detection is horseradish peroxidase—mediated ECL, and signal is net of background. Error bars are standard error. c) Electron micrograph of plant-produced τGFP particles in the presence of GFP, purified by sucrose cushion and size exclusion chromatography. Scale bar 100 nm.
    Figure Legend Snippet: Plant-produced τGFP particles bind GFP. a) Ultracentrifuge tubes containing sucrose cushions photographed under UV light after ultracentrifugation. GFP-associated fluorescence remains in the supernatant when GFP-containing plant lysate is centrifuged alone or mixed with tEL-containing plant lysate; but migrates through the cushion when GFP-containing and τGFP-containing plant lysates are mixed. b) Detection of GFP by sandwich ELISA, after coating wells with τGFP (green), τglyc (orange) or an anti-GFP polyclonal IgG (blue) and adding GFP to the wells at four different concentrations after blocking. Detection is horseradish peroxidase—mediated ECL, and signal is net of background. Error bars are standard error. c) Electron micrograph of plant-produced τGFP particles in the presence of GFP, purified by sucrose cushion and size exclusion chromatography. Scale bar 100 nm.

    Techniques Used: Produced, Fluorescence, Sandwich ELISA, Blocking Assay, Purification, Size-exclusion Chromatography

    7) Product Images from "GLIAL ANKYRINS FACILITATE PARANODAL AXOGLIAL JUNCTION ASSEMBLY"

    Article Title: GLIAL ANKYRINS FACILITATE PARANODAL AXOGLIAL JUNCTION ASSEMBLY

    Journal: Nature neuroscience

    doi: 10.1038/nn.3858

    AnkB and AnkG interact with NF155 in vivo and can be targeted to paranodes independently of paranodal junctions and NF155. ( a–d ) P8 sciatic nerve sections were stained for AnkB (N105/17), NFasc and Caspr ( a, b ), and the respective longitudinal line scans are shown ( c, d ). ( e ) Immunoprecipitation of AnkB from adult rat sciatic nerves co-precipitated NF155. Immunoprecipitation with the anti-GFP antibody served as a negative control. Heavy chain, mouse IgG heavy chain. AnkB was detected by H-300 rabbit polyclonal antibodies. ( f ) Immunoprecipitation of AnkG from P21 mouse spinal cords co-precipitated NF186 and NF155. AnkG was detected by the goat polyclonal antibodies. The immunoprecipitation in ( e, f ) was reproduced at least three times. The full blots are presented in Supplementary Fig. 3 . ( g–i ) Immunostaining of P7 sciatic nerves (AnkB, rabbit polyclonal). Arrows point to the residual AnkB and NFasc at paranodes. ( j–l ) Immunostaining of P7 spinal cords (AnkG, N106/36). Arrows point to the residual AnkG and NFasc at paranodes. ( m–o ) Immunostaining of P5 sciatic nerves (AnkB, rabbit polyclonal). Arrows point to the paranodes with residual AnkB. ( p–r ) Immunostaining of P12 Nfasc-cHet ( Cnp-Cre;Nfasc f/+ ) and Nfasc-cKO ( Cnp-Cre;Nfasc f/f ) spinal cords (AnkG, N106/36). Two mice per genotype and more than 100 nodes were examined in each mouse. 80–190 nodes per animal were quantified. Scale bars = ( a, b ) 5 μm for (a) and 3.3 μm for (b); 5 μm ( g–i , j–l , m–o , and p–r ).
    Figure Legend Snippet: AnkB and AnkG interact with NF155 in vivo and can be targeted to paranodes independently of paranodal junctions and NF155. ( a–d ) P8 sciatic nerve sections were stained for AnkB (N105/17), NFasc and Caspr ( a, b ), and the respective longitudinal line scans are shown ( c, d ). ( e ) Immunoprecipitation of AnkB from adult rat sciatic nerves co-precipitated NF155. Immunoprecipitation with the anti-GFP antibody served as a negative control. Heavy chain, mouse IgG heavy chain. AnkB was detected by H-300 rabbit polyclonal antibodies. ( f ) Immunoprecipitation of AnkG from P21 mouse spinal cords co-precipitated NF186 and NF155. AnkG was detected by the goat polyclonal antibodies. The immunoprecipitation in ( e, f ) was reproduced at least three times. The full blots are presented in Supplementary Fig. 3 . ( g–i ) Immunostaining of P7 sciatic nerves (AnkB, rabbit polyclonal). Arrows point to the residual AnkB and NFasc at paranodes. ( j–l ) Immunostaining of P7 spinal cords (AnkG, N106/36). Arrows point to the residual AnkG and NFasc at paranodes. ( m–o ) Immunostaining of P5 sciatic nerves (AnkB, rabbit polyclonal). Arrows point to the paranodes with residual AnkB. ( p–r ) Immunostaining of P12 Nfasc-cHet ( Cnp-Cre;Nfasc f/+ ) and Nfasc-cKO ( Cnp-Cre;Nfasc f/f ) spinal cords (AnkG, N106/36). Two mice per genotype and more than 100 nodes were examined in each mouse. 80–190 nodes per animal were quantified. Scale bars = ( a, b ) 5 μm for (a) and 3.3 μm for (b); 5 μm ( g–i , j–l , m–o , and p–r ).

    Techniques Used: In Vivo, Staining, Immunoprecipitation, Negative Control, Immunostaining, Mouse Assay

    Paranodal AnkB is derived from Schwann cells in the PNS. ( a ) Immunostaining of a mouse sciatic nerve for AnkG (node, rabbit polyclonal anti-AnkG) and AnkB (paranodes, N105/17). ( b ) Cultured DRG neurons were infected with adenovirus carrying a GFP and AnkB shRNA-expressing construct, and immunostained (AnkB, N105/13). Arrowheads point to the GFP-positive axon. ( c, d ) Schwann cells were added to the same culture as in ( b ) and induced to myelinate. The co-culture was labeled for myelin basic protein (MBP), GFP and AnkB (N105/13 ( c ) or N105/17 ( d )). The arrows point to paranodal AnkB. A line scan of fluorescence intensity of the paranode indicated in ( d ) is shown in the inset. ( e ) Immunoblots of lysates from rat hippocampal (Hc) neuron and purified Schwann cell (Sc) cultures (AnkB, N105/17). The full blots are presented in Supplementary Fig. 3 . ( f ) DRG neurons from the AnkB conventional KO were co-cultured with myelinating rat Schwann cells and immunostained for AnkB (N105/17), neurofilament-M (NF-M) and MBP. The arrow points to a paranode. Localization of AnkB along the inner mesaxon 13 was also observed as spiral extensions from paranodal junctions. ( g ) The scheme of the Ank2 conditional allele. The two loxP sites (red triangles) flank exon 24. After Cre recombination and removal of exon 24, a premature stop codon is generated in exon 25. ( h, i ) Immunostaining of 4-week-old AnkB-cHet ( h ) and AnkB-cKO ( i ) sciatic nerves (AnkB, rabbit polyclonal anti-AnkB). Arrows point to paranodal junctions. Scale bars = 5 μm ( a; h, i ), and 10 μm ( b–d, f ).
    Figure Legend Snippet: Paranodal AnkB is derived from Schwann cells in the PNS. ( a ) Immunostaining of a mouse sciatic nerve for AnkG (node, rabbit polyclonal anti-AnkG) and AnkB (paranodes, N105/17). ( b ) Cultured DRG neurons were infected with adenovirus carrying a GFP and AnkB shRNA-expressing construct, and immunostained (AnkB, N105/13). Arrowheads point to the GFP-positive axon. ( c, d ) Schwann cells were added to the same culture as in ( b ) and induced to myelinate. The co-culture was labeled for myelin basic protein (MBP), GFP and AnkB (N105/13 ( c ) or N105/17 ( d )). The arrows point to paranodal AnkB. A line scan of fluorescence intensity of the paranode indicated in ( d ) is shown in the inset. ( e ) Immunoblots of lysates from rat hippocampal (Hc) neuron and purified Schwann cell (Sc) cultures (AnkB, N105/17). The full blots are presented in Supplementary Fig. 3 . ( f ) DRG neurons from the AnkB conventional KO were co-cultured with myelinating rat Schwann cells and immunostained for AnkB (N105/17), neurofilament-M (NF-M) and MBP. The arrow points to a paranode. Localization of AnkB along the inner mesaxon 13 was also observed as spiral extensions from paranodal junctions. ( g ) The scheme of the Ank2 conditional allele. The two loxP sites (red triangles) flank exon 24. After Cre recombination and removal of exon 24, a premature stop codon is generated in exon 25. ( h, i ) Immunostaining of 4-week-old AnkB-cHet ( h ) and AnkB-cKO ( i ) sciatic nerves (AnkB, rabbit polyclonal anti-AnkB). Arrows point to paranodal junctions. Scale bars = 5 μm ( a; h, i ), and 10 μm ( b–d, f ).

    Techniques Used: Derivative Assay, Immunostaining, Cell Culture, Infection, shRNA, Expressing, Construct, Co-Culture Assay, Labeling, Fluorescence, Western Blot, Purification, Generated

    8) Product Images from "An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms"

    Article Title: An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

    Journal: bioRxiv

    doi: 10.1101/763789

    ssDNA homology donors are effective in S2 cells to tag organelles. (A) Schematic of drop-in cassette encoding for sfGFP artificial exon. Size of the construct including the homology arms is indicated in the right. sfGFP: superfolderGFP; SA: Splice Acceptor of mhc ; SD: Splice Donor of mhc ; L: flexible linker that consists of four copies of Gly-Gly-Ser. (B) Diagram of steps to isolate cell clones resulting from successful homologous recombination events. (C) Examples of S2R+ cells with organelles marked with GFP. Left panel, antibody staining; middle panel, GFP signal; right panel, the merge.
    Figure Legend Snippet: ssDNA homology donors are effective in S2 cells to tag organelles. (A) Schematic of drop-in cassette encoding for sfGFP artificial exon. Size of the construct including the homology arms is indicated in the right. sfGFP: superfolderGFP; SA: Splice Acceptor of mhc ; SD: Splice Donor of mhc ; L: flexible linker that consists of four copies of Gly-Gly-Ser. (B) Diagram of steps to isolate cell clones resulting from successful homologous recombination events. (C) Examples of S2R+ cells with organelles marked with GFP. Left panel, antibody staining; middle panel, GFP signal; right panel, the merge.

    Techniques Used: Construct, Clone Assay, Homologous Recombination, Staining

    FACS data of control cells (left) and ssDNA knock-in cells (right). All cell lines express mCherry::Clic, which is present in the parental cell line and thus in these derivatives. Single cell clones with GFP expression levels greater than 2 X 10 2 were retained.
    Figure Legend Snippet: FACS data of control cells (left) and ssDNA knock-in cells (right). All cell lines express mCherry::Clic, which is present in the parental cell line and thus in these derivatives. Single cell clones with GFP expression levels greater than 2 X 10 2 were retained.

    Techniques Used: FACS, Knock-In, Clone Assay, Expressing

    Detection of subcellular localization of GFP tagged proteins in S2 cells. Left panels, confocal fluorescence detection of GFP fusion proteins in single-cell isolated clones. The specific proteins tagged by GFP knock-in are indicated. Center panels, confocal fluorescence detection of mCherry::Clic, which is present in the parental cell line and thus in these derivatives. Right panels, merged image.
    Figure Legend Snippet: Detection of subcellular localization of GFP tagged proteins in S2 cells. Left panels, confocal fluorescence detection of GFP fusion proteins in single-cell isolated clones. The specific proteins tagged by GFP knock-in are indicated. Center panels, confocal fluorescence detection of mCherry::Clic, which is present in the parental cell line and thus in these derivatives. Right panels, merged image.

    Techniques Used: Fluorescence, Isolation, Clone Assay, Knock-In

    9) Product Images from "Reducing insulin via conditional partial gene ablation in adults reverses diet-induced weight gain"

    Article Title: Reducing insulin via conditional partial gene ablation in adults reverses diet-induced weight gain

    Journal: The FASEB Journal

    doi: 10.1096/fj.201700518R

    Adult reduction of Ins2 gene dose in mice consuming low- and moderate-fat diets does not reverse weight gain. A ) Schematic of our hypothesis that reduced insulin would reverse weight gain and obesity in adult male mice fed a low-fat (10%, yellow), moderate-fat (25%, light blue), or high-fat (58%, dark blue) diet. B ) Tamoxifen induction of Pdx1 Cre ERT led to near complete, as evidenced by membrane GFP expression (bottom). C ) Insulin content in mice ( n = 7, 6; control n is listed first throughout) fed a moderate-fat diet were measured from isolated islets 40 wk after tamoxifen injections. For insulin content measurements, n represents individual mice. D ) Circulating insulin at 5, 25, and 40 wk after tamoxifen injection in control littermates ( n = 10–15) and experimental mice ( n = 7–12) fed a moderate-fat diet. E , F ) Glucose tolerance ( E ) and insulin sensitivity ( F ) in mice ( n = 15, 12). Insets: area under the curve (AUC) ( E ) and area over the curve (AOC) ( F ). G ) Glucose-stimulated insulin secretion ( n = 15, 12). H ) Percentage change in body mass of male mice fed a moderate-fat diet; control littermates ( n = 20; Ins1 −/− : Ins2 f/+ :mTmG; gray dashed line) and experimental mice ( n = 20; Ins1 −/− : Ins2 f/+ : Pdx1 Cre ERT :mTmG; blue dashed line). I ) Percentage change in body mass of male mice fed a low-fat diet; control littermates ( n = 3; Ins1 −/− : Ins2 f/+ :mTmG; gray dashed line) and experimental mice ( n = 2; Ins1 −/− : Ins2 f/+ : Pdx1 Cre ERT :mTmG; yellow line). Unless otherwise indicated, measurements were conducted from samples collected from mice between 5 and 7 wk after tamoxifen injection. Data are means ± sem .
    Figure Legend Snippet: Adult reduction of Ins2 gene dose in mice consuming low- and moderate-fat diets does not reverse weight gain. A ) Schematic of our hypothesis that reduced insulin would reverse weight gain and obesity in adult male mice fed a low-fat (10%, yellow), moderate-fat (25%, light blue), or high-fat (58%, dark blue) diet. B ) Tamoxifen induction of Pdx1 Cre ERT led to near complete, as evidenced by membrane GFP expression (bottom). C ) Insulin content in mice ( n = 7, 6; control n is listed first throughout) fed a moderate-fat diet were measured from isolated islets 40 wk after tamoxifen injections. For insulin content measurements, n represents individual mice. D ) Circulating insulin at 5, 25, and 40 wk after tamoxifen injection in control littermates ( n = 10–15) and experimental mice ( n = 7–12) fed a moderate-fat diet. E , F ) Glucose tolerance ( E ) and insulin sensitivity ( F ) in mice ( n = 15, 12). Insets: area under the curve (AUC) ( E ) and area over the curve (AOC) ( F ). G ) Glucose-stimulated insulin secretion ( n = 15, 12). H ) Percentage change in body mass of male mice fed a moderate-fat diet; control littermates ( n = 20; Ins1 −/− : Ins2 f/+ :mTmG; gray dashed line) and experimental mice ( n = 20; Ins1 −/− : Ins2 f/+ : Pdx1 Cre ERT :mTmG; blue dashed line). I ) Percentage change in body mass of male mice fed a low-fat diet; control littermates ( n = 3; Ins1 −/− : Ins2 f/+ :mTmG; gray dashed line) and experimental mice ( n = 2; Ins1 −/− : Ins2 f/+ : Pdx1 Cre ERT :mTmG; yellow line). Unless otherwise indicated, measurements were conducted from samples collected from mice between 5 and 7 wk after tamoxifen injection. Data are means ± sem .

    Techniques Used: Mouse Assay, Expressing, Isolation, Injection

    10) Product Images from "Distinct effects of volatile and intravenous anaesthetics on presynaptic calcium dynamics in mouse hippocampal GABAergic neurones"

    Article Title: Distinct effects of volatile and intravenous anaesthetics on presynaptic calcium dynamics in mouse hippocampal GABAergic neurones

    Journal: BJA: British Journal of Anaesthesia

    doi: 10.1016/j.bja.2022.01.014

    Neuronal-type-specific presynaptic Ca 2+ transients. (a) Circuit illustration of GCaMP6f-targeted cell types (left) and transgenic crosses used to target specific neuronal subpopulations (right). Mouse strains with interneurone-specific promoters encoding Cre were crossed to either Cre-dependent strain Ai14 to express cytosolic tdTomato or Ai95 to express cytosolic GcaMP6f. (b) GcaMP6f amplified with anti-GFP immunolabelling in SST::Ai95. (c) GAD2-Cre::Ai14 cultures transfected with CaMKII-GcaMP6f. Boutons without RFP were defined as glutamatergic/GAD2 – . (d) GcaMP6f fluorescence in Nkx2.1-Cre::Ai95 neurones at rest (di), after a train of 20 APs (dii), and after ionomycin (diii). Fluorescence increase is shown as Δ F = F 20AP – F 0 (div). (e) Fluorescence in dendritic (purple) and punctate axonal (red) regions of interest during 20 APs. Image size is 20 × 20 μm. (f) Compartment-specific traces of Ca 2+ transients following 20 AP. AP, action potential; GFP, green fluorescent protein.
    Figure Legend Snippet: Neuronal-type-specific presynaptic Ca 2+ transients. (a) Circuit illustration of GCaMP6f-targeted cell types (left) and transgenic crosses used to target specific neuronal subpopulations (right). Mouse strains with interneurone-specific promoters encoding Cre were crossed to either Cre-dependent strain Ai14 to express cytosolic tdTomato or Ai95 to express cytosolic GcaMP6f. (b) GcaMP6f amplified with anti-GFP immunolabelling in SST::Ai95. (c) GAD2-Cre::Ai14 cultures transfected with CaMKII-GcaMP6f. Boutons without RFP were defined as glutamatergic/GAD2 – . (d) GcaMP6f fluorescence in Nkx2.1-Cre::Ai95 neurones at rest (di), after a train of 20 APs (dii), and after ionomycin (diii). Fluorescence increase is shown as Δ F = F 20AP – F 0 (div). (e) Fluorescence in dendritic (purple) and punctate axonal (red) regions of interest during 20 APs. Image size is 20 × 20 μm. (f) Compartment-specific traces of Ca 2+ transients following 20 AP. AP, action potential; GFP, green fluorescent protein.

    Techniques Used: Transgenic Assay, Amplification, Transfection, Fluorescence

    11) Product Images from "CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis"

    Article Title: CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

    Journal: bioRxiv

    doi: 10.1101/242941

    CHC22 is localized at the ER-to-Golgi Intermediate Compartment in HeLa GLUT4 and human myotubes. (A and B) Representative Structured Illumination Microscopy of a HeLa-GLUT4 cell (HeLa-G4) (A) and the human skeletal muscle cell line hSkMC-AB1190-GLUT4 (hSkMC) (B) stained for CHC22 (red) and p115 (blue). The gray circles delineate the nuclei (N). Muscle cell staining with each antibody is shown in black on white below the color images. Scale bars: 10 µm. (C and D) Representative Structured Illumination Microscopy of the perinuclear region of HeLa-GLUT4 cells and hSkMC-AB1190-GLUT4 stained for CHC22 (red) and p115 (C), ERGIC-53 (blue) (D). The solid gray lines delineate the nuclear border (N). The dashed white lines span the segment for which fluorescence intensities for GLUT4 (green), CHC22 (red) and p115, ERGIC-53 (blue) were plotted. Arrowheads indicate areas of peak overlap. Scale bars: 1 µm. In A, B, C and D, GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 or immunostained with anti-GFP antibody in hSkMC-AB1190-GLUT4. Merged images in (A, B and C) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.
    Figure Legend Snippet: CHC22 is localized at the ER-to-Golgi Intermediate Compartment in HeLa GLUT4 and human myotubes. (A and B) Representative Structured Illumination Microscopy of a HeLa-GLUT4 cell (HeLa-G4) (A) and the human skeletal muscle cell line hSkMC-AB1190-GLUT4 (hSkMC) (B) stained for CHC22 (red) and p115 (blue). The gray circles delineate the nuclei (N). Muscle cell staining with each antibody is shown in black on white below the color images. Scale bars: 10 µm. (C and D) Representative Structured Illumination Microscopy of the perinuclear region of HeLa-GLUT4 cells and hSkMC-AB1190-GLUT4 stained for CHC22 (red) and p115 (C), ERGIC-53 (blue) (D). The solid gray lines delineate the nuclear border (N). The dashed white lines span the segment for which fluorescence intensities for GLUT4 (green), CHC22 (red) and p115, ERGIC-53 (blue) were plotted. Arrowheads indicate areas of peak overlap. Scale bars: 1 µm. In A, B, C and D, GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 or immunostained with anti-GFP antibody in hSkMC-AB1190-GLUT4. Merged images in (A, B and C) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Techniques Used: Microscopy, Staining, Fluorescence

    Surface GLUT4 is recycled to the GSC in proximity to the ERGIC. (A) Representative immunofluorescence (IF) staining for internalized surface-labeled GLUT4 (HA-tag, blue) and CHC22 (red) for HeLa-GLUT4 cells at 0, 10 or 30 minutes after insulin treatment. Total GLUT4 is detected by GFP tag (green). Scale bars: 5 µm. (B) Pearson’s overlap for labeling of CHC22 and HA-tag (data expressed as mean ± SEM, N=3, 8-40 cells per experiment). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test *p
    Figure Legend Snippet: Surface GLUT4 is recycled to the GSC in proximity to the ERGIC. (A) Representative immunofluorescence (IF) staining for internalized surface-labeled GLUT4 (HA-tag, blue) and CHC22 (red) for HeLa-GLUT4 cells at 0, 10 or 30 minutes after insulin treatment. Total GLUT4 is detected by GFP tag (green). Scale bars: 5 µm. (B) Pearson’s overlap for labeling of CHC22 and HA-tag (data expressed as mean ± SEM, N=3, 8-40 cells per experiment). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test *p

    Techniques Used: Immunofluorescence, Staining, Labeling

    Depletion of CHC22 or p115, but not GM130, sortilin or IRAP disrupt perinuclear targeting of GLUT4. (A) Immunoblotting for CHC22, CHC17, p115, GM130 and β-actin after transfection of HeLa-GLUT4 cells with siRNA targeting CHC17, CHC22, p115, GM130, sortilin, IRAP or with non-targeting control siRNA. The position of molecular weight (MW) markers is indicated in kilodaltons (kDa). (B) Representative immunofluorescence (IF) staining for CHC22 (red) and p115 (blue) in HeLa-GLUT4 cells after siRNA transfection as in (A), with GLUT4 detected by GFP tag (green). N, nuclei. Arrow points to CHC22-depleted cell. Scale bars: 10 µm. (C) Representative IF staining for GM130 (yellow), p115 (red) and CHC22 (blue) in HeLa-GLUT4 cells after treatment with siRNA targeting GM130 or with control siRNA, with GLUT4 detected by GFP tag (green). Individual antibody staining is shown in black and white, while the merged image shows all four colors with overlaps in white. Scale bars: 25 µm. (D and E) Representative IF staining for GLUT4 (green), CHC22 or sortilin (red) and p115 (blue) in HeLa-GLUT4 cells after treatment with siRNA targeting sortilin or IRAP or with non-targeting control. Scale bars: 5 µm. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.
    Figure Legend Snippet: Depletion of CHC22 or p115, but not GM130, sortilin or IRAP disrupt perinuclear targeting of GLUT4. (A) Immunoblotting for CHC22, CHC17, p115, GM130 and β-actin after transfection of HeLa-GLUT4 cells with siRNA targeting CHC17, CHC22, p115, GM130, sortilin, IRAP or with non-targeting control siRNA. The position of molecular weight (MW) markers is indicated in kilodaltons (kDa). (B) Representative immunofluorescence (IF) staining for CHC22 (red) and p115 (blue) in HeLa-GLUT4 cells after siRNA transfection as in (A), with GLUT4 detected by GFP tag (green). N, nuclei. Arrow points to CHC22-depleted cell. Scale bars: 10 µm. (C) Representative IF staining for GM130 (yellow), p115 (red) and CHC22 (blue) in HeLa-GLUT4 cells after treatment with siRNA targeting GM130 or with control siRNA, with GLUT4 detected by GFP tag (green). Individual antibody staining is shown in black and white, while the merged image shows all four colors with overlaps in white. Scale bars: 25 µm. (D and E) Representative IF staining for GLUT4 (green), CHC22 or sortilin (red) and p115 (blue) in HeLa-GLUT4 cells after treatment with siRNA targeting sortilin or IRAP or with non-targeting control. Scale bars: 5 µm. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Techniques Used: Transfection, Molecular Weight, Immunofluorescence, Staining

    HeLa-GLUT4 cells have a functional GLUT4 trafficking pathway that requires CHC22. (A) Representative images of GLUT4 (exofacial haemagglutinin (HA)-tag, internal GFP tag) in HeLa-GLUT4 cells before (basal) or after insulin treatment. GLUT4 at the plasma membrane was detected by immunofluorescence (IF) after surface labeling with anti-HA monoclonal antibody (red). Total GLUT4 (green) was detected by GFP tag. Arrows show the GLUT4 storage compartment. Arrowheads point to peripheral GLUT4 vesicles. Scale bars: 7.5 µm. (B) Left panel – Representative FACS histogram of surface GLUT4 fluorescence intensities (signal from anti-HA labeling) before (basal) and after insulin treatment (Ins). Middle panel – Quantification of surface:total GLUT4 (HA:GFP mean fluorescence intensity signals). Data expressed as mean ± SEM, N=3, 10,000 cells acquired per experiment. Two-tailed unpaired Student’s t-test with equal variances, **p
    Figure Legend Snippet: HeLa-GLUT4 cells have a functional GLUT4 trafficking pathway that requires CHC22. (A) Representative images of GLUT4 (exofacial haemagglutinin (HA)-tag, internal GFP tag) in HeLa-GLUT4 cells before (basal) or after insulin treatment. GLUT4 at the plasma membrane was detected by immunofluorescence (IF) after surface labeling with anti-HA monoclonal antibody (red). Total GLUT4 (green) was detected by GFP tag. Arrows show the GLUT4 storage compartment. Arrowheads point to peripheral GLUT4 vesicles. Scale bars: 7.5 µm. (B) Left panel – Representative FACS histogram of surface GLUT4 fluorescence intensities (signal from anti-HA labeling) before (basal) and after insulin treatment (Ins). Middle panel – Quantification of surface:total GLUT4 (HA:GFP mean fluorescence intensity signals). Data expressed as mean ± SEM, N=3, 10,000 cells acquired per experiment. Two-tailed unpaired Student’s t-test with equal variances, **p

    Techniques Used: Functional Assay, Immunofluorescence, Labeling, FACS, Fluorescence, Two Tailed Test

    Immunofluorescence localization of CHC22 at the ER-to-Golgi Intermediate Compartment in HeLa-GLUT4 cells and in human skeletal muscle cells. (A) CHC17 (X22 antibody) or CHC22 (CLTCL1 antibody from Proteintech) immunoblots (IB) of clathrin coated vesicles (CCV) purified from pig brain containing only CHC17 or of cell lysate from bacteria expressing low levels of the hub fragment (residues 1074-1640) of CHC22 (hub 22). The migration of molecular weight (MW) markers is indicated in kilodaltons (kDa). Ponceau staining for proteins is shown on the right (Pro). (B) Representative confocal microscopy immunofluorescence (IF) imaging of CHC22 (red or blue), p115 (red or blue) and GLUT4 (green) in HeLa-GLUT4 cells (top panel) or LHCNM2 myotubes (bottom panel). (C) Representative IF staining for CHC22 (blue), ERGIC-53 (red) and GLUT4 (green) in HeLa-GLUT4 cells (top panel) or LHCNM2 myotubes (bottom panel). Scale bars: 5 µm for HeLa-GLUT4 cells and 7.5 µm for LHCNM2 myotubes in (B) and (C). (D) Representative IF staining for CHC22 (blue), GM130 or TGN46 (red) and GLUT4 (GFP, green) in HeLa-GLUT4 cells. Scale bars: 5 µm. (E) Representative IF staining for CHC22 (blue), GM130 or TGN46 (green) and p115 (red) in LHCNM2 myotubes. Scale bars: 7.5 µm. (F) Representative IF staining for CHC22 (blue), calreticulin (red) and GLUT4 (green) in HeLa-GLUT4 cells. Scale bars: 5 µm. (G) Representative IF staining for CHC22 (red), calnexin (CNX, blue) and GLUT4 (green) in hSkMC-AB1190-GLUT4. Scale bars: 10 µm. (H) Representative Structured Illumination Microscopy (SIM) of a HeLa-GLUT4 (HeLa-G4) cell (top panel) and human skeletal muscle cell (hSkMC-AB1190-GLUT4, bottom panel) stained for CHC22 (red) and TGN46 (blue). GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 and immunostained with an anti-GFP antibody in hSkMC-AB1190-GLUT4. Scale bar: 10 µm. Merged images in (B-H) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white. (I) Representative fluorescence intensity plots for GLUT4 (green), CHC22 (red) and p115, ERGIC-53, GM130 or TGN46 (blue) generated from SIM images of the perinuclear region of HeLa-GLUT4 cells.
    Figure Legend Snippet: Immunofluorescence localization of CHC22 at the ER-to-Golgi Intermediate Compartment in HeLa-GLUT4 cells and in human skeletal muscle cells. (A) CHC17 (X22 antibody) or CHC22 (CLTCL1 antibody from Proteintech) immunoblots (IB) of clathrin coated vesicles (CCV) purified from pig brain containing only CHC17 or of cell lysate from bacteria expressing low levels of the hub fragment (residues 1074-1640) of CHC22 (hub 22). The migration of molecular weight (MW) markers is indicated in kilodaltons (kDa). Ponceau staining for proteins is shown on the right (Pro). (B) Representative confocal microscopy immunofluorescence (IF) imaging of CHC22 (red or blue), p115 (red or blue) and GLUT4 (green) in HeLa-GLUT4 cells (top panel) or LHCNM2 myotubes (bottom panel). (C) Representative IF staining for CHC22 (blue), ERGIC-53 (red) and GLUT4 (green) in HeLa-GLUT4 cells (top panel) or LHCNM2 myotubes (bottom panel). Scale bars: 5 µm for HeLa-GLUT4 cells and 7.5 µm for LHCNM2 myotubes in (B) and (C). (D) Representative IF staining for CHC22 (blue), GM130 or TGN46 (red) and GLUT4 (GFP, green) in HeLa-GLUT4 cells. Scale bars: 5 µm. (E) Representative IF staining for CHC22 (blue), GM130 or TGN46 (green) and p115 (red) in LHCNM2 myotubes. Scale bars: 7.5 µm. (F) Representative IF staining for CHC22 (blue), calreticulin (red) and GLUT4 (green) in HeLa-GLUT4 cells. Scale bars: 5 µm. (G) Representative IF staining for CHC22 (red), calnexin (CNX, blue) and GLUT4 (green) in hSkMC-AB1190-GLUT4. Scale bars: 10 µm. (H) Representative Structured Illumination Microscopy (SIM) of a HeLa-GLUT4 (HeLa-G4) cell (top panel) and human skeletal muscle cell (hSkMC-AB1190-GLUT4, bottom panel) stained for CHC22 (red) and TGN46 (blue). GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 and immunostained with an anti-GFP antibody in hSkMC-AB1190-GLUT4. Scale bar: 10 µm. Merged images in (B-H) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white. (I) Representative fluorescence intensity plots for GLUT4 (green), CHC22 (red) and p115, ERGIC-53, GM130 or TGN46 (blue) generated from SIM images of the perinuclear region of HeLa-GLUT4 cells.

    Techniques Used: Immunofluorescence, Western Blot, Purification, Expressing, Migration, Molecular Weight, Staining, Confocal Microscopy, Imaging, Microscopy, Fluorescence, Generated

    The CHC22 compartment is localized proximal to the trans-Golgi Network and does not overlap with the cis-Golgi. (A, B and C) Representative Structured Illumination Microscopy of the perinuclear region of HeLa-GLUT4 cells (HeLa-G4) and hSkMC-AB1190-GLUT4 (hSkMC) stained for CHC22 (red) and GM130 (A), TGN46 (B) and syntaxin 6 (STX-6, blue) (C). The solid gray lines delineate the nuclear border (N). The dashed white lines span the segment over which fluorescence intensities for GLUT4 (green), CHC22 (red) and GM130, TGN46 and STX-6 (blue) were plotted. Scale bars: 1 µm. In A, B and C, GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 in HeLa-GLUT4 or immunostained using an anti-GFP antibody in hSkMC-AB1190-GLUT4. Merged images in (A, B and C) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.
    Figure Legend Snippet: The CHC22 compartment is localized proximal to the trans-Golgi Network and does not overlap with the cis-Golgi. (A, B and C) Representative Structured Illumination Microscopy of the perinuclear region of HeLa-GLUT4 cells (HeLa-G4) and hSkMC-AB1190-GLUT4 (hSkMC) stained for CHC22 (red) and GM130 (A), TGN46 (B) and syntaxin 6 (STX-6, blue) (C). The solid gray lines delineate the nuclear border (N). The dashed white lines span the segment over which fluorescence intensities for GLUT4 (green), CHC22 (red) and GM130, TGN46 and STX-6 (blue) were plotted. Scale bars: 1 µm. In A, B and C, GLUT4 (green) was detected by GFP tag in HeLa-GLUT4 in HeLa-GLUT4 or immunostained using an anti-GFP antibody in hSkMC-AB1190-GLUT4. Merged images in (A, B and C) show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white.

    Techniques Used: Microscopy, Staining, Fluorescence

    CHC22 re-distributes with p115 following Brefeldin A treatment. (A, B) Representative immunofluorescence (IF) staining for CHC22 (blue) and p115 (red) in HeLa-GLUT4 cells (A) or LHCNM2 myotubes (B) treated or not with Brefeldin A (BFA). GLUT4 (green) was detected by GFP tag in HeLa-G4 cells and by IF of endogenous protein in LHCNM2 cells. Scale bars: 5 and 25 µm for (A) and (B), respectively. (C, D) Representative immunofluorescence (IF) staining of HeLa-GLUT4 cells for CHC22 (blue) and ERGIC-53 (red) in (C) or Rab1 (red) in (D) treated or not with BFA and stimulated or not by insulin (Ins). GLUT4 (green) was detected by GFP. Scale bars: 10 µm. (E) Quantification of Pearson’s overlap values between CHC22, GLUT4, ERGIC-53 and Rab1 (data as in (C D) expressed as mean ± SEM, N=3 to 4, 5 to 42 cells per experiment). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-hoc test *p
    Figure Legend Snippet: CHC22 re-distributes with p115 following Brefeldin A treatment. (A, B) Representative immunofluorescence (IF) staining for CHC22 (blue) and p115 (red) in HeLa-GLUT4 cells (A) or LHCNM2 myotubes (B) treated or not with Brefeldin A (BFA). GLUT4 (green) was detected by GFP tag in HeLa-G4 cells and by IF of endogenous protein in LHCNM2 cells. Scale bars: 5 and 25 µm for (A) and (B), respectively. (C, D) Representative immunofluorescence (IF) staining of HeLa-GLUT4 cells for CHC22 (blue) and ERGIC-53 (red) in (C) or Rab1 (red) in (D) treated or not with BFA and stimulated or not by insulin (Ins). GLUT4 (green) was detected by GFP. Scale bars: 10 µm. (E) Quantification of Pearson’s overlap values between CHC22, GLUT4, ERGIC-53 and Rab1 (data as in (C D) expressed as mean ± SEM, N=3 to 4, 5 to 42 cells per experiment). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-hoc test *p

    Techniques Used: Immunofluorescence, Staining

    CHC22 and proteins involved in the GLUT4 pathway participate in membrane trafficking from the ERGIC. (A) Representative images of Legionella pneumophila ( L.p. )-infected A549 cells transiently transfected with GFP-tagged CHC22 or CHC17 (green). One hour after infection with either wild type (WT) or mutant ΔdotA L.p. (MOI=50), bacteria were detected by immunofluorescence (IF, red). Arrows point to L.p. and boxed inserts (upper right or left) show L.p. region at 5X magnification. Scale bars: 10 µm for cells expressing CHC22-GFP and 7.5 µm for cells expressing CHC17-GFP. (B) Quantification of the proportion of L.p. vacuoles positive for CHC22 or CHC17. Data expressed as mean ± SEM, N=3, 4 to 35 vacuoles counted per experiment performed as represented in (A). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test ***p
    Figure Legend Snippet: CHC22 and proteins involved in the GLUT4 pathway participate in membrane trafficking from the ERGIC. (A) Representative images of Legionella pneumophila ( L.p. )-infected A549 cells transiently transfected with GFP-tagged CHC22 or CHC17 (green). One hour after infection with either wild type (WT) or mutant ΔdotA L.p. (MOI=50), bacteria were detected by immunofluorescence (IF, red). Arrows point to L.p. and boxed inserts (upper right or left) show L.p. region at 5X magnification. Scale bars: 10 µm for cells expressing CHC22-GFP and 7.5 µm for cells expressing CHC17-GFP. (B) Quantification of the proportion of L.p. vacuoles positive for CHC22 or CHC17. Data expressed as mean ± SEM, N=3, 4 to 35 vacuoles counted per experiment performed as represented in (A). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-hoc test ***p

    Techniques Used: Infection, Transfection, Mutagenesis, Immunofluorescence, Expressing

    Newly synthesized GLUT4 is delayed in the early secretory pathway compared to GLUT1. (A) Representative stills extracted from Video 1 showing a HeLa cell expressing the endoplasmic reticulum (ER) Ii-hook fused to streptavidin along with HA-GLUT1-SBP-mCherry (GLUT1, red) and HA-GLUT4-SBP-GFP (GLUT4, green). The intracellular traffic of GLUT1-mCherry and GLUT4-GFP was simultaneously tracked for 1h after biotin addition released them from the ER. Upon ER exit, both GLUT1 and GLUT4 accumulated in the perinuclear region of the cell (yellow). From 26 min onwards, highly mobile GLUT1 vesicles (arrowheads) were visible (red) while GLUT4 remained perinuclear. Scale bar: 10 µm. (B, D, F, H, J) Representative immunofluorescence staining for GLUT1-SBP-GFP or GLUT4-SBP-GFP (detected with anti-GFP antibody, green), CHC22 (red) and (B) calnexin (CNX, blue), (D) ERGIC-53 (blue), (F) p115 (blue), (H) GM130 (blue) or (J) TGN46 (blue) in HeLa cells expressing HA-GLUT1-SBP-GFP or HA-GLUT4-SBP-GFP along with the ER Ii-hook. Traffic of GLUT4 and GLUT1 was tracked at 0, 15, 30 and 60 minutes after release from the ER by biotin. Arrows point to GLUT1 detected at the plasma membrane and arrowheads point to GLUT1-positive endosomal structures. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white. Scale bars: 10 µm. (C, E, G, I, K, L) Pearson’s overlap between GLUT1 or GLUT4 and CNX, ERGIC-53, p115, GM130, TGN46 or CHC22 at different time-points post-ER release. Data expressed as mean ± SEM, N=3-4, 10-46 cells per experiment. One-way analysis of variance (ANOVA) followed by Sidak’s multiple comparison post-hoc test *p
    Figure Legend Snippet: Newly synthesized GLUT4 is delayed in the early secretory pathway compared to GLUT1. (A) Representative stills extracted from Video 1 showing a HeLa cell expressing the endoplasmic reticulum (ER) Ii-hook fused to streptavidin along with HA-GLUT1-SBP-mCherry (GLUT1, red) and HA-GLUT4-SBP-GFP (GLUT4, green). The intracellular traffic of GLUT1-mCherry and GLUT4-GFP was simultaneously tracked for 1h after biotin addition released them from the ER. Upon ER exit, both GLUT1 and GLUT4 accumulated in the perinuclear region of the cell (yellow). From 26 min onwards, highly mobile GLUT1 vesicles (arrowheads) were visible (red) while GLUT4 remained perinuclear. Scale bar: 10 µm. (B, D, F, H, J) Representative immunofluorescence staining for GLUT1-SBP-GFP or GLUT4-SBP-GFP (detected with anti-GFP antibody, green), CHC22 (red) and (B) calnexin (CNX, blue), (D) ERGIC-53 (blue), (F) p115 (blue), (H) GM130 (blue) or (J) TGN46 (blue) in HeLa cells expressing HA-GLUT1-SBP-GFP or HA-GLUT4-SBP-GFP along with the ER Ii-hook. Traffic of GLUT4 and GLUT1 was tracked at 0, 15, 30 and 60 minutes after release from the ER by biotin. Arrows point to GLUT1 detected at the plasma membrane and arrowheads point to GLUT1-positive endosomal structures. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in turquoise, and red/green/blue overlap in white. Scale bars: 10 µm. (C, E, G, I, K, L) Pearson’s overlap between GLUT1 or GLUT4 and CNX, ERGIC-53, p115, GM130, TGN46 or CHC22 at different time-points post-ER release. Data expressed as mean ± SEM, N=3-4, 10-46 cells per experiment. One-way analysis of variance (ANOVA) followed by Sidak’s multiple comparison post-hoc test *p

    Techniques Used: Synthesized, Expressing, Immunofluorescence, Staining

    12) Product Images from "RNA localization and co‐translational interactions control RAB13 GTPase function and cell migration"

    Article Title: RNA localization and co‐translational interactions control RAB13 GTPase function and cell migration

    Journal: The EMBO Journal

    doi: 10.15252/embj.2020104958

    Peripheral RAB 13 RNA translation is required for RAB 13 protein activation but not steady‐state distribution or membrane association Wide‐field images of RAB13 immunofluorescence in MDA‐MB-231 cells treated with control or RAB13 (191 + 230) PMOs and ratios of peripheral/perinuclear intensity. Scale bars: 10 μm. n = 45–50 cells. Bars: mean ± s.e.m. Similar results were observed in two additional independent experiments. Fluorescence images (projections of confocal slices throughout the cell height) of cells expressing GFP‐RAB13 with the indicated UTRs. Note that in both cases the protein assumes indistinguishable distribution. Scale bars: 10 μm. Soluble/particulate fractionation of the indicated cell lines followed by Western blot to detect the indicated proteins. RhoGDI and TfRc serve as soluble and particulate markers, respectively. Graph shows quantitation from n = 3 independent experiments. Bars: mean ± s.e.m. Active RAB13 (RAB13‐GTP) was pulled down using MICAL‐L1 RBD‐GST from the indicated PMO‐treated cells (D) or GFP‐RAB13‐expressing lines (E). The amount of endogenous or exogenous RAB13 was measured by quantitative Western blot, and relative levels of active RAB13 are plotted. n = 8 (D), n = 4 (E). Bars: mean ± s.e.m. Data information: P‐ values: *
    Figure Legend Snippet: Peripheral RAB 13 RNA translation is required for RAB 13 protein activation but not steady‐state distribution or membrane association Wide‐field images of RAB13 immunofluorescence in MDA‐MB-231 cells treated with control or RAB13 (191 + 230) PMOs and ratios of peripheral/perinuclear intensity. Scale bars: 10 μm. n = 45–50 cells. Bars: mean ± s.e.m. Similar results were observed in two additional independent experiments. Fluorescence images (projections of confocal slices throughout the cell height) of cells expressing GFP‐RAB13 with the indicated UTRs. Note that in both cases the protein assumes indistinguishable distribution. Scale bars: 10 μm. Soluble/particulate fractionation of the indicated cell lines followed by Western blot to detect the indicated proteins. RhoGDI and TfRc serve as soluble and particulate markers, respectively. Graph shows quantitation from n = 3 independent experiments. Bars: mean ± s.e.m. Active RAB13 (RAB13‐GTP) was pulled down using MICAL‐L1 RBD‐GST from the indicated PMO‐treated cells (D) or GFP‐RAB13‐expressing lines (E). The amount of endogenous or exogenous RAB13 was measured by quantitative Western blot, and relative levels of active RAB13 are plotted. n = 8 (D), n = 4 (E). Bars: mean ± s.e.m. Data information: P‐ values: *

    Techniques Used: Activation Assay, Immunofluorescence, Multiple Displacement Amplification, Fluorescence, Expressing, Fractionation, Western Blot, Quantitation Assay

    Peripheral RAB 13 RNA promotes the local association of RAB 13 with the exchange factor RABIF Immunoprecipitation and Western blot analysis to detect proteins bound to GFP‐RAB13 expressed from constructs carrying wt or ΔPMO RAB13 3′UTR. GFP‐RAB13 (T22N) expresses a nucleotide‐free mutant which binds tightly to putative GEFs. RABIF panel is also shown with adjusted contrast to reveal lower binding to wtGFP‐RAB13. Quantification of RABIF binding to RAB13 from experiments as in (A). n = 6. Bars: mean ± s.e.m. Active RAB13 pull‐down assay from cells with CRISPR knockdown of RABIF using the indicated sgRNAs. n = 3. Bars: mean ± s.e.m. Quantification of RABIF‐RAB13 PLA signal from cells transfected with the indicated siRNAs. Number of observed cells is indicated within each bar. Bars: mean ± s.e.m. Similar results were observed in one additional independent experiment. Representative RABIF‐RAB13 PLA images and quantitations from cells transfected with the indicated PMOs. N > 70 cells. Bars: mean ± s.e.m. Arrows indicate PLA signal at the cell periphery. Arrowheads indicate PLA signal within the cell body. Boxed regions are magnified in the insets. Scale bars: 15 μm; 5 μm in insets. Representative RABIF‐GFP PLA images and quantitations from cells expressing GFP‐RAB13(T22N) carrying wt or ΔPMO RAB13 3′UTR. N > 70 cells. Bars: mean ± s.e.m. Arrows indicate PLA signal at the cell periphery. Arrowheads indicate PLA signal within the cell body. Boxed regions are magnified in the insets. Scale bars: 10 μm; 4 μm in insets. Data information: P ‐values: *
    Figure Legend Snippet: Peripheral RAB 13 RNA promotes the local association of RAB 13 with the exchange factor RABIF Immunoprecipitation and Western blot analysis to detect proteins bound to GFP‐RAB13 expressed from constructs carrying wt or ΔPMO RAB13 3′UTR. GFP‐RAB13 (T22N) expresses a nucleotide‐free mutant which binds tightly to putative GEFs. RABIF panel is also shown with adjusted contrast to reveal lower binding to wtGFP‐RAB13. Quantification of RABIF binding to RAB13 from experiments as in (A). n = 6. Bars: mean ± s.e.m. Active RAB13 pull‐down assay from cells with CRISPR knockdown of RABIF using the indicated sgRNAs. n = 3. Bars: mean ± s.e.m. Quantification of RABIF‐RAB13 PLA signal from cells transfected with the indicated siRNAs. Number of observed cells is indicated within each bar. Bars: mean ± s.e.m. Similar results were observed in one additional independent experiment. Representative RABIF‐RAB13 PLA images and quantitations from cells transfected with the indicated PMOs. N > 70 cells. Bars: mean ± s.e.m. Arrows indicate PLA signal at the cell periphery. Arrowheads indicate PLA signal within the cell body. Boxed regions are magnified in the insets. Scale bars: 15 μm; 5 μm in insets. Representative RABIF‐GFP PLA images and quantitations from cells expressing GFP‐RAB13(T22N) carrying wt or ΔPMO RAB13 3′UTR. N > 70 cells. Bars: mean ± s.e.m. Arrows indicate PLA signal at the cell periphery. Arrowheads indicate PLA signal within the cell body. Boxed regions are magnified in the insets. Scale bars: 10 μm; 4 μm in insets. Data information: P ‐values: *

    Techniques Used: Immunoprecipitation, Western Blot, Construct, Mutagenesis, Binding Assay, Pull Down Assay, CRISPR, Proximity Ligation Assay, Transfection, Expressing

    RAB 13 RNA mislocalization does not affect RAB 13 binding to membranes or association with REP ‐1 or Rab GDI Cells treated with the indicated PMOs were fractionated into soluble and particulate fractions, and the indicated proteins were detected by Western blot. RhoGDI and TfRc serve as soluble and particulate markers, respectively. Lysates from the indicated GFP or GFP‐RAB13‐expressing cell lines were immunoprecipitated with anti‐GFP antibodies and blotted to detect the indicated proteins. Relative REP‐1 and RabGDI binding are quantified in the graphs from n = 3 (REP‐1) and n = 5 (RabGDI) independent experiments. Bars: mean ± s.e.m. No significant differences were detected by Wilcoxon matched‐pairs signed‐rank test.
    Figure Legend Snippet: RAB 13 RNA mislocalization does not affect RAB 13 binding to membranes or association with REP ‐1 or Rab GDI Cells treated with the indicated PMOs were fractionated into soluble and particulate fractions, and the indicated proteins were detected by Western blot. RhoGDI and TfRc serve as soluble and particulate markers, respectively. Lysates from the indicated GFP or GFP‐RAB13‐expressing cell lines were immunoprecipitated with anti‐GFP antibodies and blotted to detect the indicated proteins. Relative REP‐1 and RabGDI binding are quantified in the graphs from n = 3 (REP‐1) and n = 5 (RabGDI) independent experiments. Bars: mean ± s.e.m. No significant differences were detected by Wilcoxon matched‐pairs signed‐rank test.

    Techniques Used: Binding Assay, Western Blot, Expressing, Immunoprecipitation

    RAB 13 protein levels do not change upon serum stimulation MDA‐MB‐231 cells were stimulated with serum for the indicated times, in the presence or absence of cycloheximide (CHX). The cells were also treated with control or RAB13‐mislocalizing PMOs. Representative Western blot analysis of whole‐cell lysates and corresponding quantitations of RAB13 levels from n = 4–5 replicates. Bars: mean ± s.e.m. No significant differences by Friedman's test. Increase in pY397‐FAK levels attests to serum stimulation. RAB13 immunofluorescence at representative protrusive regions. A cell line expressing GFP was used to delineate cell borders and provide an internal cytosolic control. RAB13 signal at front lamellipodial regions was quantified. n = 35–51 protrusions. Bars: mean ± s.e.m. No increase is detected upon stimulation. By contrast, at early time points a decrease is detected (5 and 20 min, P
    Figure Legend Snippet: RAB 13 protein levels do not change upon serum stimulation MDA‐MB‐231 cells were stimulated with serum for the indicated times, in the presence or absence of cycloheximide (CHX). The cells were also treated with control or RAB13‐mislocalizing PMOs. Representative Western blot analysis of whole‐cell lysates and corresponding quantitations of RAB13 levels from n = 4–5 replicates. Bars: mean ± s.e.m. No significant differences by Friedman's test. Increase in pY397‐FAK levels attests to serum stimulation. RAB13 immunofluorescence at representative protrusive regions. A cell line expressing GFP was used to delineate cell borders and provide an internal cytosolic control. RAB13 signal at front lamellipodial regions was quantified. n = 35–51 protrusions. Bars: mean ± s.e.m. No increase is detected upon stimulation. By contrast, at early time points a decrease is detected (5 and 20 min, P

    Techniques Used: Multiple Displacement Amplification, Western Blot, Immunofluorescence, Expressing

    Peripheral localization of exogenous RAB 13 RNA does not affect RAB 13 RNA stability or translation Schematics depict GFP or GFP‐RAB13 constructs stably expressed in MDA‐MB-231 cells. RAB13 coding sequence is followed either by the wild‐type RAB13 UTR or by the RAB13 UTR carrying a 52‐nt deletion corresponding to the region targeted by PMOs 191 and 230 (ΔPMO UTR). Exogenous RNA is detected by FISH against the GFP sequence. Arrows point to RNA localized at protrusions. Scale bars: 10 μm. Graphs show PDI measurements of GFP or NET1 RNA from multiple cells. n = 42–54 cells in 4 independent experiments (for GFP); n = 27 cells in 2 experiments (for Net1). Bars: mean ± s.e.m. **** P
    Figure Legend Snippet: Peripheral localization of exogenous RAB 13 RNA does not affect RAB 13 RNA stability or translation Schematics depict GFP or GFP‐RAB13 constructs stably expressed in MDA‐MB-231 cells. RAB13 coding sequence is followed either by the wild‐type RAB13 UTR or by the RAB13 UTR carrying a 52‐nt deletion corresponding to the region targeted by PMOs 191 and 230 (ΔPMO UTR). Exogenous RNA is detected by FISH against the GFP sequence. Arrows point to RNA localized at protrusions. Scale bars: 10 μm. Graphs show PDI measurements of GFP or NET1 RNA from multiple cells. n = 42–54 cells in 4 independent experiments (for GFP); n = 27 cells in 2 experiments (for Net1). Bars: mean ± s.e.m. **** P

    Techniques Used: Construct, Stable Transfection, Multiple Displacement Amplification, Sequencing, Fluorescence In Situ Hybridization

    RABIF associates with RAB 13 co‐translationally Schematic depicting experimental strategy for assessing co‐translational association of RABIF with nascent RAB13. Quantification of GFP RNA levels associating with RABIF in immunoprecipitation assays from the indicated cell lines. Note that even though RABIF binds several‐fold more to RAB13(T22N) (see Fig 7 A and B), it binds similarly to the wild‐type and T22N RAB13 RNA, indicating that RABIF binds similarly to nascent RAB13. After translation, it is likely displaced upon GTP loading of wild‐type RAB13, while it remains more stably bound to the nucleotide‐free (T22N) form. N = 6. Bars: mean ± s.e.m. P ‐values: *
    Figure Legend Snippet: RABIF associates with RAB 13 co‐translationally Schematic depicting experimental strategy for assessing co‐translational association of RABIF with nascent RAB13. Quantification of GFP RNA levels associating with RABIF in immunoprecipitation assays from the indicated cell lines. Note that even though RABIF binds several‐fold more to RAB13(T22N) (see Fig 7 A and B), it binds similarly to the wild‐type and T22N RAB13 RNA, indicating that RABIF binds similarly to nascent RAB13. After translation, it is likely displaced upon GTP loading of wild‐type RAB13, while it remains more stably bound to the nucleotide‐free (T22N) form. N = 6. Bars: mean ± s.e.m. P ‐values: *

    Techniques Used: Immunoprecipitation, Stable Transfection

    Loss of peripheral RAB 13 RNA localization disrupts cell migration and phenocopies acute RAB 13 protein loss Transwell migration of MDA-MB‐231 cells treated with control PMOs or RAB13 PMOs (191 + 230). Cells reaching the bottom surface after 4 h were counted. n = 25 fields of view in each of 6 independent experiments. Bars: mean ± s.e.m. Cells expressing Cherry‐NLS, and treated with the indicated PMOs, were tracked every 5 min for 10 h to derive average migration speed. n = 65 cells. Bars: mean ± s.e.m. PMO‐treated cells were induced to invade through a Matrigel plug. Cell staining intensity in arbitrary units (a.u.) was used to quantify relative invasion from n = 4 independent experiments. Bars: mean ± s.e.m. Lifeact‐GFP-expressing cells were treated with the indicated PMOs and imaged every minute over 1 h. Sequential image frames, from a control cell, highlight edge retraction (red arrowheads) or protrusion (yellow arrowheads). Corresponding edge velocity is shown, with negative values indicating retraction and positive values indicating extension. Average protrusion and retraction speeds were calculated from n = 11–13 cells. Bars: mean ± s.e.m. See also Movies EV2 and EV3 . Cells treated with the indicated PMOs or siRNAs were analyzed by Western blot (upper panels) to detect RAB13 and GAPDH protein levels. Migration speed was assessed as in (B) from n = 55–78 cells (bottom graph). Bars: mean ± s.e.m. Protrusion and retraction speed of cells treated with the indicated PMOs or siRNAs were assessed as in (D). Graphs show average normalized values from n = 10–13 cells imaged for 1 h. Bars: mean ± s.e.m. Migration speed of RAB13 knockdown cells re‐expressing GFP‐RAB13 or GFP‐RAB13 with a frameshift (fs) mutation at the beginning of the RAB13 coding region. 28–52 cells were analyzed. Bars: mean ± s.e.m. Similar results were obtained in two additional independent experiments. Re‐expressing cells were identified through GFP fluorescence, and cells with similar, low GFP signal were tracked. All cells are expressing cherry‐NLS for accurate tracking. Data information: P ‐values: *
    Figure Legend Snippet: Loss of peripheral RAB 13 RNA localization disrupts cell migration and phenocopies acute RAB 13 protein loss Transwell migration of MDA-MB‐231 cells treated with control PMOs or RAB13 PMOs (191 + 230). Cells reaching the bottom surface after 4 h were counted. n = 25 fields of view in each of 6 independent experiments. Bars: mean ± s.e.m. Cells expressing Cherry‐NLS, and treated with the indicated PMOs, were tracked every 5 min for 10 h to derive average migration speed. n = 65 cells. Bars: mean ± s.e.m. PMO‐treated cells were induced to invade through a Matrigel plug. Cell staining intensity in arbitrary units (a.u.) was used to quantify relative invasion from n = 4 independent experiments. Bars: mean ± s.e.m. Lifeact‐GFP-expressing cells were treated with the indicated PMOs and imaged every minute over 1 h. Sequential image frames, from a control cell, highlight edge retraction (red arrowheads) or protrusion (yellow arrowheads). Corresponding edge velocity is shown, with negative values indicating retraction and positive values indicating extension. Average protrusion and retraction speeds were calculated from n = 11–13 cells. Bars: mean ± s.e.m. See also Movies EV2 and EV3 . Cells treated with the indicated PMOs or siRNAs were analyzed by Western blot (upper panels) to detect RAB13 and GAPDH protein levels. Migration speed was assessed as in (B) from n = 55–78 cells (bottom graph). Bars: mean ± s.e.m. Protrusion and retraction speed of cells treated with the indicated PMOs or siRNAs were assessed as in (D). Graphs show average normalized values from n = 10–13 cells imaged for 1 h. Bars: mean ± s.e.m. Migration speed of RAB13 knockdown cells re‐expressing GFP‐RAB13 or GFP‐RAB13 with a frameshift (fs) mutation at the beginning of the RAB13 coding region. 28–52 cells were analyzed. Bars: mean ± s.e.m. Similar results were obtained in two additional independent experiments. Re‐expressing cells were identified through GFP fluorescence, and cells with similar, low GFP signal were tracked. All cells are expressing cherry‐NLS for accurate tracking. Data information: P ‐values: *

    Techniques Used: Migration, Multiple Displacement Amplification, Expressing, Staining, Western Blot, Mutagenesis, Fluorescence

    13) Product Images from "BBSome trains remove activated GPCRs from cilia by enabling passage through the transition zone"

    Article Title: BBSome trains remove activated GPCRs from cilia by enabling passage through the transition zone

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201709041

    Activated GPCRs undergo processive retrograde movements and confinements at base and tip. (A) Diagram of the Qdot labeling strategy. IMCD3-[pEF1αΔ- AP SSTR3 NG ] cells stably expressing BirA-ER were first treated with unlabeled mSA to passivate the surface-exposed biotinylated AP SSTR3. SA Qdots were then added to the medium to label the GPCRs newly arrived at the surface. Finally, biotin was added to the medium to passivate the excess SA on Qdots. (B) The diffusive properties of SSTR3 are not altered by Qdot labeling. The instantaneous velocities of mSA647-labeled AP SSTR3 NG and Qdot655-labeled AP SSTR3 NG were measured by single-molecule tracking in the absence of sst. n = 1,223–3,032 instantaneous velocities. (C) Qdot labeling does not alter the exit rate of SSTR3. IMCD3-[pEF1α- AP SSTR3 GFP ] cells were sparsely labeled with Qdot655 as described in Materials and methods and treated with vehicle or sst for 2 h before fixation. The number of Qdots per cilium was counted in both vehicle- and sst-treated conditions. n = 263–303 cilia from three independent experiments. Error bars represent SEM. (D) Representative kymographs showing the movements of SA Qdot-labeled ciliary AP SSTR3 NG ( QD SSTR3) in vehicle- or sst-treated cells. Red labels and line coloring highlight four characteristic movement behaviors. Bar, 2 µm. b, base; t, tip. (E) Centroid mapping of QD SSTR3. Left: The contour of the cilium was traced as a dotted line that captures all QD SSTR3 positions. (i) Diffusive movement, (ii) tip confinement followed by retrograde movement, (iii) retrograde movement followed by base confinement and return into the cilium, and (iv) tip confinement followed by fully processive retrograde movement and base confinement. The time dimension is color coded from red to purple. (F and G) Signaling increases tip confinement and processive retrograde movement of SSTR3. Cells were treated with sst (green, sst) or vehicle (gray, control) for 40 min before imaging was initiated for 10–20 min. (F) Durations of persistent movement events for QD SSTR3 in anterograde and retrograde directions. (G) The durations of confinement events for QD SSTR3 at ciliary tip or base were binned into two categories. n = 12 cilia for each condition. (H) Representative kymographs showing the comovement between a single QD SSTR3 and a BBSome retrograde train. IMCD3-[pEF1α- NG3 BBS5; pEF1αΔ- AP SSTR3] cells were treated with sst for 40 min before imaging. Bar, 2 μm. B, base; T, tip.
    Figure Legend Snippet: Activated GPCRs undergo processive retrograde movements and confinements at base and tip. (A) Diagram of the Qdot labeling strategy. IMCD3-[pEF1αΔ- AP SSTR3 NG ] cells stably expressing BirA-ER were first treated with unlabeled mSA to passivate the surface-exposed biotinylated AP SSTR3. SA Qdots were then added to the medium to label the GPCRs newly arrived at the surface. Finally, biotin was added to the medium to passivate the excess SA on Qdots. (B) The diffusive properties of SSTR3 are not altered by Qdot labeling. The instantaneous velocities of mSA647-labeled AP SSTR3 NG and Qdot655-labeled AP SSTR3 NG were measured by single-molecule tracking in the absence of sst. n = 1,223–3,032 instantaneous velocities. (C) Qdot labeling does not alter the exit rate of SSTR3. IMCD3-[pEF1α- AP SSTR3 GFP ] cells were sparsely labeled with Qdot655 as described in Materials and methods and treated with vehicle or sst for 2 h before fixation. The number of Qdots per cilium was counted in both vehicle- and sst-treated conditions. n = 263–303 cilia from three independent experiments. Error bars represent SEM. (D) Representative kymographs showing the movements of SA Qdot-labeled ciliary AP SSTR3 NG ( QD SSTR3) in vehicle- or sst-treated cells. Red labels and line coloring highlight four characteristic movement behaviors. Bar, 2 µm. b, base; t, tip. (E) Centroid mapping of QD SSTR3. Left: The contour of the cilium was traced as a dotted line that captures all QD SSTR3 positions. (i) Diffusive movement, (ii) tip confinement followed by retrograde movement, (iii) retrograde movement followed by base confinement and return into the cilium, and (iv) tip confinement followed by fully processive retrograde movement and base confinement. The time dimension is color coded from red to purple. (F and G) Signaling increases tip confinement and processive retrograde movement of SSTR3. Cells were treated with sst (green, sst) or vehicle (gray, control) for 40 min before imaging was initiated for 10–20 min. (F) Durations of persistent movement events for QD SSTR3 in anterograde and retrograde directions. (G) The durations of confinement events for QD SSTR3 at ciliary tip or base were binned into two categories. n = 12 cilia for each condition. (H) Representative kymographs showing the comovement between a single QD SSTR3 and a BBSome retrograde train. IMCD3-[pEF1α- NG3 BBS5; pEF1αΔ- AP SSTR3] cells were treated with sst for 40 min before imaging. Bar, 2 μm. B, base; T, tip.

    Techniques Used: Labeling, Stable Transfection, Expressing, Imaging

    Reconstitution of signal-dependent retrieval of SSTR3 and GPR161. (A) Diagram of the signal-dependent retrieval systems under study. Left: Addition of sst triggers SSTR3 exit from cilia by directly activating SSTR3. Right: Addition of SAG activates the Hedgehog pathway and promotes GPR161 retrieval. SMO, Smoothened. (B) Kinetics of SSTR3 disappearance from cilia of primary hippocampal neurons and of IMCD3 stably expressing AP SSTR3 NG under the control of the TATA-less EF1α promoter were estimated by quantitation of immunofluorescence signals after addition of sst. The entire dataset for the sst condition is shown in Fig. S1 B. Data were fitted to a single exponential. Error bars indicate 95% confidence interval (CI). n = 280–424 cilia (neurons) and 57–80 cilia (IMCD3). (C) High-level expression of SSTR3 drives elongation of primary cilia. Top: AP SSTR3 GFP driven by various promoters or AP SSTR3 NG driven by EF1αΔ promoter was expressed stably at the FlpIn locus of IMCD3 cells, and ciliary fluorescence levels were measured and compared to a GFP calibrator ( Breslow et al., 2013 ) or an NG calibrator (see Materials and methods). Endogenous SSTR3 levels were estimated by comparative immunostaining (see Materials and methods). A Mann-Whitney test was used for pairwise comparisons of the number of SSTR3 molecules per cilia in neurons and in IMCD3 cells expressing AP SSTR3 NG or AP SSTR3 GFP under the control of pEF1αΔ. P > 0.05. n = 10–38 cilia. Error bars represent SD. Bottom: Effect of AP SSTR3 GFP expression on cilium length. Cilia lengths were measured in the GFP channel by live-cell imaging. n = 10–38 cilia. Error bars represent SD. Cilium lengthening upon GPCR overexpression was previously reported by Guadiana et al. (2013) . (D) IMCD3-[pCrys- AP GPR161 NG3 ] were treated for 2 h with either SAG or vehicle. AP GPR161 NG3 was visualized by NG fluorescence, and basal bodies of cilia were stained with ninein. All cells were pretreated with the translation inhibitor emetine to eliminate signals from new protein synthesis. Bar, 4 µm. (E) Absolute quantitation of ciliary GPCR abundance. Top: Calibration of single-molecule fluorescence intensity. Bacterially expressed NG3 protein was spotted on glass coverslips (inset), and the fluorescent intensity of each individual NG3 was measured. n = 1,257 particles measured. Bottom: The three-step photobleaching of a representative spot shows that the fluorescence was emitted by a single NG3 molecule. The measured fluorescence intensity of NG3 was used to calibrate NG- and NG3-tagged SSTR3, GPR161, BBS5, and IFT88. Bar, 0.5 μm. (F) IMCD3-[pEF1αΔ- AP SSTR3 NG ] cells were treated with vehicle or sst for 2 h. Stable expression of an ER-localized biotin ligase BirA enables the biotinylation of AP SSTR3 with the biotin existing in the DMEM/F-12 cell culture medium. Ciliary AP SSTR3 was pulse-labeled by Alexa Fluor 647–conjugated mSA (mSA647) for 5–10 min before imaging (see Materials and methods for details). Bar, 1 μm. The absolute number of AP SSTR3 NG molecules per cilia at t 0 was calculated by measuring the NG signal and using the NG3 calibrator. For all other time points, the ratio in ciliary mSA647 signal compared with t 0 was used to calculate the absolute number of molecules (see Materials and methods for details). Data were fitted to a single exponential. Error bars indicate 95% CI. n = 14 cilia. (G) IMCD3-[pCrys-GPR161 NG3 ] cells were treated with SAG or vehicle for 2 h. NG fluorescence was tracked in individual cilia, and the ratio of GPR161 NG3 to endogenous GPR161 was used to calculate the total levels of GPR161 as detailed in Materials and methods. Bar, 1 μm. Data were fitted to a single exponential. Error bars indicate 95% CI. n = 12–20 cilia.
    Figure Legend Snippet: Reconstitution of signal-dependent retrieval of SSTR3 and GPR161. (A) Diagram of the signal-dependent retrieval systems under study. Left: Addition of sst triggers SSTR3 exit from cilia by directly activating SSTR3. Right: Addition of SAG activates the Hedgehog pathway and promotes GPR161 retrieval. SMO, Smoothened. (B) Kinetics of SSTR3 disappearance from cilia of primary hippocampal neurons and of IMCD3 stably expressing AP SSTR3 NG under the control of the TATA-less EF1α promoter were estimated by quantitation of immunofluorescence signals after addition of sst. The entire dataset for the sst condition is shown in Fig. S1 B. Data were fitted to a single exponential. Error bars indicate 95% confidence interval (CI). n = 280–424 cilia (neurons) and 57–80 cilia (IMCD3). (C) High-level expression of SSTR3 drives elongation of primary cilia. Top: AP SSTR3 GFP driven by various promoters or AP SSTR3 NG driven by EF1αΔ promoter was expressed stably at the FlpIn locus of IMCD3 cells, and ciliary fluorescence levels were measured and compared to a GFP calibrator ( Breslow et al., 2013 ) or an NG calibrator (see Materials and methods). Endogenous SSTR3 levels were estimated by comparative immunostaining (see Materials and methods). A Mann-Whitney test was used for pairwise comparisons of the number of SSTR3 molecules per cilia in neurons and in IMCD3 cells expressing AP SSTR3 NG or AP SSTR3 GFP under the control of pEF1αΔ. P > 0.05. n = 10–38 cilia. Error bars represent SD. Bottom: Effect of AP SSTR3 GFP expression on cilium length. Cilia lengths were measured in the GFP channel by live-cell imaging. n = 10–38 cilia. Error bars represent SD. Cilium lengthening upon GPCR overexpression was previously reported by Guadiana et al. (2013) . (D) IMCD3-[pCrys- AP GPR161 NG3 ] were treated for 2 h with either SAG or vehicle. AP GPR161 NG3 was visualized by NG fluorescence, and basal bodies of cilia were stained with ninein. All cells were pretreated with the translation inhibitor emetine to eliminate signals from new protein synthesis. Bar, 4 µm. (E) Absolute quantitation of ciliary GPCR abundance. Top: Calibration of single-molecule fluorescence intensity. Bacterially expressed NG3 protein was spotted on glass coverslips (inset), and the fluorescent intensity of each individual NG3 was measured. n = 1,257 particles measured. Bottom: The three-step photobleaching of a representative spot shows that the fluorescence was emitted by a single NG3 molecule. The measured fluorescence intensity of NG3 was used to calibrate NG- and NG3-tagged SSTR3, GPR161, BBS5, and IFT88. Bar, 0.5 μm. (F) IMCD3-[pEF1αΔ- AP SSTR3 NG ] cells were treated with vehicle or sst for 2 h. Stable expression of an ER-localized biotin ligase BirA enables the biotinylation of AP SSTR3 with the biotin existing in the DMEM/F-12 cell culture medium. Ciliary AP SSTR3 was pulse-labeled by Alexa Fluor 647–conjugated mSA (mSA647) for 5–10 min before imaging (see Materials and methods for details). Bar, 1 μm. The absolute number of AP SSTR3 NG molecules per cilia at t 0 was calculated by measuring the NG signal and using the NG3 calibrator. For all other time points, the ratio in ciliary mSA647 signal compared with t 0 was used to calculate the absolute number of molecules (see Materials and methods for details). Data were fitted to a single exponential. Error bars indicate 95% CI. n = 14 cilia. (G) IMCD3-[pCrys-GPR161 NG3 ] cells were treated with SAG or vehicle for 2 h. NG fluorescence was tracked in individual cilia, and the ratio of GPR161 NG3 to endogenous GPR161 was used to calculate the total levels of GPR161 as detailed in Materials and methods. Bar, 1 μm. Data were fitted to a single exponential. Error bars indicate 95% CI. n = 12–20 cilia.

    Techniques Used: Stable Transfection, Expressing, Quantitation Assay, Immunofluorescence, Fluorescence, Immunostaining, MANN-WHITNEY, Live Cell Imaging, Over Expression, Staining, Cell Culture, Labeling, Imaging

    14) Product Images from "Locomotor-related propriospinal V3 neurons produce primary afferent depolarization and modulate sensory transmission to motoneurons"

    Article Title: Locomotor-related propriospinal V3 neurons produce primary afferent depolarization and modulate sensory transmission to motoneurons

    Journal: bioRxiv

    doi: 10.1101/2022.07.04.498712

    Connections between V3 neurons and GABAergic neurons that produce PAD. A - B : V3 neurons (tdTom) contacts onto GABAergic neurons, where presynaptic V3 neuron contacts are VGLUT2 + (red), the vesicular transporter expressed in these glutamatergic V3 neurons. Spinal cord from mouse expressing Sim1//tdTom (magenta, V3) and GAD1-GFP (green, GABAergic neurons). C : Close up of V3 neuron contacts (Sim1//tdTom) onto GABAergic neuron (GAD1-GFP)), with V3 presynaptic terminal labelled with bassoon and GABAergic neuron labelled with GAD2, the latter to show that it is a GAD2 + neuron which uniquely innervates afferents (Todd refs). Bassoon is also expressed in the GABAergic neuron boutons near the GAD2 clusters and the V3 contacts. D : GABAergic (GAD1-GFP) neurons also innervate V3 neurons, with GAD2 + presynaptic contacts. E : Distribution and incidence of contacts between V3 neurons and GABAergic neurons in the dorsal, deep dorsal, intermediate and ventral laminae. F : Schematic summarizing trisynaptic circuit mediating PAD with the addition of a contact from GABAergic neurons onto V3 neurons that inhibits the circuit.
    Figure Legend Snippet: Connections between V3 neurons and GABAergic neurons that produce PAD. A - B : V3 neurons (tdTom) contacts onto GABAergic neurons, where presynaptic V3 neuron contacts are VGLUT2 + (red), the vesicular transporter expressed in these glutamatergic V3 neurons. Spinal cord from mouse expressing Sim1//tdTom (magenta, V3) and GAD1-GFP (green, GABAergic neurons). C : Close up of V3 neuron contacts (Sim1//tdTom) onto GABAergic neuron (GAD1-GFP)), with V3 presynaptic terminal labelled with bassoon and GABAergic neuron labelled with GAD2, the latter to show that it is a GAD2 + neuron which uniquely innervates afferents (Todd refs). Bassoon is also expressed in the GABAergic neuron boutons near the GAD2 clusters and the V3 contacts. D : GABAergic (GAD1-GFP) neurons also innervate V3 neurons, with GAD2 + presynaptic contacts. E : Distribution and incidence of contacts between V3 neurons and GABAergic neurons in the dorsal, deep dorsal, intermediate and ventral laminae. F : Schematic summarizing trisynaptic circuit mediating PAD with the addition of a contact from GABAergic neurons onto V3 neurons that inhibits the circuit.

    Techniques Used: Expressing

    15) Product Images from "A viral toolbox for conditional and transneuronal gene expression in zebrafish"

    Article Title: A viral toolbox for conditional and transneuronal gene expression in zebrafish

    Journal: eLife

    doi: 10.7554/eLife.77153

    Analysis of gene expression in GABAergic neurons. ( A ) Schematic: injection of EnvA-RVΔG-GFP into the OB of adult Tg[ gad1b :Gal4; UAS :TVA-mCherry] fish. ( B ) Example of FACS analysis of GFP and mCherry expression. Boxes depict cells selected as mCherry+/GFP+ (EnvA-RVΔG-GFP infected gad1b neurons), mCherry+/GFP- (non-infected gad1b neurons), mCherry-/GFP- (negative control containing other OB cells). gad1b is one of two isoforms of gad1 that are expressed differentially in GABAergic neurons. ( C ) Expression of marker genes (x-axis) in infected gad1b neurons (mCherry+/GFP+; green), non-infected gad1b neurons (mCherry+/GFP-; magenta), and other OB cells (mCherry-/GFP-; black). Cells classified as gad1b-positive by fluorescence markers, but not other cells, expressed gad1b but not gad1a , the other gad1 isoform. Expression of fluorescent marker genes followed the detection of fluorescent markers by FACS. The neuronal marker elav3 was present in all three pools. Plot symbols represent data from individual samples; box plots show median and 25 th and 75 th percentiles, circles and error bars indicate mean and s.d. over individual samples (N=8 samples). ( D ) Expression of negative markers for GABAergic neurons. The selected marker genes ( slc17a6a , slc17a6b , tbx21 , lhx2b, and lhx9 ) should be expressed in mitral cells of the OB and other excitatory neurons but not in GABAergic neurons. Consistent with this expectation, expression of all negative markers was low or absent in pools of gad1b cells selected by FACS (N=8 samples).
    Figure Legend Snippet: Analysis of gene expression in GABAergic neurons. ( A ) Schematic: injection of EnvA-RVΔG-GFP into the OB of adult Tg[ gad1b :Gal4; UAS :TVA-mCherry] fish. ( B ) Example of FACS analysis of GFP and mCherry expression. Boxes depict cells selected as mCherry+/GFP+ (EnvA-RVΔG-GFP infected gad1b neurons), mCherry+/GFP- (non-infected gad1b neurons), mCherry-/GFP- (negative control containing other OB cells). gad1b is one of two isoforms of gad1 that are expressed differentially in GABAergic neurons. ( C ) Expression of marker genes (x-axis) in infected gad1b neurons (mCherry+/GFP+; green), non-infected gad1b neurons (mCherry+/GFP-; magenta), and other OB cells (mCherry-/GFP-; black). Cells classified as gad1b-positive by fluorescence markers, but not other cells, expressed gad1b but not gad1a , the other gad1 isoform. Expression of fluorescent marker genes followed the detection of fluorescent markers by FACS. The neuronal marker elav3 was present in all three pools. Plot symbols represent data from individual samples; box plots show median and 25 th and 75 th percentiles, circles and error bars indicate mean and s.d. over individual samples (N=8 samples). ( D ) Expression of negative markers for GABAergic neurons. The selected marker genes ( slc17a6a , slc17a6b , tbx21 , lhx2b, and lhx9 ) should be expressed in mitral cells of the OB and other excitatory neurons but not in GABAergic neurons. Consistent with this expectation, expression of all negative markers was low or absent in pools of gad1b cells selected by FACS (N=8 samples).

    Techniques Used: Expressing, Injection, Fluorescence In Situ Hybridization, FACS, Infection, Negative Control, Marker, Fluorescence

    Co-packaging of two different viruses does not facilitate co-infection of two viruses. ( A ) Expression of GFP and mCherry after injection of HSV1[ UAS :GFP UAS :TVA-mCherry] into the cerebellum of Tg[ gad1b :Gal4] fish. In this virus, two expression constructs, UAS :GFP and UAS :TVA-mCherry, are packaged into the same virus particles. Expression is observed in Purkinje neurons and in putative Golgi cells. Note high rate of co-expression of GFP and mCherry. ML: molecular layer; PL: Purkinje layer; GL: granular layer. ( B ) Expression of GFP and mCherry in the Purkinje layer after co-injection of two independent viruses (HSV1[ UAS :GFP] and HSV1[ UAS :TVA-mCherry]) into the cerebellum of Tg[ gad1b :Gal4] fish. Note that the rate of co-expression was high even though GFP and mCherry were delivered by separate viruses. Note also that the overall expression was sparse, implying that co-expression was unlikely to occur by chance. ( C ) Percentage of GFP and mCherry-expressing neurons among all fluorescent neurons. Filled circles represent data from individual fish, box plot indicates median and the 25th and 75th percentiles, and open circles indicate mean over individual fish. N: number of fish.
    Figure Legend Snippet: Co-packaging of two different viruses does not facilitate co-infection of two viruses. ( A ) Expression of GFP and mCherry after injection of HSV1[ UAS :GFP UAS :TVA-mCherry] into the cerebellum of Tg[ gad1b :Gal4] fish. In this virus, two expression constructs, UAS :GFP and UAS :TVA-mCherry, are packaged into the same virus particles. Expression is observed in Purkinje neurons and in putative Golgi cells. Note high rate of co-expression of GFP and mCherry. ML: molecular layer; PL: Purkinje layer; GL: granular layer. ( B ) Expression of GFP and mCherry in the Purkinje layer after co-injection of two independent viruses (HSV1[ UAS :GFP] and HSV1[ UAS :TVA-mCherry]) into the cerebellum of Tg[ gad1b :Gal4] fish. Note that the rate of co-expression was high even though GFP and mCherry were delivered by separate viruses. Note also that the overall expression was sparse, implying that co-expression was unlikely to occur by chance. ( C ) Percentage of GFP and mCherry-expressing neurons among all fluorescent neurons. Filled circles represent data from individual fish, box plot indicates median and the 25th and 75th percentiles, and open circles indicate mean over individual fish. N: number of fish.

    Techniques Used: Infection, Expressing, Injection, Fluorescence In Situ Hybridization, Construct

    Transneuronal tracing using pseudotyped rabies virus in zebrafish larvae. ( A ) Expression of GFP (green) and TVA-mCherry (red) 6 days after injection of EnvA-RVΔG-GFP into the spinal cord of Tg[ gad1b :Gal4; UAS :TVA-mCherry] fish at 7 dpf. Boxed region is enlarged on the right. ( B ) Same after co-injection of EnvA-RVΔG-GFP and HSV1[ UAS :zoSADG] into the spinal cord of Tg[ gad1b :Gal4; UAS :TVA-mCherry] fish at 7 dpf.
    Figure Legend Snippet: Transneuronal tracing using pseudotyped rabies virus in zebrafish larvae. ( A ) Expression of GFP (green) and TVA-mCherry (red) 6 days after injection of EnvA-RVΔG-GFP into the spinal cord of Tg[ gad1b :Gal4; UAS :TVA-mCherry] fish at 7 dpf. Boxed region is enlarged on the right. ( B ) Same after co-injection of EnvA-RVΔG-GFP and HSV1[ UAS :zoSADG] into the spinal cord of Tg[ gad1b :Gal4; UAS :TVA-mCherry] fish at 7 dpf.

    Techniques Used: Expressing, Injection, Fluorescence In Situ Hybridization

    HSV1-mediated gene expression. ( A ) Expression of DsRed (magenta) 6 days after injection of HSV1[ LTCMV :DsRed] into the OB (arrow) of an adult Tg[ vglut1 :GFP] fish kept at 26 °C (maximum projection of confocal stack). Boxed areas (OB and Dp) are enlarged below. The number of DsRed-expressing neurons is low compared to DsRed expression at 36 °C ( Figure 1 ). ( B ) DsRed expression in the dorsal telencephalon at different timepoints after injection of HSV1[ LTCMV :DsRed] into the ipsilateral OB. Fish were kept at 36 °C. Black dots represent data from individual fish, box plot indicates median and 25th and 75th percentiles, circles and error bars indicate mean and s.d. over individual fish. N: number of fish.
    Figure Legend Snippet: HSV1-mediated gene expression. ( A ) Expression of DsRed (magenta) 6 days after injection of HSV1[ LTCMV :DsRed] into the OB (arrow) of an adult Tg[ vglut1 :GFP] fish kept at 26 °C (maximum projection of confocal stack). Boxed areas (OB and Dp) are enlarged below. The number of DsRed-expressing neurons is low compared to DsRed expression at 36 °C ( Figure 1 ). ( B ) DsRed expression in the dorsal telencephalon at different timepoints after injection of HSV1[ LTCMV :DsRed] into the ipsilateral OB. Fish were kept at 36 °C. Black dots represent data from individual fish, box plot indicates median and 25th and 75th percentiles, circles and error bars indicate mean and s.d. over individual fish. N: number of fish.

    Techniques Used: Expressing, Injection, Fluorescence In Situ Hybridization

    Expression pattern of vglut1 and vglut2 in olfactory bulb and Dp. ( A ) Coronal cross sections through the OB and anterior telencephalon from Tg[ vglut2a :RFP; vglut1 :GFP] double transgenic fish. Note that vglut2a (magenta) is expressed by axons of olfactory sensory neurons innervating glomeruli in the OB and by a subset of mitral cells, while expression of vglut1 (green) in the OB is weak or absent. Dotted lines outline OBs. ( B ) More posterior coronal cross sections through the telencephalon of the same fish at the level of Dp. Note that expression of vglut2a and vglut1 in the telencephalon are largely complementary. Neurons in Dp express primarily vglut1 . Dotted areas indicate the dorsal lateral telencephalic area (Dl) and Dp.
    Figure Legend Snippet: Expression pattern of vglut1 and vglut2 in olfactory bulb and Dp. ( A ) Coronal cross sections through the OB and anterior telencephalon from Tg[ vglut2a :RFP; vglut1 :GFP] double transgenic fish. Note that vglut2a (magenta) is expressed by axons of olfactory sensory neurons innervating glomeruli in the OB and by a subset of mitral cells, while expression of vglut1 (green) in the OB is weak or absent. Dotted lines outline OBs. ( B ) More posterior coronal cross sections through the telencephalon of the same fish at the level of Dp. Note that expression of vglut2a and vglut1 in the telencephalon are largely complementary. Neurons in Dp express primarily vglut1 . Dotted areas indicate the dorsal lateral telencephalic area (Dl) and Dp.

    Techniques Used: Expressing, Transgenic Assay, Fluorescence In Situ Hybridization

    Targeting of GABAergic neurons in the telencephalon. ( A ) Injection of HSV1[ UAS :TVA-mCherry] into the telencephalon of adult wildtype fish. No expression of TVA-mCherry was detectable (granular particles are autofluorescent endogenous objects). ( B ) Coronal section through the telencephalon at the level of Dp after injection of HSV1[ UAS :TVA-mCherry] into Tg[ gad1b :Gal4; gad1b :GFP] double transgenic fish. The injection was targeted to a volume around Dp. mCherry was expressed predominantly in a cluster of GFP-positive neurons associated with Dp. Note long-range projections of mCherry-expressing neurons to multiple telencephalic areas. ( C ) Enlargements of boxed region in ( a ). Arrowheads indicate GFP+/mCherry +neurons.
    Figure Legend Snippet: Targeting of GABAergic neurons in the telencephalon. ( A ) Injection of HSV1[ UAS :TVA-mCherry] into the telencephalon of adult wildtype fish. No expression of TVA-mCherry was detectable (granular particles are autofluorescent endogenous objects). ( B ) Coronal section through the telencephalon at the level of Dp after injection of HSV1[ UAS :TVA-mCherry] into Tg[ gad1b :Gal4; gad1b :GFP] double transgenic fish. The injection was targeted to a volume around Dp. mCherry was expressed predominantly in a cluster of GFP-positive neurons associated with Dp. Note long-range projections of mCherry-expressing neurons to multiple telencephalic areas. ( C ) Enlargements of boxed region in ( a ). Arrowheads indicate GFP+/mCherry +neurons.

    Techniques Used: Injection, Fluorescence In Situ Hybridization, Expressing, Transgenic Assay

    Injection of pseudotyped rabies virus does not infect neurons in the absence of TVA. ( A ) Absence of expression after injection of EnvA-RVΔG-GFP into the telencephalon of adult wildtype fish.( B ) Absence of expression after injection of EnvA-RVΔG-GFP into the optic tectum of wildtype fish at 7 dpf.
    Figure Legend Snippet: Injection of pseudotyped rabies virus does not infect neurons in the absence of TVA. ( A ) Absence of expression after injection of EnvA-RVΔG-GFP into the telencephalon of adult wildtype fish.( B ) Absence of expression after injection of EnvA-RVΔG-GFP into the optic tectum of wildtype fish at 7 dpf.

    Techniques Used: Injection, Expressing, Fluorescence In Situ Hybridization

    Sequential injection of HSV1 and rabies virus. Expression of GFP in the cerebellum after sequential injection of (1) HSV1[ UAS :TVA-mCherry] and HSV1[ UAS :zoSADG] and (2) EnvA-RVΔG-GFP into the cerebellum of Tg[ gad1b :Gal4] fish. Outlined regions are enlarged. Only few labeled neurons were detected. Left: EnvA-RVΔG-GFP was injected 2 days after HSV1 injection. Right: EnvA-RVΔG-GFP was injected 4 days after HSV1 injection.
    Figure Legend Snippet: Sequential injection of HSV1 and rabies virus. Expression of GFP in the cerebellum after sequential injection of (1) HSV1[ UAS :TVA-mCherry] and HSV1[ UAS :zoSADG] and (2) EnvA-RVΔG-GFP into the cerebellum of Tg[ gad1b :Gal4] fish. Outlined regions are enlarged. Only few labeled neurons were detected. Left: EnvA-RVΔG-GFP was injected 2 days after HSV1 injection. Right: EnvA-RVΔG-GFP was injected 4 days after HSV1 injection.

    Techniques Used: Injection, Expressing, Fluorescence In Situ Hybridization, Labeling

    Temperature-dependence of infection by rabies virus. ( A ) Experimental scheme: Rabies virus (EnvA-RVΔG-GFP) was injected into the telencephalon of transgenic fish expressing TVA-mCherry in GABAergic neurons (Tg[ gad1b :Gal4; UAS :TVA-mCherry]). ( B ) Expression of TVA-mCherry and GFP when fish were kept at 26 °C for 6 days after injection. Note almost complete absence of GFP expression.( C ) Expression of TVA-mCherry and GFP when fish were kept at 36 °C for 6 days after injection. Note strong GFP expression.( D ) Expression of TVA-mCherry and GFP six days after injection when the housing temperature was increased from 26 °C to 36 °C 3 days after injection. GFP expression was weak and sparse.
    Figure Legend Snippet: Temperature-dependence of infection by rabies virus. ( A ) Experimental scheme: Rabies virus (EnvA-RVΔG-GFP) was injected into the telencephalon of transgenic fish expressing TVA-mCherry in GABAergic neurons (Tg[ gad1b :Gal4; UAS :TVA-mCherry]). ( B ) Expression of TVA-mCherry and GFP when fish were kept at 26 °C for 6 days after injection. Note almost complete absence of GFP expression.( C ) Expression of TVA-mCherry and GFP when fish were kept at 36 °C for 6 days after injection. Note strong GFP expression.( D ) Expression of TVA-mCherry and GFP six days after injection when the housing temperature was increased from 26 °C to 36 °C 3 days after injection. GFP expression was weak and sparse.

    Techniques Used: Infection, Injection, Transgenic Assay, Fluorescence In Situ Hybridization, Expressing

    Functional manipulation using HSV1. ( A ) Top: Expression of GFP in the cerebellum after injection of HSV1[ UAS :GFP] into the cerebellum of Tg[ gad1b :Gal4] fish. Bottom: Expression of TeNT-GFP after injection of HSV1[ UAS :TeNT-GFP] into the cerebellum of Tg[ gad1b :Gal4] fish. ( B ) Examples of swimming trajectories (15 min) of individual Tg[ gad1b :Gal4] fish that received injections of HSV1[ UAS :GFP] (left) or HSV1[ UAS :TeNT-GFP] into the cerebellum. Note that the fish injected with HSV1[ UAS :TeNT-GFP] covered less territory, showed fewer long straight swims, and showed a tendency to stay lower in the water column. ( C ) Mean swimming speed and 2D space occupancy of Tg[ gad1b :Gal4] fish that received injections of HSV1[ UAS :GFP] (left) or HSV1[ UAS :TeNT-GFP] into the cerebellum. Plot symbols represent data from individual fish; box plots show median and 25th and 75th percentiles, circles and error bars indicate mean and s.d. over individual fish. N: number of fish. p=0.03 for swimming speed, p=0.0012 for space occupancy, Wilcoxon rank sum test.
    Figure Legend Snippet: Functional manipulation using HSV1. ( A ) Top: Expression of GFP in the cerebellum after injection of HSV1[ UAS :GFP] into the cerebellum of Tg[ gad1b :Gal4] fish. Bottom: Expression of TeNT-GFP after injection of HSV1[ UAS :TeNT-GFP] into the cerebellum of Tg[ gad1b :Gal4] fish. ( B ) Examples of swimming trajectories (15 min) of individual Tg[ gad1b :Gal4] fish that received injections of HSV1[ UAS :GFP] (left) or HSV1[ UAS :TeNT-GFP] into the cerebellum. Note that the fish injected with HSV1[ UAS :TeNT-GFP] covered less territory, showed fewer long straight swims, and showed a tendency to stay lower in the water column. ( C ) Mean swimming speed and 2D space occupancy of Tg[ gad1b :Gal4] fish that received injections of HSV1[ UAS :GFP] (left) or HSV1[ UAS :TeNT-GFP] into the cerebellum. Plot symbols represent data from individual fish; box plots show median and 25th and 75th percentiles, circles and error bars indicate mean and s.d. over individual fish. N: number of fish. p=0.03 for swimming speed, p=0.0012 for space occupancy, Wilcoxon rank sum test.

    Techniques Used: Functional Assay, Expressing, Injection, Fluorescence In Situ Hybridization

    HSV1-mediated gene delivery in larvae zebrafish and Gal4/UAS. ( A ) Expression of GFP 48 hr after injection of HSV1[ LTCMV :GFP] into the optic tectum of zebrafish larvae (3 dpf; maximum intensity projection of confocal stack). Larvae were kept after the injection at 28.5, 32, or 35 °C. ( B ) Expression of GFP 48 hr after injection of HSV1[ LTCMV :GFP] into the optic tectum of a larva at 5 dpf (maximum intensity projection of confocal stack). The larva was kept after the injection at 35 °C. ( C ) Expression of GFP 48 hr after injection of HSV1[ LTCMV :GFP] into the optic tectum of a larva at 14 dpf (maximum intensity projection of confocal stack). The larva was kept after the injection at 35 °C. ( D ) Expression of GFP 48 hr after injection of HSV1[ LTCMV :GFP] into trunk muscles at 7 dpf (maximum intensity projection of confocal stack). The larva was kept after the injection at 35 °C. Note retrograde labeling of motor neurons (M.N.). ( E ) Expression of GFP 48 hr after injection of HSV1[ UAS :GFP] into the hindbrain of a Tg[ gad1b :Gal4; gad1b :DsRed] larva at 7 dpf (maximum intensity projection of confocal stack). The larva was kept after the injection at 35 °C. Note co-localization of DsRed and GFP in hindbrain and cerebellum.
    Figure Legend Snippet: HSV1-mediated gene delivery in larvae zebrafish and Gal4/UAS. ( A ) Expression of GFP 48 hr after injection of HSV1[ LTCMV :GFP] into the optic tectum of zebrafish larvae (3 dpf; maximum intensity projection of confocal stack). Larvae were kept after the injection at 28.5, 32, or 35 °C. ( B ) Expression of GFP 48 hr after injection of HSV1[ LTCMV :GFP] into the optic tectum of a larva at 5 dpf (maximum intensity projection of confocal stack). The larva was kept after the injection at 35 °C. ( C ) Expression of GFP 48 hr after injection of HSV1[ LTCMV :GFP] into the optic tectum of a larva at 14 dpf (maximum intensity projection of confocal stack). The larva was kept after the injection at 35 °C. ( D ) Expression of GFP 48 hr after injection of HSV1[ LTCMV :GFP] into trunk muscles at 7 dpf (maximum intensity projection of confocal stack). The larva was kept after the injection at 35 °C. Note retrograde labeling of motor neurons (M.N.). ( E ) Expression of GFP 48 hr after injection of HSV1[ UAS :GFP] into the hindbrain of a Tg[ gad1b :Gal4; gad1b :DsRed] larva at 7 dpf (maximum intensity projection of confocal stack). The larva was kept after the injection at 35 °C. Note co-localization of DsRed and GFP in hindbrain and cerebellum.

    Techniques Used: Expressing, Injection, Labeling

    Transneuronal tracing using pseudotyped rabies virus from vglut1 +neurons in Dp in adult zebrafish. ( A ) Co-injection of EnvA-RVΔG-GFP and HSV1[ UAS :TVA-mCherry] into Dp of Tg[ vglut1 :Gal4] fish in the absence of glycoprotein. Coronal section through the injected telencephalic hemisphere at the level of Dp. Area outlined by dashed rectangle is enlarged below and red channel is enhanced. Co-expression of GFP (green) and mCherry (magenta) indicates starter cells. ( B ) Same as in ( A ) but with trans-complementation of zoSADG in starter neurons by co-injection of HSV1[ UAS :zoSADG]. Left: coronal section through the injected telencephalic hemisphere. Right: coronal section through the ipsilateral olfactory bulb. Expression of GFP only (green) indicates transneuronally labeled neurons.
    Figure Legend Snippet: Transneuronal tracing using pseudotyped rabies virus from vglut1 +neurons in Dp in adult zebrafish. ( A ) Co-injection of EnvA-RVΔG-GFP and HSV1[ UAS :TVA-mCherry] into Dp of Tg[ vglut1 :Gal4] fish in the absence of glycoprotein. Coronal section through the injected telencephalic hemisphere at the level of Dp. Area outlined by dashed rectangle is enlarged below and red channel is enhanced. Co-expression of GFP (green) and mCherry (magenta) indicates starter cells. ( B ) Same as in ( A ) but with trans-complementation of zoSADG in starter neurons by co-injection of HSV1[ UAS :zoSADG]. Left: coronal section through the injected telencephalic hemisphere. Right: coronal section through the ipsilateral olfactory bulb. Expression of GFP only (green) indicates transneuronally labeled neurons.

    Techniques Used: Injection, Fluorescence In Situ Hybridization, Expressing, Labeling

    16) Product Images from "CRL5-dependent regulation of Arl4c and Arf6 controls hippocampal morphogenesis"

    Article Title: CRL5-dependent regulation of Arl4c and Arf6 controls hippocampal morphogenesis

    Journal: bioRxiv

    doi: 10.1101/2020.01.10.902221

    CRL5 regulates pyramidal neuron position and apical dendrite morphology in a cell-autonomous fashion. (A-B) In utero electroporation of GFP (A) or GFP/shCul5 (B) expressing plasmids at E15.5 embryos and samples collected at P10. shCul5 electroporated neurons over-migrated into sr / slm and had “V-shaped” multiple apical dendrites. (a’-b’) High-magnification images of A-B shows detailed PN morphology and dendritic formation. Pink arrowheads indicate PNs with correct position and apical dendrite. Yellow arrowheads indicate over-migrated PNs with abnormal apical dendrites. Yellow hollow arrowheads indicate over-migrated PNs with normal apical dendrite. (C-D) Quantification of the percentage number of displaced GFP+ cells/total GFP+ cells (C), and percentage number of GFP+ cells with normal, disrupted (single apical dendrite with abnormal orientation), and multiple (2 or more) apical dendrites in electroporated hippocampal samples. At least 3 sections were quantified per electroporated brain. Mean ± SD. Statistics, multiple t-test with Bonferroni-Dunn method adjusted p value.
    Figure Legend Snippet: CRL5 regulates pyramidal neuron position and apical dendrite morphology in a cell-autonomous fashion. (A-B) In utero electroporation of GFP (A) or GFP/shCul5 (B) expressing plasmids at E15.5 embryos and samples collected at P10. shCul5 electroporated neurons over-migrated into sr / slm and had “V-shaped” multiple apical dendrites. (a’-b’) High-magnification images of A-B shows detailed PN morphology and dendritic formation. Pink arrowheads indicate PNs with correct position and apical dendrite. Yellow arrowheads indicate over-migrated PNs with abnormal apical dendrites. Yellow hollow arrowheads indicate over-migrated PNs with normal apical dendrite. (C-D) Quantification of the percentage number of displaced GFP+ cells/total GFP+ cells (C), and percentage number of GFP+ cells with normal, disrupted (single apical dendrite with abnormal orientation), and multiple (2 or more) apical dendrites in electroporated hippocampal samples. At least 3 sections were quantified per electroporated brain. Mean ± SD. Statistics, multiple t-test with Bonferroni-Dunn method adjusted p value.

    Techniques Used: In Utero, Electroporation, Expressing

    Arl4c and Arf6 have opposite effects on CRL5-dependent PN over-migration but same effects on PN dendrite extension into the stratum lacunosum moleculare. (A-D) Representative images of hippocampus electroporated at E15 and collected at P10 showing (A) control (GFP), (B) Cul5 KD (shCul5), (C) double Cul5/Arl4c KD (shCul5/shArl4c), and (D) double Cul5/Arf6 KD (shCul5/shArf6) transfected PNs. Whereas Arl4c KD rescue the PN over-migration phenotype caused by Cul5 KD, Arf6 KD exacerbated it. Both Cul5/Arl4c- or Cul5/Arf6-KD PNs fail to extend their apical dendrites into the slm in comparison to control or Cul5-KD PNs. The striped lines represent the hippocampal fissure. (E) Quantification of GFP+ cells in each strata (left) and apical dendrite morphology (right). Statistics, multiple Student t-test with Bonferroni-Dunn method adjusted p value. (F-G) Single (F) Arl4c (shArl4c) or (G) Arf6 (shArf6) KD constructs were co-transfected with GFP by in utero electroporation in E15 hippocampi and brains were collected at P10. In both conditions, PNs migrated normally, had a single radially-oriented apical dendrite, but fail to extend their dendrites into the slm . The striped lines represent the hippocampal fissure.
    Figure Legend Snippet: Arl4c and Arf6 have opposite effects on CRL5-dependent PN over-migration but same effects on PN dendrite extension into the stratum lacunosum moleculare. (A-D) Representative images of hippocampus electroporated at E15 and collected at P10 showing (A) control (GFP), (B) Cul5 KD (shCul5), (C) double Cul5/Arl4c KD (shCul5/shArl4c), and (D) double Cul5/Arf6 KD (shCul5/shArf6) transfected PNs. Whereas Arl4c KD rescue the PN over-migration phenotype caused by Cul5 KD, Arf6 KD exacerbated it. Both Cul5/Arl4c- or Cul5/Arf6-KD PNs fail to extend their apical dendrites into the slm in comparison to control or Cul5-KD PNs. The striped lines represent the hippocampal fissure. (E) Quantification of GFP+ cells in each strata (left) and apical dendrite morphology (right). Statistics, multiple Student t-test with Bonferroni-Dunn method adjusted p value. (F-G) Single (F) Arl4c (shArl4c) or (G) Arf6 (shArf6) KD constructs were co-transfected with GFP by in utero electroporation in E15 hippocampi and brains were collected at P10. In both conditions, PNs migrated normally, had a single radially-oriented apical dendrite, but fail to extend their dendrites into the slm . The striped lines represent the hippocampal fissure.

    Techniques Used: Migration, Transfection, Construct, In Utero, Electroporation

    Dab1 accumulation is not sufficient to disrupt pyramidal neuron positioning or apical dendrite morphology. (A) Dab1 accumulated in both Rbx2cKO-Emx1 and SOCS7-/- hippocampi in comparison to control (Rbx2 fl/fl). Coronal sections of P21 hippocampi were stained with anti-Dab1 and counterstained with DAPI. (B) Representative images of western blotting experiments indicating that Dab1 accumulated in both Rbx2cKO-Emx1 and SOCS7-/- hippocampal samples, but Fyn only accumulated in Rbx2cKO-Emx1 P21 hippocampal lysates. (C) Quantification of displaced DKK3+ cells. No difference was observed between genotypes. At least 3 sections quantified per brain. Mean ± SD. Statistics, multiple t-test with Bonferroni-Dunn method adjusted p value. (D) Coronal sections of P21 SOCS7+/- and SOCS7-/- hippocampi were stained with an anti-DKK3 antibody and counterstained with DAPI. (d’-d’’) High-magnification images of the CA1 indicating that normal PN localization in SOCS7-/- hippocampi. (E) In utero electroporation of GFP in SOCS7+/- and SOCS7-/- embryos at E15.5 and collected at P10. Electroporated GFP+ PNs show normal positioning and apical dendrites.
    Figure Legend Snippet: Dab1 accumulation is not sufficient to disrupt pyramidal neuron positioning or apical dendrite morphology. (A) Dab1 accumulated in both Rbx2cKO-Emx1 and SOCS7-/- hippocampi in comparison to control (Rbx2 fl/fl). Coronal sections of P21 hippocampi were stained with anti-Dab1 and counterstained with DAPI. (B) Representative images of western blotting experiments indicating that Dab1 accumulated in both Rbx2cKO-Emx1 and SOCS7-/- hippocampal samples, but Fyn only accumulated in Rbx2cKO-Emx1 P21 hippocampal lysates. (C) Quantification of displaced DKK3+ cells. No difference was observed between genotypes. At least 3 sections quantified per brain. Mean ± SD. Statistics, multiple t-test with Bonferroni-Dunn method adjusted p value. (D) Coronal sections of P21 SOCS7+/- and SOCS7-/- hippocampi were stained with an anti-DKK3 antibody and counterstained with DAPI. (d’-d’’) High-magnification images of the CA1 indicating that normal PN localization in SOCS7-/- hippocampi. (E) In utero electroporation of GFP in SOCS7+/- and SOCS7-/- embryos at E15.5 and collected at P10. Electroporated GFP+ PNs show normal positioning and apical dendrites.

    Techniques Used: Staining, Western Blot, In Utero, Electroporation

    Arl4c participates in dendritic development of cultured hippocampal neurons. (A-B) Arl4c expression negative regulate dendritic complexity of hippocampal neurons. E16 hippocampal primary neurons were obtained and transfected in suspension with GFP (Control) or human ARL4C /GFP (hARL4C) expression plasmids, or a shRNA against Arl4c/GFP (shArl4c) plasmids. Representative images (A) and Sholl analysis (B) from independent experiments are shown. 20-50 neurons were quantified per experiment. ARL4C over-expression decreased and Arl4c knockdown increased dendritic complexity within 60 μm of soma. Yellow arrowheads indicate neurites. Mean ± SEM. Statistics, two-way ANOVA. (C) Length of the longest neurites were measured in each condition and no statistical differences were observe between conditions (n=3). Number of neurons quantified: 125- Control; 119-ARL4C; 168-shArl4c. Mean ± SEM. Statistics, one-way ANOVA.
    Figure Legend Snippet: Arl4c participates in dendritic development of cultured hippocampal neurons. (A-B) Arl4c expression negative regulate dendritic complexity of hippocampal neurons. E16 hippocampal primary neurons were obtained and transfected in suspension with GFP (Control) or human ARL4C /GFP (hARL4C) expression plasmids, or a shRNA against Arl4c/GFP (shArl4c) plasmids. Representative images (A) and Sholl analysis (B) from independent experiments are shown. 20-50 neurons were quantified per experiment. ARL4C over-expression decreased and Arl4c knockdown increased dendritic complexity within 60 μm of soma. Yellow arrowheads indicate neurites. Mean ± SEM. Statistics, two-way ANOVA. (C) Length of the longest neurites were measured in each condition and no statistical differences were observe between conditions (n=3). Number of neurons quantified: 125- Control; 119-ARL4C; 168-shArl4c. Mean ± SEM. Statistics, one-way ANOVA.

    Techniques Used: Cell Culture, Expressing, Transfection, shRNA, Over Expression

    17) Product Images from "iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins"

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.139261

    Modification of the crucial steps of the DamID protocol. (A) Medaka zygotes were injected with mRNA coding for Dam-f-GFP or Dam-f-TF (Medaka Rx2). Embryos were maintained in ERM supplemented with an antibiotic solution and gDNA was isolated at stage 22. (B) Medaka embryos (stage 22) expressing Dam-f-GFP. (C) DamID LM-PCR at 25 cycles using the modifications presented in the main text generates only Dpn I-dependent amplification (see Materials and Methods, iDamIDseq protocol). (D) Flowchart comparing the standard DamID-seq protocol (based on Wu et al., 2016 ) with the iDamIDseq protocol (improvements are underlined).
    Figure Legend Snippet: Modification of the crucial steps of the DamID protocol. (A) Medaka zygotes were injected with mRNA coding for Dam-f-GFP or Dam-f-TF (Medaka Rx2). Embryos were maintained in ERM supplemented with an antibiotic solution and gDNA was isolated at stage 22. (B) Medaka embryos (stage 22) expressing Dam-f-GFP. (C) DamID LM-PCR at 25 cycles using the modifications presented in the main text generates only Dpn I-dependent amplification (see Materials and Methods, iDamIDseq protocol). (D) Flowchart comparing the standard DamID-seq protocol (based on Wu et al., 2016 ) with the iDamIDseq protocol (improvements are underlined).

    Techniques Used: Modification, Injection, Isolation, Expressing, Polymerase Chain Reaction, Amplification

    Analysis of iDamIDseq results on Rx2. (A) Samples showed high correlations between replicates and low correlations between Rx2 and GFP, based on the genome-wide read coverage. (B) The most overrepresented motif found de novo has a consensus sequence BYAATTA, very similar to the binding motif known for the mammalian Rax protein. (C) Abundance of the RAX motif in the identified sites, with respect to random sequence, correlates with the score of these regions. The Rx2-enriched sites identified by iDEAR show a higher enrichment than those represented by MACS2 and the Marshall and Brand pipeline.
    Figure Legend Snippet: Analysis of iDamIDseq results on Rx2. (A) Samples showed high correlations between replicates and low correlations between Rx2 and GFP, based on the genome-wide read coverage. (B) The most overrepresented motif found de novo has a consensus sequence BYAATTA, very similar to the binding motif known for the mammalian Rax protein. (C) Abundance of the RAX motif in the identified sites, with respect to random sequence, correlates with the score of these regions. The Rx2-enriched sites identified by iDEAR show a higher enrichment than those represented by MACS2 and the Marshall and Brand pipeline.

    Techniques Used: Genome Wide, Sequencing, Binding Assay

    Optimization of the bacterial Dam gene is necessary for proper expression of Dam fusion proteins to avoid aberrant splicing. (A) Plasmids containing GFP, Dam-f-GFP and cMyc-Dam-f-GFP cassettes driven by the 3.5 kb ubiquitin promoter (Ubi) were co-injected with Tol2 transposase into medaka zygotes. Successfully injected larvae expressing EGFP in the heart were selected for further studies. Only Ubi::GFP is expressed ubiquitously in the body of the larvae. (B) RNA was isolated from pools of larvae from the experimental groups. RT-PCR was performed using a forward primer (orange arrowhead in A) annealing in the non-coding exon included in the ubiquitin promoter (NoE) and the reverse primer (green arrowhead in A) in the body of the GFP-coding sequence. Proper splicing occurs between NoE and GFP in the Ubi::GFP larvae. In Ubi::Dam-f-GFP larvae, incorrect splicing occurs between the NoE and a cryptic acceptor site in the GFP-coding region (red arrowheads). In the Ubi::cMyc-Dam-f-GFP, NoE is spliced to the proper acceptor upstream of the cMyc sequence, but after that the cMyc sequence is aberrantly spliced, using a cryptic donor site, to the same cryptic acceptor sequence in GFP as for Ubi::Dam-f-GFP (see also Fig. S2 ). The prokaryotic Dam ORF carries a strong splicing enhancer recognized in the eukaryotic context. (C) Optimization of the Dam ORF removed this potential, facilitating proper expression of the fusion proteins.
    Figure Legend Snippet: Optimization of the bacterial Dam gene is necessary for proper expression of Dam fusion proteins to avoid aberrant splicing. (A) Plasmids containing GFP, Dam-f-GFP and cMyc-Dam-f-GFP cassettes driven by the 3.5 kb ubiquitin promoter (Ubi) were co-injected with Tol2 transposase into medaka zygotes. Successfully injected larvae expressing EGFP in the heart were selected for further studies. Only Ubi::GFP is expressed ubiquitously in the body of the larvae. (B) RNA was isolated from pools of larvae from the experimental groups. RT-PCR was performed using a forward primer (orange arrowhead in A) annealing in the non-coding exon included in the ubiquitin promoter (NoE) and the reverse primer (green arrowhead in A) in the body of the GFP-coding sequence. Proper splicing occurs between NoE and GFP in the Ubi::GFP larvae. In Ubi::Dam-f-GFP larvae, incorrect splicing occurs between the NoE and a cryptic acceptor site in the GFP-coding region (red arrowheads). In the Ubi::cMyc-Dam-f-GFP, NoE is spliced to the proper acceptor upstream of the cMyc sequence, but after that the cMyc sequence is aberrantly spliced, using a cryptic donor site, to the same cryptic acceptor sequence in GFP as for Ubi::Dam-f-GFP (see also Fig. S2 ). The prokaryotic Dam ORF carries a strong splicing enhancer recognized in the eukaryotic context. (C) Optimization of the Dam ORF removed this potential, facilitating proper expression of the fusion proteins.

    Techniques Used: Expressing, Injection, Isolation, Reverse Transcription Polymerase Chain Reaction, Sequencing

    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).
    Figure Legend Snippet: Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).

    Techniques Used: Injection, Agarose Gel Electrophoresis, Isolation, Activity Assay, Transformation Assay, Functional Assay, Construct

    18) Product Images from "Leucine-Rich Repeat Kinase 2 limits dopamine D1 receptor signaling in striatum and biases against heavy persistent alcohol drinking"

    Article Title: Leucine-Rich Repeat Kinase 2 limits dopamine D1 receptor signaling in striatum and biases against heavy persistent alcohol drinking

    Journal: bioRxiv

    doi: 10.1101/2022.05.26.493614

    Assessment of D1R, Glutamatergic, and GABAergic function in D1-Lrrk2-KO mice. A , Quantification of c-Fos positive cells in the striatum of Lrrk2 floxP/floxP mice injected with either Cre(eGFP) or GFP after systemic administration of saline or SKF81297 (2 mg/kg). B , Average AMPAR/NMDAR ratio. C , Paired pulse ratio of the synaptic responses before and after (shaded area) bath application of D1-like agonist. For all panels, bars represent mean ± S.E.M and symbols represent values from individual mice. * denotes P
    Figure Legend Snippet: Assessment of D1R, Glutamatergic, and GABAergic function in D1-Lrrk2-KO mice. A , Quantification of c-Fos positive cells in the striatum of Lrrk2 floxP/floxP mice injected with either Cre(eGFP) or GFP after systemic administration of saline or SKF81297 (2 mg/kg). B , Average AMPAR/NMDAR ratio. C , Paired pulse ratio of the synaptic responses before and after (shaded area) bath application of D1-like agonist. For all panels, bars represent mean ± S.E.M and symbols represent values from individual mice. * denotes P

    Techniques Used: Mouse Assay, Injection

    19) Product Images from "KIF1Bβ mutations detected in hereditary neuropathy impair IGF1R transport and axon growth"

    Article Title: KIF1Bβ mutations detected in hereditary neuropathy impair IGF1R transport and axon growth

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201801085

    IGF1R is a novel cargo of KIF1Bβ. (A) Yeast two-hybrid assays assessing the direct binding between the ICD (961–1,373 aa) of mouse IGF1Rβ and various deletion mutants of mouse KIF1Bβ. Note that the 885–1,410-aa region within the KIF1Bβ stalk domain is the minimal essential region for its binding. PH, Pleckstrin homology. (B) Endogenous vesicle IP. IB of the brain lysates (Lys) of Kif1bβ GFP/GFP and Kif1bβ +/+ (WT) mice and their immunoprecipitates using an anti-GFP antibody (IP by anti-GFP) labeled with the indicated antibodies. (C) Nycodenz gradient vesicle flotation assay of adult mouse brain lysates labeled with the indicated antibodies. Note that KIF1Bβ, IGF1Rα, IGF1Rβ, and synaptophysin are detected in the same fractions 14–15. (D) A Coomassie Brilliant Blue–stained gel for a pulldown assay between purified GST–KIF1Bβ885–1,410 and IGF1RβICD–3×FLAG showing their direct interaction. (E and F) Double-color time-lapse fluorescence images of a dissociated hippocampal neuron at DIV7 transfected with KIF1Bβ-EGFP and SP-IGF1Rβ-TagRFP using spinning-disk confocal microscopy equipped with 100×/1.46 Plan Apochromat oil-immersion objective lens (E) and its kymograph (F). The cell body is on the left side. Bars, 10 µm. Note that the speed of the comigrating vesicle (arrows in E) is ∼1 µm/s. See Video 2.
    Figure Legend Snippet: IGF1R is a novel cargo of KIF1Bβ. (A) Yeast two-hybrid assays assessing the direct binding between the ICD (961–1,373 aa) of mouse IGF1Rβ and various deletion mutants of mouse KIF1Bβ. Note that the 885–1,410-aa region within the KIF1Bβ stalk domain is the minimal essential region for its binding. PH, Pleckstrin homology. (B) Endogenous vesicle IP. IB of the brain lysates (Lys) of Kif1bβ GFP/GFP and Kif1bβ +/+ (WT) mice and their immunoprecipitates using an anti-GFP antibody (IP by anti-GFP) labeled with the indicated antibodies. (C) Nycodenz gradient vesicle flotation assay of adult mouse brain lysates labeled with the indicated antibodies. Note that KIF1Bβ, IGF1Rα, IGF1Rβ, and synaptophysin are detected in the same fractions 14–15. (D) A Coomassie Brilliant Blue–stained gel for a pulldown assay between purified GST–KIF1Bβ885–1,410 and IGF1RβICD–3×FLAG showing their direct interaction. (E and F) Double-color time-lapse fluorescence images of a dissociated hippocampal neuron at DIV7 transfected with KIF1Bβ-EGFP and SP-IGF1Rβ-TagRFP using spinning-disk confocal microscopy equipped with 100×/1.46 Plan Apochromat oil-immersion objective lens (E) and its kymograph (F). The cell body is on the left side. Bars, 10 µm. Note that the speed of the comigrating vesicle (arrows in E) is ∼1 µm/s. See Video 2.

    Techniques Used: Binding Assay, Mouse Assay, Labeling, Staining, Purification, Fluorescence, Transfection, Confocal Microscopy

    20) Product Images from "FUS inclusions disrupt RNA localization by sequestering kinesin-1 and inhibiting microtubule detyrosination"

    Article Title: FUS inclusions disrupt RNA localization by sequestering kinesin-1 and inhibiting microtubule detyrosination

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201608022

    Overexpression of KIF5B rescues Glu-MTs and RNA localization in the presence of FUS granules. (A) NIH/3T3 cells transfected with RFP and GFP (first lane), RFP-FUS(P525L) and GFP (second lane), or RFP-FUS(P525L) and YFP-KIF5B (third lane) were analyzed by WB to detect the indicated proteins. The asterisk indicates a nonspecific band. (B) Representative images of cells transfected as in A and immunostained to detect Glu-tubulin. For RFP-FUS(P525L)–expressing cells, only cells exhibiting cytoplasmic FUS granules were imaged. (C) Percentages of cells with Glu-MTs from B were calculated. (D) Representative images of cells transfected as in A and analyzed by FISH to detect the Ddr2 RNA. Cells exhibiting cytoplasmic RFP-FUS(P525L) granules were imaged. White lines indicate cell outlines. (E) Edge ratio values of cells from D. (C and E) Total numbers of cells observed in three independent trials is indicated within each bar. *, P = 0.003; **, P
    Figure Legend Snippet: Overexpression of KIF5B rescues Glu-MTs and RNA localization in the presence of FUS granules. (A) NIH/3T3 cells transfected with RFP and GFP (first lane), RFP-FUS(P525L) and GFP (second lane), or RFP-FUS(P525L) and YFP-KIF5B (third lane) were analyzed by WB to detect the indicated proteins. The asterisk indicates a nonspecific band. (B) Representative images of cells transfected as in A and immunostained to detect Glu-tubulin. For RFP-FUS(P525L)–expressing cells, only cells exhibiting cytoplasmic FUS granules were imaged. (C) Percentages of cells with Glu-MTs from B were calculated. (D) Representative images of cells transfected as in A and analyzed by FISH to detect the Ddr2 RNA. Cells exhibiting cytoplasmic RFP-FUS(P525L) granules were imaged. White lines indicate cell outlines. (E) Edge ratio values of cells from D. (C and E) Total numbers of cells observed in three independent trials is indicated within each bar. *, P = 0.003; **, P

    Techniques Used: Over Expression, Transfection, Western Blot, Expressing, Fluorescence In Situ Hybridization

    FUS inclusions do not affect TTL activity or MT stabilization but rather impact the function of TCP. (A) Edge ratio of Ddr2 RNA from cells expressing GFP-FUS(R521C) and additionally transfected with control siRNAs or siRNAs against TTL. Only cells exhibiting cytoplasmic GFP-FUS(R521C) granules were analyzed. (B) Glu-tubulin levels were detected by WB and normalized to α-tubulin levels. The graph shows the increase in Glu-tubulin levels upon TTL knockdown (siTTL/siControl) from cells additionally expressing the indicated constructs or siRNAs. Results were expressed relative to the control sample of each trial. (C) Cells expressing GFP or GFP-FUS(P525L) were stained for α-tubulin after being exposed to nocodazole for the indicated minutes. Graph shows total tubulin intensity measured in > 35 cells for each time point. Similar results were observed in two independent trials. Yellow lines indicate cell outlines. (D) Immunostaining of Glu-tubulin and α-tubulin in GFP- or GFP-FUS(P525L)–expressing cells treated or not treated with taxol. Glu-tubulin intensity was normalized to α-tubulin and expressed relative to the GFP sample. (C and D) Arrows indicate cytoplasmic FUS inclusions. (A, B, and D) Numbers in bars indicate the amounts of cells observed in two (A and D) or two to five (B) independent experiments. (A–D) *, P
    Figure Legend Snippet: FUS inclusions do not affect TTL activity or MT stabilization but rather impact the function of TCP. (A) Edge ratio of Ddr2 RNA from cells expressing GFP-FUS(R521C) and additionally transfected with control siRNAs or siRNAs against TTL. Only cells exhibiting cytoplasmic GFP-FUS(R521C) granules were analyzed. (B) Glu-tubulin levels were detected by WB and normalized to α-tubulin levels. The graph shows the increase in Glu-tubulin levels upon TTL knockdown (siTTL/siControl) from cells additionally expressing the indicated constructs or siRNAs. Results were expressed relative to the control sample of each trial. (C) Cells expressing GFP or GFP-FUS(P525L) were stained for α-tubulin after being exposed to nocodazole for the indicated minutes. Graph shows total tubulin intensity measured in > 35 cells for each time point. Similar results were observed in two independent trials. Yellow lines indicate cell outlines. (D) Immunostaining of Glu-tubulin and α-tubulin in GFP- or GFP-FUS(P525L)–expressing cells treated or not treated with taxol. Glu-tubulin intensity was normalized to α-tubulin and expressed relative to the GFP sample. (C and D) Arrows indicate cytoplasmic FUS inclusions. (A, B, and D) Numbers in bars indicate the amounts of cells observed in two (A and D) or two to five (B) independent experiments. (A–D) *, P

    Techniques Used: Activity Assay, Expressing, Transfection, Western Blot, Construct, Staining, Immunostaining

    FUS mutants disrupt KIF5C distribution, Glu-MTs, and axonal RNA localization in primary neuronal cells. (A) Representative whole-cell images and enlarged soma views of DIV6 primary hippocampal neurons expressing GFP or GFP-FUS(R495X). Arrows indicate cytoplasmic FUS inclusions in soma; yellow lines indicate cell outlines. (B) Representative WBs and quantification of KIF5C levels in cells expressing GFP or GFP-FUS(R495X). The asterisk indicates a nonspecific band. (C) KIF5C immunostaining of DIV6 primary hippocampal neurons expressing GFP or GFP-FUS(R495X). KIF5C intensity in growth cones (yellow outlines) was normalized to growth cone area and expressed relative to the GFP sample. (D) DIV6 primary hippocampal neurons expressing GFP or GFP-FUS(R495X) or treated with parthenolide were immunostained for Glu-tubulin and Tyr-tubulin. Glu-tubulin intensity in growth cones was normalized to Tyr-tubulin intensity and expressed relative to GFP samples. (E) FISH of Dynll2 RNA and polyA RNA on DIV6 primary hippocampal neurons expressing GFP or GFP-FUS(R495X) or treated with parthenolide. Axonal intensity of Dynll2 RNA was normalized to the axonal area and expressed relative to GFP samples. (C–E) *, P
    Figure Legend Snippet: FUS mutants disrupt KIF5C distribution, Glu-MTs, and axonal RNA localization in primary neuronal cells. (A) Representative whole-cell images and enlarged soma views of DIV6 primary hippocampal neurons expressing GFP or GFP-FUS(R495X). Arrows indicate cytoplasmic FUS inclusions in soma; yellow lines indicate cell outlines. (B) Representative WBs and quantification of KIF5C levels in cells expressing GFP or GFP-FUS(R495X). The asterisk indicates a nonspecific band. (C) KIF5C immunostaining of DIV6 primary hippocampal neurons expressing GFP or GFP-FUS(R495X). KIF5C intensity in growth cones (yellow outlines) was normalized to growth cone area and expressed relative to the GFP sample. (D) DIV6 primary hippocampal neurons expressing GFP or GFP-FUS(R495X) or treated with parthenolide were immunostained for Glu-tubulin and Tyr-tubulin. Glu-tubulin intensity in growth cones was normalized to Tyr-tubulin intensity and expressed relative to GFP samples. (E) FISH of Dynll2 RNA and polyA RNA on DIV6 primary hippocampal neurons expressing GFP or GFP-FUS(R495X) or treated with parthenolide. Axonal intensity of Dynll2 RNA was normalized to the axonal area and expressed relative to GFP samples. (C–E) *, P

    Techniques Used: Expressing, Immunostaining, Fluorescence In Situ Hybridization

    Kinesin-1 RNA and protein are recruited in FUS granules. (A) WBs of KIF5B expression in NIH/3T3 cells expressing GFP or GFP-FUS mutants. The asterisk indicates a nonspecific band. (B) Immunostaining of KIF5B or KIF5C in NIH/3T3 cells exhibiting mutant FUS cytoplasmic granules and corresponding quantitations. The number of cells observed from more than two independent trials is indicated within each bar. (C) Kif5b or polyA RNA FISH in cells with FUS granules. Yellow lines indicate cell outlines. (D) Signal intensity of Kif5b or polyA RNA within FUS granules normalized to total RNA signal intensity in the cytoplasm. (E) GFP- or GFP-FUS(P525L)–expressing cells were sorted based on GFP to increase the proportion of transfected cells. The Kif5b RNA amount was detected using the NanoString nCounter platform and normalized against the Ldha , Rsp12 , and Rpl35 RNA amounts. (D and E) *, P
    Figure Legend Snippet: Kinesin-1 RNA and protein are recruited in FUS granules. (A) WBs of KIF5B expression in NIH/3T3 cells expressing GFP or GFP-FUS mutants. The asterisk indicates a nonspecific band. (B) Immunostaining of KIF5B or KIF5C in NIH/3T3 cells exhibiting mutant FUS cytoplasmic granules and corresponding quantitations. The number of cells observed from more than two independent trials is indicated within each bar. (C) Kif5b or polyA RNA FISH in cells with FUS granules. Yellow lines indicate cell outlines. (D) Signal intensity of Kif5b or polyA RNA within FUS granules normalized to total RNA signal intensity in the cytoplasm. (E) GFP- or GFP-FUS(P525L)–expressing cells were sorted based on GFP to increase the proportion of transfected cells. The Kif5b RNA amount was detected using the NanoString nCounter platform and normalized against the Ldha , Rsp12 , and Rpl35 RNA amounts. (D and E) *, P

    Techniques Used: Expressing, Immunostaining, Mutagenesis, Fluorescence In Situ Hybridization, Transfection

    TDP-43 inclusions and stress granules, but not Dcp1 bodies, disrupt Glu-MTs and RNA localization at protrusions. (A) Representative IF images of total α-tubulin and Glu-tubulin of cells expressing RFP or RFP–TDP-43(A315T) and quantification of percentages of cells with Glu-MTs. (B) Ddr2 or Arpc3 RNA edge ratios of cells expressing GFP or GFP–TDP-43(A315T) with or without cytoplasmic granules. Numbers within each bar indicate the total number of cells observed in more than four independent experiments. (C and D) Cells treated or not treated with sodium arsenite were immunostained to detect Glu-tubulin and TIA-1–containing stress granules (arrows; C) or analyzed by FISH (D). (E) Representative IF images of total α-tubulin and Glu-tubulin of cells expressing GFP or GFP-Dcp1α and percentages of cells with Glu-MTs. (A and C–E) Numbers within each bar indicate the total number of cells observed in three independent experiments. (A–E) *, P = 0.0005; Student’s t test (A); and *, P
    Figure Legend Snippet: TDP-43 inclusions and stress granules, but not Dcp1 bodies, disrupt Glu-MTs and RNA localization at protrusions. (A) Representative IF images of total α-tubulin and Glu-tubulin of cells expressing RFP or RFP–TDP-43(A315T) and quantification of percentages of cells with Glu-MTs. (B) Ddr2 or Arpc3 RNA edge ratios of cells expressing GFP or GFP–TDP-43(A315T) with or without cytoplasmic granules. Numbers within each bar indicate the total number of cells observed in more than four independent experiments. (C and D) Cells treated or not treated with sodium arsenite were immunostained to detect Glu-tubulin and TIA-1–containing stress granules (arrows; C) or analyzed by FISH (D). (E) Representative IF images of total α-tubulin and Glu-tubulin of cells expressing GFP or GFP-Dcp1α and percentages of cells with Glu-MTs. (A and C–E) Numbers within each bar indicate the total number of cells observed in three independent experiments. (A–E) *, P = 0.0005; Student’s t test (A); and *, P

    Techniques Used: Expressing, Fluorescence In Situ Hybridization

    ALS-associated FUS mutants mislocalize RNAs from cell protrusions in an inclusion-dependent manner. (A) FISH of NIH/3T3 fibroblast cells to detect the indicated RNAs. The cells adopt a triangular shape on Y-shaped micropatterned substrates. The cell outline was used to derive the cell mask as well as a peripheral mask extending 2 µm from the outer cell perimeter. Edge ratios from a cell population were calculated as the fraction of RNA intensity in the peripheral mask normalized to the fraction of area in the peripheral mask. The APC-dependent localized RNA Ddr2 exhibits a higher edge ratio than the nonlocalized control RNA Arpc3 . (B) Representative images of Ddr2 RNA FISH of cells expressing GFP, GFP-FUS(R521C), or GFP-FUS(R495X), without or with cytoplasmic granules (arrowheads), and edge ratio quantitations. Numbers within each bar indicate the total number of cells observed in independent experiments (five for GFP and GFP-FUS(R521C), one for GFP-FUS(R495X), and two for GFP-FUS(P525L)). (C) Schematic of experimental strategy for Hsp104-induced disaggregation. (D) WBs detecting the indicated proteins from induced or uninduced cells expressing Hsp104 variants. GFP-FUS levels, normalized to GAPDH, were expressed relative to doxycycline (Dox)-negative samples. There were no statistical differences between Dox – and Dox + groups by paired t test. n = 3–6. The asterisk indicates a nonspecific band. (E) Percentages of GFP-FUS(P525L)–disaggregated cells 24 h after induction of the indicated Hsp104 variants. Numbers within each bar indicate the total number of cells observed in four to five independent experiments apart from the Hsp104-uninduced wild-type (wt) sample, which was performed once. (F) Representative pre- and postinduction images of GFP-FUS(P525L) cells showing aggregation or disaggregation as well as Ddr2 RNA FISH from same cells. Graphs are edge ratios of Ddr2 or Arpc3 RNAs. The total numbers of cells observed in two independent experiments are indicated within each bar. Similar results were obtained in two additional experiments in which Hsp104 expression was induced for shorter periods (12 or 18 h). (B, E, and F) *, P
    Figure Legend Snippet: ALS-associated FUS mutants mislocalize RNAs from cell protrusions in an inclusion-dependent manner. (A) FISH of NIH/3T3 fibroblast cells to detect the indicated RNAs. The cells adopt a triangular shape on Y-shaped micropatterned substrates. The cell outline was used to derive the cell mask as well as a peripheral mask extending 2 µm from the outer cell perimeter. Edge ratios from a cell population were calculated as the fraction of RNA intensity in the peripheral mask normalized to the fraction of area in the peripheral mask. The APC-dependent localized RNA Ddr2 exhibits a higher edge ratio than the nonlocalized control RNA Arpc3 . (B) Representative images of Ddr2 RNA FISH of cells expressing GFP, GFP-FUS(R521C), or GFP-FUS(R495X), without or with cytoplasmic granules (arrowheads), and edge ratio quantitations. Numbers within each bar indicate the total number of cells observed in independent experiments (five for GFP and GFP-FUS(R521C), one for GFP-FUS(R495X), and two for GFP-FUS(P525L)). (C) Schematic of experimental strategy for Hsp104-induced disaggregation. (D) WBs detecting the indicated proteins from induced or uninduced cells expressing Hsp104 variants. GFP-FUS levels, normalized to GAPDH, were expressed relative to doxycycline (Dox)-negative samples. There were no statistical differences between Dox – and Dox + groups by paired t test. n = 3–6. The asterisk indicates a nonspecific band. (E) Percentages of GFP-FUS(P525L)–disaggregated cells 24 h after induction of the indicated Hsp104 variants. Numbers within each bar indicate the total number of cells observed in four to five independent experiments apart from the Hsp104-uninduced wild-type (wt) sample, which was performed once. (F) Representative pre- and postinduction images of GFP-FUS(P525L) cells showing aggregation or disaggregation as well as Ddr2 RNA FISH from same cells. Graphs are edge ratios of Ddr2 or Arpc3 RNAs. The total numbers of cells observed in two independent experiments are indicated within each bar. Similar results were obtained in two additional experiments in which Hsp104 expression was induced for shorter periods (12 or 18 h). (B, E, and F) *, P

    Techniques Used: Fluorescence In Situ Hybridization, Expressing

    Kinesin-1 promotes Glu-MT formation through directing TCP activity. (A) Immunostaining of Glu-tubulin and α-tubulin in GFP-, GFP-KIF5C(T93N)–, or GFP-KIF5B(ΔMD)–expressing cells. (B) Glu-tubulin intensity of cells from A was normalized to α-tubulin intensity and expressed relative to the GFP sample. Numbers of cells observed in more than three independent trials are indicated. *, P
    Figure Legend Snippet: Kinesin-1 promotes Glu-MT formation through directing TCP activity. (A) Immunostaining of Glu-tubulin and α-tubulin in GFP-, GFP-KIF5C(T93N)–, or GFP-KIF5B(ΔMD)–expressing cells. (B) Glu-tubulin intensity of cells from A was normalized to α-tubulin intensity and expressed relative to the GFP sample. Numbers of cells observed in more than three independent trials are indicated. *, P

    Techniques Used: Activity Assay, Immunostaining, Expressing

    Kinesin-1 is required for the formation of Glu-MTs and RNA localization at cell protrusions. (A) Representative images of Glu-tubulin and α-tubulin staining of cells transfected with the indicated siRNAs. (B) Percentage of cells with Glu-MTs upon transfection with the indicated siRNAs or with a dominant-negative kinesin-1 construct. (C) Representative images of Ddr2 RNA FISH of cells transfected with the indicated siRNAs or expressing GFP or GFP-KIF5B(ΔMD). (D) Ddr2 RNA edge ratio of cells expressing the indicated siRNAs or GFP-tagged constructs. (B and D) Numbers of cells observed in two or more independent trials is indicated within each bar. *, P
    Figure Legend Snippet: Kinesin-1 is required for the formation of Glu-MTs and RNA localization at cell protrusions. (A) Representative images of Glu-tubulin and α-tubulin staining of cells transfected with the indicated siRNAs. (B) Percentage of cells with Glu-MTs upon transfection with the indicated siRNAs or with a dominant-negative kinesin-1 construct. (C) Representative images of Ddr2 RNA FISH of cells transfected with the indicated siRNAs or expressing GFP or GFP-KIF5B(ΔMD). (D) Ddr2 RNA edge ratio of cells expressing the indicated siRNAs or GFP-tagged constructs. (B and D) Numbers of cells observed in two or more independent trials is indicated within each bar. *, P

    Techniques Used: Staining, Transfection, Dominant Negative Mutation, Construct, Fluorescence In Situ Hybridization, Expressing

    21) Product Images from "Generation of anisotropic strain dysregulates wild-type cell division at the interface between host and oncogenic tissue"

    Article Title: Generation of anisotropic strain dysregulates wild-type cell division at the interface between host and oncogenic tissue

    Journal: Current Biology

    doi: 10.1016/j.cub.2021.05.023

    The wild-type epithelium responds to oncogene-expressing clusters with altered cell division (A and B) Dot plots showing the percentage of wild-type cells that divided per minute of time lapse at different distances from GFP, GFP-kRas V12 , or GFP-cMYC clusters. (A) Kruskal-Wallis test: ∗ p
    Figure Legend Snippet: The wild-type epithelium responds to oncogene-expressing clusters with altered cell division (A and B) Dot plots showing the percentage of wild-type cells that divided per minute of time lapse at different distances from GFP, GFP-kRas V12 , or GFP-cMYC clusters. (A) Kruskal-Wallis test: ∗ p

    Techniques Used: Expressing

    Actomyosin contraction in cluster is required to generate strain and alter cell division in wild-type tissue (A and B) Confocal images of fixed, stage 10 embryos with a GFP-kRas V12 cluster, stained for (A) phosphorylated myosin II (magenta), single-headed arrows highlight tricellular junctions with increased phospho-myosin II in GFP-kRas V12 cells compared to wild-type tissue (double-headed arrows), and (B) F-actin (phalloidin; magenta), single-headed arrows highlight increased F-actin at the cell cortex in the GFP-kRas V12 cluster compared to wild-type tissue (double-headed arrows). (C) Rose histograms showing the orientation of wild-type cells’ long axes up to 6 cells from GFP-control (red) or GFP-RhoA Q63L (orange) cell clusters, relative to the cluster, in 10° bins. Kolmogorov-Smirnov test: p
    Figure Legend Snippet: Actomyosin contraction in cluster is required to generate strain and alter cell division in wild-type tissue (A and B) Confocal images of fixed, stage 10 embryos with a GFP-kRas V12 cluster, stained for (A) phosphorylated myosin II (magenta), single-headed arrows highlight tricellular junctions with increased phospho-myosin II in GFP-kRas V12 cells compared to wild-type tissue (double-headed arrows), and (B) F-actin (phalloidin; magenta), single-headed arrows highlight increased F-actin at the cell cortex in the GFP-kRas V12 cluster compared to wild-type tissue (double-headed arrows). (C) Rose histograms showing the orientation of wild-type cells’ long axes up to 6 cells from GFP-control (red) or GFP-RhoA Q63L (orange) cell clusters, relative to the cluster, in 10° bins. Kolmogorov-Smirnov test: p

    Techniques Used: Staining

    Modeling early-stage carcinoma in Xenopus laevis (A) Schematic of the microinjection protocol. Xenopus embryos were injected with Cherry-histone-H2B and BFP-CAAX mRNA at the 2-cell stage. At the 32-cell stage, a single cell was injected with GFP, GFP-kRas V12 , or GFP-cMYC mRNA. Embryos were developed to early gastrula stage 10 and imaged. (B–D) Confocal microscopy images of Xenopus embryos developed to early gastrula stage 10, following injection of a single cell at the 32-cell stage with (B) GFP, (C) GFP-kRas V12 , or (D) GFP-cMYC mRNA. Scale bars represent 100 μm. (E) Western blot showing phosphorylated ERK, unphosphorylated ERK, and α-tubulin expression in uninjected control embryos and embryos injected with GFP, GFP-kRas V12 , or GFP-cMYC mRNA. (F) Bar chart showing the average percentage of cells that divided per minute of time lapse, in either GFP, GFP-kRas V12 , or GFP-cMYC overexpression clusters ( ∗ p
    Figure Legend Snippet: Modeling early-stage carcinoma in Xenopus laevis (A) Schematic of the microinjection protocol. Xenopus embryos were injected with Cherry-histone-H2B and BFP-CAAX mRNA at the 2-cell stage. At the 32-cell stage, a single cell was injected with GFP, GFP-kRas V12 , or GFP-cMYC mRNA. Embryos were developed to early gastrula stage 10 and imaged. (B–D) Confocal microscopy images of Xenopus embryos developed to early gastrula stage 10, following injection of a single cell at the 32-cell stage with (B) GFP, (C) GFP-kRas V12 , or (D) GFP-cMYC mRNA. Scale bars represent 100 μm. (E) Western blot showing phosphorylated ERK, unphosphorylated ERK, and α-tubulin expression in uninjected control embryos and embryos injected with GFP, GFP-kRas V12 , or GFP-cMYC mRNA. (F) Bar chart showing the average percentage of cells that divided per minute of time lapse, in either GFP, GFP-kRas V12 , or GFP-cMYC overexpression clusters ( ∗ p

    Techniques Used: Injection, Confocal Microscopy, Western Blot, Expressing, Over Expression

    kRas V12 cell cluster imposes a mechanical strain on the wild-type epithelium (A and B) Cropped regions of confocal time-lapse stills showing laser ablation at a cell edge (highlighted by cherry-UtrCH: F-actin) in a GFP-kRas V12 cluster (A) and a surrounding wild-type cell (B). Ablation occurs at t = 0, yellow lines show the original positions of cell vertices before laser ablation, and red lines show the real-time positions of cell vertices. (C) Recoil measurements for cells in GFP-control (red), GFP-kRas V12 (green), and GFP-cMYC (yellow) clusters and areas of wild-type tissue around GFP-kRas V12 clusters (wild type; light green); n = 10 cells for each sample; error bars are SEM. (D) Initial recoil velocity calculated from recoil measurements in (C); one-way ANOVA: ∗∗ p
    Figure Legend Snippet: kRas V12 cell cluster imposes a mechanical strain on the wild-type epithelium (A and B) Cropped regions of confocal time-lapse stills showing laser ablation at a cell edge (highlighted by cherry-UtrCH: F-actin) in a GFP-kRas V12 cluster (A) and a surrounding wild-type cell (B). Ablation occurs at t = 0, yellow lines show the original positions of cell vertices before laser ablation, and red lines show the real-time positions of cell vertices. (C) Recoil measurements for cells in GFP-control (red), GFP-kRas V12 (green), and GFP-cMYC (yellow) clusters and areas of wild-type tissue around GFP-kRas V12 clusters (wild type; light green); n = 10 cells for each sample; error bars are SEM. (D) Initial recoil velocity calculated from recoil measurements in (C); one-way ANOVA: ∗∗ p

    Techniques Used:

    22) Product Images from "Spatial regulation of Drosophila ovarian Follicle Stem Cell division rates and cell cycle transitions"

    Article Title: Spatial regulation of Drosophila ovarian Follicle Stem Cell division rates and cell cycle transitions

    Journal: bioRxiv

    doi: 10.1101/2022.06.22.497017

    FUCCI reporter responses to key cell cycle regulators and signals. Examples of responses of FUCCI reporters to conditional expression of indicated transgenes, showing Fas3 (blue) staining to infer FSC identity and location, (A-H) GFP and RFP or (A’-H”) only RFP. All samples were also stained with DAPI to be able to count all cells, including those in S-phase (red arrows). All G1 (green arrows) and G2/M (yellow arrows) FSCs in the range of z-sections shown are indicated, as is the anterior Fas3 border (purple arrows). (A) Excess Stg increased G1 and decreased G2 FSC numbers. (B) Excess Stg together with CycE greatly increased S and decreased G2 FSC numbers. (C) Dominant-negative PI3K decreased G1 and increased G2 FSC numbers. (D) Excess JAK-STAT activity decreased G2 and increased S FSC numbers, but (E) principally increased G1 and decreased G2 FSC numbers together with excess Dacapo, Cdk2 inhibitor. (F) Decreased JAK-STAT activity increased G2 and decreased S FSC numbers. ECs were mainly in G1 in all samples but (D) and (E) illustrate conversion of some to G2 by excess JAK-STAT activity. Scale bar is 10μm.
    Figure Legend Snippet: FUCCI reporter responses to key cell cycle regulators and signals. Examples of responses of FUCCI reporters to conditional expression of indicated transgenes, showing Fas3 (blue) staining to infer FSC identity and location, (A-H) GFP and RFP or (A’-H”) only RFP. All samples were also stained with DAPI to be able to count all cells, including those in S-phase (red arrows). All G1 (green arrows) and G2/M (yellow arrows) FSCs in the range of z-sections shown are indicated, as is the anterior Fas3 border (purple arrows). (A) Excess Stg increased G1 and decreased G2 FSC numbers. (B) Excess Stg together with CycE greatly increased S and decreased G2 FSC numbers. (C) Dominant-negative PI3K decreased G1 and increased G2 FSC numbers. (D) Excess JAK-STAT activity decreased G2 and increased S FSC numbers, but (E) principally increased G1 and decreased G2 FSC numbers together with excess Dacapo, Cdk2 inhibitor. (F) Decreased JAK-STAT activity increased G2 and decreased S FSC numbers. ECs were mainly in G1 in all samples but (D) and (E) illustrate conversion of some to G2 by excess JAK-STAT activity. Scale bar is 10μm.

    Techniques Used: Expressing, Staining, Dominant Negative Mutation, Activity Assay

    Live FUCCI reporter imaging shows cell cycle transitions and phase durations. (A, B) Time-stamped frames from live imaging of (A) control and (B) C587 > Hop germaria. (A) A yellow G2 cell (white arrow) changes to M-phase morphology at 20min, producing two G1 daughters (white arrows) with low GFP (no RFP) signal at 30min, strengthening by 40min. One daughter (red arrow) has lost GFP, indicating S-phase (red arrow) at 50min. The second daughter lost GFP, entering S-phase at 60min. The highlighted cell and its daughters are scored as layer 1 FSCs because they are at the posterior margin of strong C587-GAL4 expression driving the FUCCI reporter transgenes. (B) A yellow G2 cell (white arrow) changes to M-phase morphology at 45min, and then produced two G1 (green) daughters (white arrows) by 1h. The highlighted cells are two diameters away from the posterior edge of C587-GAL4 expression, indicating that they are layer 3 FSCs. Cell cycle transitions for layer 3 FSCs were observed only for cells with increased JAK-STAT activity, consistent with slow cycling of anterior FSCs resulting partly from insufficient JAK-STAT activity. Scale bar is 10μm. (C) Summary of calculated duration of G1 (green), S (red), G2 (yellow) and M (white) phases of the cell cycle from the sum of all live imaging assays for control (left), C587 > Hop (middle) and C587 > CycE (right) germaria. For each genotype, the ratio of the whole cell cycle length for layer 2 relative to layer 1 FSCs is shown. The lengths of cell cycles as a percentage of controls are shown in red for excess JAK and CycE.
    Figure Legend Snippet: Live FUCCI reporter imaging shows cell cycle transitions and phase durations. (A, B) Time-stamped frames from live imaging of (A) control and (B) C587 > Hop germaria. (A) A yellow G2 cell (white arrow) changes to M-phase morphology at 20min, producing two G1 daughters (white arrows) with low GFP (no RFP) signal at 30min, strengthening by 40min. One daughter (red arrow) has lost GFP, indicating S-phase (red arrow) at 50min. The second daughter lost GFP, entering S-phase at 60min. The highlighted cell and its daughters are scored as layer 1 FSCs because they are at the posterior margin of strong C587-GAL4 expression driving the FUCCI reporter transgenes. (B) A yellow G2 cell (white arrow) changes to M-phase morphology at 45min, and then produced two G1 (green) daughters (white arrows) by 1h. The highlighted cells are two diameters away from the posterior edge of C587-GAL4 expression, indicating that they are layer 3 FSCs. Cell cycle transitions for layer 3 FSCs were observed only for cells with increased JAK-STAT activity, consistent with slow cycling of anterior FSCs resulting partly from insufficient JAK-STAT activity. Scale bar is 10μm. (C) Summary of calculated duration of G1 (green), S (red), G2 (yellow) and M (white) phases of the cell cycle from the sum of all live imaging assays for control (left), C587 > Hop (middle) and C587 > CycE (right) germaria. For each genotype, the ratio of the whole cell cycle length for layer 2 relative to layer 1 FSCs is shown. The lengths of cell cycles as a percentage of controls are shown in red for excess JAK and CycE.

    Techniques Used: Imaging, Expressing, Produced, Activity Assay

    FUCCI reporter and EdU labeling to score cell cycle phase of all FSCs and ECs. (A) Cartoon representation of a germarium based on ( 18 ). Cap cells (CCs) at the anterior (left) contact germline stem cells (not highlighted), which produce cystoblast daughters that mature into 16-cell germline cysts (white) as they progress posteriorly. Quiescent Escort cells (ECs) extend processes around germline cysts and support their differentiation. Follicle Stem Cells (FSCs) occupy three AP rings ( 3 , 2 , 1 ) around the germarial circumference and immediately anterior to strong Fas3 expression (red) on the surface of all early Follicle Cells (FCs). FCs proliferate to form a monolayer epithelium, including specialized terminal Polar cells (PCs), which secrete the Upd ligand responsible for generating a JAK-STAT pathway gradient (green). Wnt pathway (red) and Hh pathway (blue) gradients have opposite polarity and are generated by ligands produced in CCs and ECs. (B) Percentage of cells in the designated locations in G1 (green), S (red) and G2/M (yellow), deduced from FUCCI reporters in wild-type germaria. SEM is shown from scoring 28 germaria. (C, E-H) A combination of consecutive z-sections capturing just over half of the FSCs and ECs in a single C587 > FUCCI germarium after EdU labeling, to illustrate comprehensive scoring. Cells expressing GFP-only (green arrows), RFP-only (red arrows) or both (yellow arrows) in (C) are clarified by images showing only (E, F) GFP or only (G, H) RFP channels. Likewise, FSCs with EdU incorporation (white) are clarified by images with (C, E, F) and without (F, H) the EdU channel. All three FSCs in S-phase (red arrows) express neither GFP nor RFP. Many FCs, posterior (right) to FSCs, and a germline cyst (large clustered nuclei; orange arrow) have EdU label. The anterior border of strong Fas3 staining is indicated by purple arrows. Sample are scored by examining each z-section, as shown in (D), in order to identify the Fas3 border and consequently assign FSCs to different layers (indicated as 1, 2 or 3 for all images). Scale bar is 10μm.
    Figure Legend Snippet: FUCCI reporter and EdU labeling to score cell cycle phase of all FSCs and ECs. (A) Cartoon representation of a germarium based on ( 18 ). Cap cells (CCs) at the anterior (left) contact germline stem cells (not highlighted), which produce cystoblast daughters that mature into 16-cell germline cysts (white) as they progress posteriorly. Quiescent Escort cells (ECs) extend processes around germline cysts and support their differentiation. Follicle Stem Cells (FSCs) occupy three AP rings ( 3 , 2 , 1 ) around the germarial circumference and immediately anterior to strong Fas3 expression (red) on the surface of all early Follicle Cells (FCs). FCs proliferate to form a monolayer epithelium, including specialized terminal Polar cells (PCs), which secrete the Upd ligand responsible for generating a JAK-STAT pathway gradient (green). Wnt pathway (red) and Hh pathway (blue) gradients have opposite polarity and are generated by ligands produced in CCs and ECs. (B) Percentage of cells in the designated locations in G1 (green), S (red) and G2/M (yellow), deduced from FUCCI reporters in wild-type germaria. SEM is shown from scoring 28 germaria. (C, E-H) A combination of consecutive z-sections capturing just over half of the FSCs and ECs in a single C587 > FUCCI germarium after EdU labeling, to illustrate comprehensive scoring. Cells expressing GFP-only (green arrows), RFP-only (red arrows) or both (yellow arrows) in (C) are clarified by images showing only (E, F) GFP or only (G, H) RFP channels. Likewise, FSCs with EdU incorporation (white) are clarified by images with (C, E, F) and without (F, H) the EdU channel. All three FSCs in S-phase (red arrows) express neither GFP nor RFP. Many FCs, posterior (right) to FSCs, and a germline cyst (large clustered nuclei; orange arrow) have EdU label. The anterior border of strong Fas3 staining is indicated by purple arrows. Sample are scored by examining each z-section, as shown in (D), in order to identify the Fas3 border and consequently assign FSCs to different layers (indicated as 1, 2 or 3 for all images). Scale bar is 10μm.

    Techniques Used: Labeling, Expressing, Generated, Produced, Staining

    Restoration of normal division rates to stat mutant FSCs requires both excess G1/S and G2/M regulators. (A) Illustration of separately measured parameters of FSC behavior from multiple control MARCM experiments reported here and previously ( 18 ); EdU indices in each FSC layer, average number and percentage of all FSCs in each layer, ECs produced per anterior FSC from 0-6d, and inferred probability (p) of conversion of a single layer 1 FSC to an FC in each 12h budding cycle. (B, C) Control MARCM samples, illustrating GFP-marked (green) layer 1 FSCs (red arrows), immediate FCs (filled yellow arrowhead in B, none in C; empty arrowhead) just posterior to the anterior border (white arrows) of strong Fas3 (red) staining, r2a (pink arrowheads) and r1 ECs (orange arrowheads, only in C). (D) Percentage of marked cells of the indicated genotypes that incorporated EdU for cells in the location of r1 ECs (purple), r2a ECs (pink), layer 3 (blue), layer 2 (green) and layer 1 (red) FSCs. SEMs and significant differences from control values are indicated (*, p
    Figure Legend Snippet: Restoration of normal division rates to stat mutant FSCs requires both excess G1/S and G2/M regulators. (A) Illustration of separately measured parameters of FSC behavior from multiple control MARCM experiments reported here and previously ( 18 ); EdU indices in each FSC layer, average number and percentage of all FSCs in each layer, ECs produced per anterior FSC from 0-6d, and inferred probability (p) of conversion of a single layer 1 FSC to an FC in each 12h budding cycle. (B, C) Control MARCM samples, illustrating GFP-marked (green) layer 1 FSCs (red arrows), immediate FCs (filled yellow arrowhead in B, none in C; empty arrowhead) just posterior to the anterior border (white arrows) of strong Fas3 (red) staining, r2a (pink arrowheads) and r1 ECs (orange arrowheads, only in C). (D) Percentage of marked cells of the indicated genotypes that incorporated EdU for cells in the location of r1 ECs (purple), r2a ECs (pink), layer 3 (blue), layer 2 (green) and layer 1 (red) FSCs. SEMs and significant differences from control values are indicated (*, p

    Techniques Used: Mutagenesis, Produced, Staining

    23) Product Images from "Programmed Cell Death Recruits Macrophages Into the Developing Mouse Cochlea"

    Article Title: Programmed Cell Death Recruits Macrophages Into the Developing Mouse Cochlea

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.777836

    Progression of developmental apoptosis and macrophage recruitment is not affected by genetic deletion of CX3CR1. (A–D′) Sensory epithelium of the developing cochlea of CX3CR1-null mice. The patterns of cell death (white arrows, cleaved caspase-3) and macrophage recruitment (magenta arrows, GFP) are similar to those observed in CX3CR1 GFP/+ mice (e.g., Figure 1 ). (E) Total number of pyknotic nuclei in the GER region of CX3CR1-null mice vs CX3CR1 GFP/+ mice. (F) Number of macrophages in the GER region of the developing cochlea of CX3CR1-null mice vs. CX3CR1 GFP/+ mice. Images in (A–D) , (F–G) were taken using a ×20 objective lens. Labels (A–D′) Red-cleaved caspase 3, Green-GFP + macrophages, Blue-DAPI. White arrow-cleaved caspase-3-labeled cells, and magenta arrows-GFP + macrophages. Abbreviations: OC: organ of Corti, GER: Greater Epithelial Ridge, ns: not significant. (N = 3–6).
    Figure Legend Snippet: Progression of developmental apoptosis and macrophage recruitment is not affected by genetic deletion of CX3CR1. (A–D′) Sensory epithelium of the developing cochlea of CX3CR1-null mice. The patterns of cell death (white arrows, cleaved caspase-3) and macrophage recruitment (magenta arrows, GFP) are similar to those observed in CX3CR1 GFP/+ mice (e.g., Figure 1 ). (E) Total number of pyknotic nuclei in the GER region of CX3CR1-null mice vs CX3CR1 GFP/+ mice. (F) Number of macrophages in the GER region of the developing cochlea of CX3CR1-null mice vs. CX3CR1 GFP/+ mice. Images in (A–D) , (F–G) were taken using a ×20 objective lens. Labels (A–D′) Red-cleaved caspase 3, Green-GFP + macrophages, Blue-DAPI. White arrow-cleaved caspase-3-labeled cells, and magenta arrows-GFP + macrophages. Abbreviations: OC: organ of Corti, GER: Greater Epithelial Ridge, ns: not significant. (N = 3–6).

    Techniques Used: Mouse Assay, Labeling

    Progression of apoptosis and macrophage infiltration in the basal region of the developing cochlea. (A–D) Mid-modiolar sections of the developing cochlea. Apoptotic cells (white arrows, cleaved caspase-3, red) were observed at P5-10. Such cells were often engulfed by macrophages (magenta arrow, GFP, green) (E) High magnification image taken from organ of Corti (red box shown in C) showing GFP-labeled macrophage engulfing pyknotic nuclei at P10. (F–G) Presence of ameboid (red arrow) and ramified (white arrow) macrophages in cochlear whole mounts. Note: GER region is present just below the dotted white line. Images in (A–D) , (F–G) were taken using a ×20 objective lens and the image in (E) was taken using a ×63 objective lens. Labels: Red-cleaved caspase- 3, Green-GFP + macrophages, Blue-DAPI. Abbreviations: OC: organ of Corti, GER: Greater Epithelial Ridge, IHC: Inner Hair Cell, SC: Supporting Cell (N = 3–6).
    Figure Legend Snippet: Progression of apoptosis and macrophage infiltration in the basal region of the developing cochlea. (A–D) Mid-modiolar sections of the developing cochlea. Apoptotic cells (white arrows, cleaved caspase-3, red) were observed at P5-10. Such cells were often engulfed by macrophages (magenta arrow, GFP, green) (E) High magnification image taken from organ of Corti (red box shown in C) showing GFP-labeled macrophage engulfing pyknotic nuclei at P10. (F–G) Presence of ameboid (red arrow) and ramified (white arrow) macrophages in cochlear whole mounts. Note: GER region is present just below the dotted white line. Images in (A–D) , (F–G) were taken using a ×20 objective lens and the image in (E) was taken using a ×63 objective lens. Labels: Red-cleaved caspase- 3, Green-GFP + macrophages, Blue-DAPI. Abbreviations: OC: organ of Corti, GER: Greater Epithelial Ridge, IHC: Inner Hair Cell, SC: Supporting Cell (N = 3–6).

    Techniques Used: Labeling, Immunohistochemistry

    BLZ945 treatment eliminates macrophages in the developing cochlea. (A) Treatment and sample collection timeline. (B–D′) Cochlear whole mounts showing depletion of macrophages following treatment with the CSF1R antagonist BLZ945 (Green-GFP + macrophages). (E–L′) Sensory epithelium of the developing cochlea showing apoptotic cells (red-cleaved caspase-3) and macrophages (green-GFP). Normal patterns of dying cells (white arrows) were observed in BLZ945-treated cochleae. (M) Number of macrophages in the GER region of the developing cochlea. Note that BLZ945 sharply reduced the number of macrophages in the GER. (N) Number of pyknotic nuclei in the GER region of the developing cochlea. Images in B- (D′) were taken using a ×5 objective lens and images in (E-L’) were taken using ×20 objective lens. Abbreviations: OC: Organ of Corti, GER: Greater Epithelial Ridge, P: Postnatal Day. (*p: p
    Figure Legend Snippet: BLZ945 treatment eliminates macrophages in the developing cochlea. (A) Treatment and sample collection timeline. (B–D′) Cochlear whole mounts showing depletion of macrophages following treatment with the CSF1R antagonist BLZ945 (Green-GFP + macrophages). (E–L′) Sensory epithelium of the developing cochlea showing apoptotic cells (red-cleaved caspase-3) and macrophages (green-GFP). Normal patterns of dying cells (white arrows) were observed in BLZ945-treated cochleae. (M) Number of macrophages in the GER region of the developing cochlea. Note that BLZ945 sharply reduced the number of macrophages in the GER. (N) Number of pyknotic nuclei in the GER region of the developing cochlea. Images in B- (D′) were taken using a ×5 objective lens and images in (E-L’) were taken using ×20 objective lens. Abbreviations: OC: Organ of Corti, GER: Greater Epithelial Ridge, P: Postnatal Day. (*p: p

    Techniques Used:

    24) Product Images from "Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila"

    Article Title: Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201607098

    Toll-6 promotes cell survival via MyD88. (A) Coimmunoprecipitations showing that MyD88-V5 bound Toll-6–Flag and Toll-7–Flag and activated Toll-6 CY –Flag and Toll-7 CY –Flag. IB, immunoblot; IP, immunoprecipitation. Molecular masses are given in kilodaltons. (B) Anti-MyD88 and exon trap reporters Dorsal-GFP and Dif-GFP visualized with anti-GFP are distributed throughout the embryonic CNS neuropile. Left, horizontal views; right, transverse sections; white arrows indicate reporter distribution within the neuropile. (C) Loss of Eve + neurons (arrows) in the CNS in MyD88 kra56 Toll-7 P8 Toll-6 26 triple mutant embryos. For quantification, see Fig. S4. aCC, anterior corner cell; EL, Eve lateral. (D) Altering MyD88 signaling affects Eve + neuron number. Dashed lines indicate 50% (left graph) or 100% (right graph) data distirbution in controls. Box plots: larvae, one-way analysis of variance, P
    Figure Legend Snippet: Toll-6 promotes cell survival via MyD88. (A) Coimmunoprecipitations showing that MyD88-V5 bound Toll-6–Flag and Toll-7–Flag and activated Toll-6 CY –Flag and Toll-7 CY –Flag. IB, immunoblot; IP, immunoprecipitation. Molecular masses are given in kilodaltons. (B) Anti-MyD88 and exon trap reporters Dorsal-GFP and Dif-GFP visualized with anti-GFP are distributed throughout the embryonic CNS neuropile. Left, horizontal views; right, transverse sections; white arrows indicate reporter distribution within the neuropile. (C) Loss of Eve + neurons (arrows) in the CNS in MyD88 kra56 Toll-7 P8 Toll-6 26 triple mutant embryos. For quantification, see Fig. S4. aCC, anterior corner cell; EL, Eve lateral. (D) Altering MyD88 signaling affects Eve + neuron number. Dashed lines indicate 50% (left graph) or 100% (right graph) data distirbution in controls. Box plots: larvae, one-way analysis of variance, P

    Techniques Used: Immunoprecipitation, Mutagenesis

    25) Product Images from "Role of a versatile peptide motif in controlling Hox nuclear export and autophagy in the Drosophila fat body"

    Article Title: Role of a versatile peptide motif in controlling Hox nuclear export and autophagy in the Drosophila fat body

    Journal: bioRxiv

    doi: 10.1101/843383

    The non-canonical NES controls Ubx nuclear export in the Drosophila fat body. A. Cloned are recognized by the GFP expression (green) and Ubx constructs are revealed with an anti-Ubx (full length Ubx constructs) or anti-HA (truncated Ubx constructs) antibody (gray). Dapi (blue) stains for nuclei and ATG8-mCherry for autophagy (red). A’. Graphs showing statistical distribution of VC-Ubx constructs and ATG8-mCherry, as described in the Figure 5 . The NES mutation blocks the nuclear export induced by the dN235dC deletion, allowing the constructs to reside in the nucleus and inhibit the autophagy. The minimal dN282dC form is localized in the nucleus but is not able to inhibit autophagy.
    Figure Legend Snippet: The non-canonical NES controls Ubx nuclear export in the Drosophila fat body. A. Cloned are recognized by the GFP expression (green) and Ubx constructs are revealed with an anti-Ubx (full length Ubx constructs) or anti-HA (truncated Ubx constructs) antibody (gray). Dapi (blue) stains for nuclei and ATG8-mCherry for autophagy (red). A’. Graphs showing statistical distribution of VC-Ubx constructs and ATG8-mCherry, as described in the Figure 5 . The NES mutation blocks the nuclear export induced by the dN235dC deletion, allowing the constructs to reside in the nucleus and inhibit the autophagy. The minimal dN282dC form is localized in the nucleus but is not able to inhibit autophagy.

    Techniques Used: Clone Assay, Expressing, Construct, Mutagenesis

    Ubx is actively exported from fat body nuclei and degraded in the L3-wandering (W) stage. A. Immunostaining of endogenous Ubx (gray) in the fat body of L3-Feeding (F) and L3-W larva. Cloned are recognized with the GFP expression (green). The ATG8-mCherry reporter (red) allows following the off (weak nuclear expression) and on (dotted staining corresponds to autophagosomes) states of autophagy in the fat body of L3-F and L3-W larvae, respectively. A’. Graphs showing the statistical distribution of Ubx staining in the nucleus (N) or cytoplasm (C), together with the number (n) and size (s) of ATG8-mCherry vesicles in fat body cells (see also Materials and Methods). B. Blocking the nuclear export by inhibiting the expression of Emb with RNAi leads to nuclear accumulation of Ubx in the nucleus of L3-W fat body cells. Note that ATG8-mCherry also accumulates in the nucleus. B’. Statistical quantification of Ubx and Atg8-mCherry distribution as described in A’. C. Blocking the proteasome activity by the expression of Cullin RNAi reveals accumulation of Ubx in L3-W fat body cells. C’. Statistical quantification of Ubx and Atg8-mCherry distribution as described in A’.
    Figure Legend Snippet: Ubx is actively exported from fat body nuclei and degraded in the L3-wandering (W) stage. A. Immunostaining of endogenous Ubx (gray) in the fat body of L3-Feeding (F) and L3-W larva. Cloned are recognized with the GFP expression (green). The ATG8-mCherry reporter (red) allows following the off (weak nuclear expression) and on (dotted staining corresponds to autophagosomes) states of autophagy in the fat body of L3-F and L3-W larvae, respectively. A’. Graphs showing the statistical distribution of Ubx staining in the nucleus (N) or cytoplasm (C), together with the number (n) and size (s) of ATG8-mCherry vesicles in fat body cells (see also Materials and Methods). B. Blocking the nuclear export by inhibiting the expression of Emb with RNAi leads to nuclear accumulation of Ubx in the nucleus of L3-W fat body cells. Note that ATG8-mCherry also accumulates in the nucleus. B’. Statistical quantification of Ubx and Atg8-mCherry distribution as described in A’. C. Blocking the proteasome activity by the expression of Cullin RNAi reveals accumulation of Ubx in L3-W fat body cells. C’. Statistical quantification of Ubx and Atg8-mCherry distribution as described in A’.

    Techniques Used: Immunostaining, Clone Assay, Expressing, Staining, Blocking Assay, Activity Assay

    26) Product Images from "The stem-like Stat3-responsive cells of zebrafish intestine are Wnt/β-catenin dependent"

    Article Title: The stem-like Stat3-responsive cells of zebrafish intestine are Wnt/β-catenin dependent

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.188987

    Tcf7l2 (Tcf4) is required for development of Stat3-responsive cells of zebrafish larvae intestine and the Stat3 pathway is activated ectopically in intestinal adenomas of apc hu745 mutants. (A,A′) In vivo EGFP fluorescence in the intestine of 6 dpf Tg(7xStat3:EGFP) / tcf7l2 hu892 / hu892 , Tg(7xStat3:EGFP) / tcf7l2 +/ hu892 and Tg(7xStat3:EGFP) / tcf7l2 +/+ siblings (A) and measurement of integrated density (A′) ( n =16). (B) qPCR analysis of il6 , gp130 , jak2a , jak2b and stat3 mRNA expression from tcf7l2 +/+ and tcf7l2 hu892/hu892 sibling larvae. (C,C′) Effect of XAV treatment on Tg(7xStat3:EGFP) embryos from 48 to 78 hpf: measurement of the integrated density of the fluorescence (C) and measurement of the number of GFP + cells (C′). (D,E) Haematoxylin-eosin staining on paraffin embedded transversal section of zebrafish apc hu745 intestine at 12 months post fertilization, displaying both normal tissue (n) and hyperplastic adenomas (a). (D′,D″) Staining of a sequential intestinal section of D using α-EGFP (green) (D′) and α-PCNA (red) (D″) Abs. (E′,E″) Double staining using α-EGFP Ab (green) and α-PCNA Ab (red) of a sequential intestinal section of E. All statistical analyses were performed by unpaired t -test. * P
    Figure Legend Snippet: Tcf7l2 (Tcf4) is required for development of Stat3-responsive cells of zebrafish larvae intestine and the Stat3 pathway is activated ectopically in intestinal adenomas of apc hu745 mutants. (A,A′) In vivo EGFP fluorescence in the intestine of 6 dpf Tg(7xStat3:EGFP) / tcf7l2 hu892 / hu892 , Tg(7xStat3:EGFP) / tcf7l2 +/ hu892 and Tg(7xStat3:EGFP) / tcf7l2 +/+ siblings (A) and measurement of integrated density (A′) ( n =16). (B) qPCR analysis of il6 , gp130 , jak2a , jak2b and stat3 mRNA expression from tcf7l2 +/+ and tcf7l2 hu892/hu892 sibling larvae. (C,C′) Effect of XAV treatment on Tg(7xStat3:EGFP) embryos from 48 to 78 hpf: measurement of the integrated density of the fluorescence (C) and measurement of the number of GFP + cells (C′). (D,E) Haematoxylin-eosin staining on paraffin embedded transversal section of zebrafish apc hu745 intestine at 12 months post fertilization, displaying both normal tissue (n) and hyperplastic adenomas (a). (D′,D″) Staining of a sequential intestinal section of D using α-EGFP (green) (D′) and α-PCNA (red) (D″) Abs. (E′,E″) Double staining using α-EGFP Ab (green) and α-PCNA Ab (red) of a sequential intestinal section of E. All statistical analyses were performed by unpaired t -test. * P

    Techniques Used: In Vivo, Fluorescence, Real-time Polymerase Chain Reaction, Expressing, Staining, Double Staining

    27) Product Images from "Differential interactions of missing in metastasis and insulin receptor tyrosine kinase substrate with RAB proteins in the endocytosis of CXCR4"

    Article Title: Differential interactions of missing in metastasis and insulin receptor tyrosine kinase substrate with RAB proteins in the endocytosis of CXCR4

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.006071

    RAB7 is essential for MIM to promote CXCR4 sorting into late endosomes. A–D , HeLa cells expressing GFP ( A and B ) or MIM-GFP ( C and D ) were plated in 6-well plates, treated with Ct-siRNA ( A and C ) or siRAB7 ( B and D ) for 48 h, and stimulated with 500 ng/ml SDF-1 for 30 min. The treated cells were costained with monoclonal CXCR4 antibody ( green ) and polyclonal CD63 antibody ( red ) and inspected by confocal microscopy. The boxed areas of images were amplified and are presented below. E , co-localization of CXCR4 and CD63 was quantitively analyzed based on Manders' coefficient, which represents the proportion of CD63 colocalized with CXCR4. F , the amount of CD63 puncta of the acquired images was also quantified. Scale bars = 10 μm. ***, p
    Figure Legend Snippet: RAB7 is essential for MIM to promote CXCR4 sorting into late endosomes. A–D , HeLa cells expressing GFP ( A and B ) or MIM-GFP ( C and D ) were plated in 6-well plates, treated with Ct-siRNA ( A and C ) or siRAB7 ( B and D ) for 48 h, and stimulated with 500 ng/ml SDF-1 for 30 min. The treated cells were costained with monoclonal CXCR4 antibody ( green ) and polyclonal CD63 antibody ( red ) and inspected by confocal microscopy. The boxed areas of images were amplified and are presented below. E , co-localization of CXCR4 and CD63 was quantitively analyzed based on Manders' coefficient, which represents the proportion of CD63 colocalized with CXCR4. F , the amount of CD63 puncta of the acquired images was also quantified. Scale bars = 10 μm. ***, p

    Techniques Used: Expressing, Confocal Microscopy, Amplification

    28) Product Images from "Probenecid Disrupts a Novel Pannexin 1-Collapsin Response Mediator Protein 2 Interaction and Increases Microtubule Stability"

    Article Title: Probenecid Disrupts a Novel Pannexin 1-Collapsin Response Mediator Protein 2 Interaction and Increases Microtubule Stability

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00124

    Probenecid treatment decreases Crmp2 co-precipitation with Panx1. (Ai) Western blot of a GFP IP from N2a cells expressing Panx1-EGFP, or EGFP control, treated (16 h) with probenecid or vehicle control. (ii) Probenecid treatment (16 h) significantly decreased the amount of Crmp2 that precipitated with Panx1-EGFP. Crmp2 signal in the IP fraction was normalized to GFP signal intensity from the same lane (Panx1-EGFP [vehicle] vs. Panx1-EGFP [probenecid]: 100.0 ± 0.8 vs. 39.4 ± 19.6, p = 0.0368, n = 3, unpaired t -test). (B) The same assay was performed with a shorter 30 min treatment (i) . (ii) Short probenecid treatment (30 min) revealed a ~30% decrease in Crmp2 co-precipitation (Panx1-EGFP [vehicle] vs. Panx1-EGFP [probenecid]: 100.0 ± 11.1 vs. 67.7 ± 19.6, p = 0.0826, n = 3, unpaired t -test). (C) PNGase F treatment was performed to confirm the upper Panx1EGFP immunoreactive band was indeed glycosylated Panx1 and assessed by Western blotting. Note that both immunoreactive bands shifted in response to N-glycosidase F treatment, suggesting that both major bands are mature glycosylated Panx1EGFP of differing molecular weights. (D) Analysis of the input intensities revealed that probenecid treatment did not change the overall expression of (i) Crmp2 ( p = 0.2000, Mann-Whitney test, n = 3) or (ii) Panx1 ( p = 0.578, unpaired t -test, n = 3). Normalized to Gapdh (not shown), n.s., non-significant.
    Figure Legend Snippet: Probenecid treatment decreases Crmp2 co-precipitation with Panx1. (Ai) Western blot of a GFP IP from N2a cells expressing Panx1-EGFP, or EGFP control, treated (16 h) with probenecid or vehicle control. (ii) Probenecid treatment (16 h) significantly decreased the amount of Crmp2 that precipitated with Panx1-EGFP. Crmp2 signal in the IP fraction was normalized to GFP signal intensity from the same lane (Panx1-EGFP [vehicle] vs. Panx1-EGFP [probenecid]: 100.0 ± 0.8 vs. 39.4 ± 19.6, p = 0.0368, n = 3, unpaired t -test). (B) The same assay was performed with a shorter 30 min treatment (i) . (ii) Short probenecid treatment (30 min) revealed a ~30% decrease in Crmp2 co-precipitation (Panx1-EGFP [vehicle] vs. Panx1-EGFP [probenecid]: 100.0 ± 11.1 vs. 67.7 ± 19.6, p = 0.0826, n = 3, unpaired t -test). (C) PNGase F treatment was performed to confirm the upper Panx1EGFP immunoreactive band was indeed glycosylated Panx1 and assessed by Western blotting. Note that both immunoreactive bands shifted in response to N-glycosidase F treatment, suggesting that both major bands are mature glycosylated Panx1EGFP of differing molecular weights. (D) Analysis of the input intensities revealed that probenecid treatment did not change the overall expression of (i) Crmp2 ( p = 0.2000, Mann-Whitney test, n = 3) or (ii) Panx1 ( p = 0.578, unpaired t -test, n = 3). Normalized to Gapdh (not shown), n.s., non-significant.

    Techniques Used: Western Blot, Expressing, MANN-WHITNEY

    Probenecid decreases proximity ligation assay (PLA) between Panx1EGFP and Crmp2. (A) Representative confocal maximum intensity projections of vehicle- (left) and probenecid-treated (right) Panx1-EGFP-expressing N2a cells demonstrating PLA-positive Crmp2-Panx1 clusters (white). (B) Probenecid treatment significantly decreased mean fluorescence intensity of the PLA signal (vehicle: 26.0 ± 2.7 arb.u. per 100 μm 2 vs. probenecid: 17.1 ± 2.1 arb.u. per 100 μm 2 , p = 0.0037, n = 77 cells and n = 70 cells, respectively, Mann-Whitney test). (C) Controls for PLA included (i) cells were incubated with α-GFP anti-mouse and α-GFP anti-rabbit to serve as a positive control. (ii) No primary antibodies were included to serve as a negative control. Hoechst 33342 was used as a nuclear counterstain. Scale bars, 10 μm.
    Figure Legend Snippet: Probenecid decreases proximity ligation assay (PLA) between Panx1EGFP and Crmp2. (A) Representative confocal maximum intensity projections of vehicle- (left) and probenecid-treated (right) Panx1-EGFP-expressing N2a cells demonstrating PLA-positive Crmp2-Panx1 clusters (white). (B) Probenecid treatment significantly decreased mean fluorescence intensity of the PLA signal (vehicle: 26.0 ± 2.7 arb.u. per 100 μm 2 vs. probenecid: 17.1 ± 2.1 arb.u. per 100 μm 2 , p = 0.0037, n = 77 cells and n = 70 cells, respectively, Mann-Whitney test). (C) Controls for PLA included (i) cells were incubated with α-GFP anti-mouse and α-GFP anti-rabbit to serve as a positive control. (ii) No primary antibodies were included to serve as a negative control. Hoechst 33342 was used as a nuclear counterstain. Scale bars, 10 μm.

    Techniques Used: Proximity Ligation Assay, Expressing, Fluorescence, MANN-WHITNEY, Incubation, Positive Control, Negative Control

    29) Product Images from "A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag"

    Article Title: A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag

    Journal: eLife

    doi: 10.7554/eLife.78923

    HeLa cells stably expressing pSu9-Halo-GFP in growing medium with 200 nM SF650 ligand.
    Figure Legend Snippet: HeLa cells stably expressing pSu9-Halo-GFP in growing medium with 200 nM SF650 ligand.

    Techniques Used: Stable Transfection, Expressing

    HaloTag can be combined with GFP- and/or RFP-based autophagy reporters. ( a ) Fluorescence images of wild-type HeLa cells stably expressing lysosomal marker LAMP1-mRuby3 and HaloTag (Halo)-LC3B in nutrient-rich medium or after 6 hr in starvation medium containing 200 nM SF650-conjugated ligand. ( b ) Fluorescence images of wild-type HeLa cells stably expressing mRFP-mGFP-LC3B in nutrient-rich medium or after 2 hr in starvation medium containing 75 nM LysoTracker Deep Red, with or without 100 nm of bafilomycin A 1 (BafA). Arrowheads point to mRFP + mGFP + puncta that represent autophagosomes; arrows point to mRFP + mGFP – puncta that should represent autolysosomes. ( c ) Quantification of mRFP + mGFP + puncta and mRFP + mGFP – puncta in cells shown in ( b ). n=91–100 cells. ( d ) Fluorescence images of wild-type HeLa cells stably expressing the lysosomal marker LAMP1-mRuby3 and Halo-mGFP-LC3B in nutrient-rich medium or after 2 hr in starvation medium with and withoutBafA. The media contained 200 nM of SF650-conjugated ligand. Arrowheads point to Halo + mGFP + puncta that represent autophagosomes; arrows point to Halo + mGFP – puncta that represent autolysosomes. ( e ) Immunoblotting and in-gel fluorescence detection of total cell lysates from wild-type and FIP200 knockout (KO) HeLa cells and mouse embryonic fibroblasts (MEFs) stably expressing Halo-mGFP-LC3B-mRFP, pulse-labeled for 20 min with 100 nM TMR-conjugated ligand in nutrient-rich medium. The cells were then collected or incubated for 6 hr in starvation medium. ( f ) Fluorescence images of cells described in ( e ) in nutrient-rich medium or after 6 hr incubation in starvation medium. The media contained 200 μM of SF650-conjugated ligand. ( g ) mGFP and mRFP intensities of cells described in ( e ) were determined with flow cytometry under nutrient-rich conditions with or without 250 nM Torin1 for 24 hr. A reduction in the mRFP:mGFP fluorescence ratio indicates autophagic flux. n=5000 cells. In box plots, solid bars indicate medians, boxes indicate the interquartile range (25th–75th percentile), and whiskers indicate the largest and smallest values within 1.5 times the interquartile range ( c, g ). Scale bar = 10 µm (main), 2 µm (inset) ( a, b, d, f ). Uncropped blot images of Figure 2—figure supplement 1e .
    Figure Legend Snippet: HaloTag can be combined with GFP- and/or RFP-based autophagy reporters. ( a ) Fluorescence images of wild-type HeLa cells stably expressing lysosomal marker LAMP1-mRuby3 and HaloTag (Halo)-LC3B in nutrient-rich medium or after 6 hr in starvation medium containing 200 nM SF650-conjugated ligand. ( b ) Fluorescence images of wild-type HeLa cells stably expressing mRFP-mGFP-LC3B in nutrient-rich medium or after 2 hr in starvation medium containing 75 nM LysoTracker Deep Red, with or without 100 nm of bafilomycin A 1 (BafA). Arrowheads point to mRFP + mGFP + puncta that represent autophagosomes; arrows point to mRFP + mGFP – puncta that should represent autolysosomes. ( c ) Quantification of mRFP + mGFP + puncta and mRFP + mGFP – puncta in cells shown in ( b ). n=91–100 cells. ( d ) Fluorescence images of wild-type HeLa cells stably expressing the lysosomal marker LAMP1-mRuby3 and Halo-mGFP-LC3B in nutrient-rich medium or after 2 hr in starvation medium with and withoutBafA. The media contained 200 nM of SF650-conjugated ligand. Arrowheads point to Halo + mGFP + puncta that represent autophagosomes; arrows point to Halo + mGFP – puncta that represent autolysosomes. ( e ) Immunoblotting and in-gel fluorescence detection of total cell lysates from wild-type and FIP200 knockout (KO) HeLa cells and mouse embryonic fibroblasts (MEFs) stably expressing Halo-mGFP-LC3B-mRFP, pulse-labeled for 20 min with 100 nM TMR-conjugated ligand in nutrient-rich medium. The cells were then collected or incubated for 6 hr in starvation medium. ( f ) Fluorescence images of cells described in ( e ) in nutrient-rich medium or after 6 hr incubation in starvation medium. The media contained 200 μM of SF650-conjugated ligand. ( g ) mGFP and mRFP intensities of cells described in ( e ) were determined with flow cytometry under nutrient-rich conditions with or without 250 nM Torin1 for 24 hr. A reduction in the mRFP:mGFP fluorescence ratio indicates autophagic flux. n=5000 cells. In box plots, solid bars indicate medians, boxes indicate the interquartile range (25th–75th percentile), and whiskers indicate the largest and smallest values within 1.5 times the interquartile range ( c, g ). Scale bar = 10 µm (main), 2 µm (inset) ( a, b, d, f ). Uncropped blot images of Figure 2—figure supplement 1e .

    Techniques Used: Fluorescence, Stable Transfection, Expressing, Marker, Knock-Out, Labeling, Incubation, Flow Cytometry

    Halo-GFP-KDEL and pSu9-Halo-GFP localize to endoplasmic reticulum (ER) and mitochondria, respectively. ( a ) Fluorescence images of wild-type HeLa cells stably expressing HaloTag (Halo)-mGFP-KDEL and ER marker mRuby3-cytochrome b 5 in nutrient-rich medium containing 200 nM SF650-conjugated ligand. ( b ) Fluorescence images of wild-type HeLa cells stably expressing pSu9-Halo-mGFP and mitochondrial marker mRuby3-OMP25 in nutrient-rich medium containing 200 nM SF650-conjugated ligand. ( c ) Fluorescence images of wild-type HeLa cells stably expressing lysosomal marker LAMP1-mRuby3 and Halo-mGFP-KDEL in nutrient-rich medium or after 6 hr incubation in starvation medium. The media contained 200 nM SF650-conjugated ligand. ( d ) Fluorescence images of wild-type HeLa cells stably expressing HA-Parkin, LAMP1-mRuby3, and pSu9-Halo-mGFP in nutrient-rich medium containing 200 nM SF650-conjugated ligand with or without 1 µM oligomycin and 2 µM antimycin (OA, collectively), which were added for 6 hr. Scale bar = 10 µm (main), 2 µm (inset) ( a, b, c, d ).
    Figure Legend Snippet: Halo-GFP-KDEL and pSu9-Halo-GFP localize to endoplasmic reticulum (ER) and mitochondria, respectively. ( a ) Fluorescence images of wild-type HeLa cells stably expressing HaloTag (Halo)-mGFP-KDEL and ER marker mRuby3-cytochrome b 5 in nutrient-rich medium containing 200 nM SF650-conjugated ligand. ( b ) Fluorescence images of wild-type HeLa cells stably expressing pSu9-Halo-mGFP and mitochondrial marker mRuby3-OMP25 in nutrient-rich medium containing 200 nM SF650-conjugated ligand. ( c ) Fluorescence images of wild-type HeLa cells stably expressing lysosomal marker LAMP1-mRuby3 and Halo-mGFP-KDEL in nutrient-rich medium or after 6 hr incubation in starvation medium. The media contained 200 nM SF650-conjugated ligand. ( d ) Fluorescence images of wild-type HeLa cells stably expressing HA-Parkin, LAMP1-mRuby3, and pSu9-Halo-mGFP in nutrient-rich medium containing 200 nM SF650-conjugated ligand with or without 1 µM oligomycin and 2 µM antimycin (OA, collectively), which were added for 6 hr. Scale bar = 10 µm (main), 2 µm (inset) ( a, b, c, d ).

    Techniques Used: Fluorescence, Stable Transfection, Expressing, Marker, Incubation

    Bulk nonselective autophagic flux can be detected with Halo-GFP. ( a ) Fluorescence images of wild-type HeLa cells stably expressing lysosomal marker LAMP1-mRuby3 and HaloTag (Halo)-mGFP under nutrient-rich conditions or after 6 hr under starvation conditions in the presence of 200 nM SF650-conjugated ligand. ( b ) Time-lapse montage of wild-type HeLa cells stably expressing Halo-mGFP cells in starvation medium containing 200 nM SF650-conjugated ligand and 75 nM LysoTracker Red. Images were taken after the indicated durations of starvation. Scale bar = 10 µm (main), 2 µm (inset, inset montage) ( a, b ).
    Figure Legend Snippet: Bulk nonselective autophagic flux can be detected with Halo-GFP. ( a ) Fluorescence images of wild-type HeLa cells stably expressing lysosomal marker LAMP1-mRuby3 and HaloTag (Halo)-mGFP under nutrient-rich conditions or after 6 hr under starvation conditions in the presence of 200 nM SF650-conjugated ligand. ( b ) Time-lapse montage of wild-type HeLa cells stably expressing Halo-mGFP cells in starvation medium containing 200 nM SF650-conjugated ligand and 75 nM LysoTracker Red. Images were taken after the indicated durations of starvation. Scale bar = 10 µm (main), 2 µm (inset, inset montage) ( a, b ).

    Techniques Used: Fluorescence, Stable Transfection, Expressing, Marker

    30) Product Images from "DNA damage signaling regulates age-dependent proliferative capacity of quiescent inner ear supporting cells"

    Article Title: DNA damage signaling regulates age-dependent proliferative capacity of quiescent inner ear supporting cells

    Journal: Aging (Albany NY)

    doi:

    Utricular supporting cells show γH2AX and 53BP1 foci upon forced cell cycle re-entry, as revealed by confocal imaging. ( A,B ) At 3 DIV, AdcD1-infected P6 and P50 utricles display EdU+ SCs with numerous, small γH2AX foci. ( C,D ) Similar γH2AX profiles are seen 1 h post-irradiation in Sox2+ utricular SCs of both ages. ( E-G ) At 3 DIV, Sox2+ SCs in both AdβGal-infected P6 and P50 utricles, and in non-infected P6 utricle show one or two large γH2AX foci per nucleus. ( H,H' ) At 3 DIV, EdU+ SCs in AdcD1-infected P50 utricle display 53BP1 foci. Edu+ SC nuclei containing 53BP1 foci are outlined ( H' ). ( I ) Four hours post-irradiation, P50 utricular Sox2+ SCs show 53BP1 foci. ( J ) At 3 DIV, AdGFP-infected P50 utricle is devoid of 53BP1 foci. A Sox2+/GFP+/53BP1- nucleus is outlined. Abbreviations: AdcD1, adenovirus encoding cyclin D1; AdβGal, adenovirus encoding β-galactosidase; AdGFP, adenovirus encoding green fluorescent protein; Gy, gray; γH2AX, Ser 139 phosphorylated histone H2AX; utr, utricle. Scale bar, shown in J: A-J, 5 µm.
    Figure Legend Snippet: Utricular supporting cells show γH2AX and 53BP1 foci upon forced cell cycle re-entry, as revealed by confocal imaging. ( A,B ) At 3 DIV, AdcD1-infected P6 and P50 utricles display EdU+ SCs with numerous, small γH2AX foci. ( C,D ) Similar γH2AX profiles are seen 1 h post-irradiation in Sox2+ utricular SCs of both ages. ( E-G ) At 3 DIV, Sox2+ SCs in both AdβGal-infected P6 and P50 utricles, and in non-infected P6 utricle show one or two large γH2AX foci per nucleus. ( H,H' ) At 3 DIV, EdU+ SCs in AdcD1-infected P50 utricle display 53BP1 foci. Edu+ SC nuclei containing 53BP1 foci are outlined ( H' ). ( I ) Four hours post-irradiation, P50 utricular Sox2+ SCs show 53BP1 foci. ( J ) At 3 DIV, AdGFP-infected P50 utricle is devoid of 53BP1 foci. A Sox2+/GFP+/53BP1- nucleus is outlined. Abbreviations: AdcD1, adenovirus encoding cyclin D1; AdβGal, adenovirus encoding β-galactosidase; AdGFP, adenovirus encoding green fluorescent protein; Gy, gray; γH2AX, Ser 139 phosphorylated histone H2AX; utr, utricle. Scale bar, shown in J: A-J, 5 µm.

    Techniques Used: Imaging, Infection, Irradiation

    Adenoviruses transduce inner ear supporting cells in explant cultures. AdGFP- and AdβGal-infected utricles and cochleas analyzed after 3 DIV. ( A,B ) Schematic representation of the utricular ( A ) and cochlear ( B ) sensory epithelium, viewed from above (whole mount specimens) and in transverse plane. Utricular hair cells with the apical stereociliary bundle (grey) are located on top of a layer SCs (red). The cochlear sensory epithelium consists of one row of inner hair cells and three rows of outer hair cells (grey). Deiters' cells (red) are located underneath outer hair cells. Inner and outer pillar cells (pink) are positioned between the inner and outer hair cell rows. ( C,D ) AdGFP-infected P6 and P50 utricles double-labeled for GFP and Sox2 show transduction in SCs. The views are focused to the level of Sox2+ SC nuclei. ( E,E' ) In AdGFP-infected P6 utricle, a small part of parvalbumin+ hair cells are transduced (arrow), in addition to SCs (arrowheads). ( F,F' ) In P6 cochlea, Deiters' cells show AdGFP transduction, as opposed to the adjacent outer and inner pillar cells. ( G ) X-Gal histochemical staining shows a patchy pattern of AdβGal transduction in the area of Deiters' cells (dotted) along the length of the cochlear duct. The boxed area represents the region used for analysis. Abbreviations: utr, utricle; co, cochlea; AdβGal, adenovirus encoding β-galactosidase; AdGFP, adenovirus encoding green fluorescent protein; parv, parvalbumin; DCs, Deiters' cells; IP, inner pillar cell; OP, outer pillar cell; IHC, inner hair cell; OHCs, outer hair cells. Scale bar, shown in G: C-F', 20 µm; G, 180 µm.
    Figure Legend Snippet: Adenoviruses transduce inner ear supporting cells in explant cultures. AdGFP- and AdβGal-infected utricles and cochleas analyzed after 3 DIV. ( A,B ) Schematic representation of the utricular ( A ) and cochlear ( B ) sensory epithelium, viewed from above (whole mount specimens) and in transverse plane. Utricular hair cells with the apical stereociliary bundle (grey) are located on top of a layer SCs (red). The cochlear sensory epithelium consists of one row of inner hair cells and three rows of outer hair cells (grey). Deiters' cells (red) are located underneath outer hair cells. Inner and outer pillar cells (pink) are positioned between the inner and outer hair cell rows. ( C,D ) AdGFP-infected P6 and P50 utricles double-labeled for GFP and Sox2 show transduction in SCs. The views are focused to the level of Sox2+ SC nuclei. ( E,E' ) In AdGFP-infected P6 utricle, a small part of parvalbumin+ hair cells are transduced (arrow), in addition to SCs (arrowheads). ( F,F' ) In P6 cochlea, Deiters' cells show AdGFP transduction, as opposed to the adjacent outer and inner pillar cells. ( G ) X-Gal histochemical staining shows a patchy pattern of AdβGal transduction in the area of Deiters' cells (dotted) along the length of the cochlear duct. The boxed area represents the region used for analysis. Abbreviations: utr, utricle; co, cochlea; AdβGal, adenovirus encoding β-galactosidase; AdGFP, adenovirus encoding green fluorescent protein; parv, parvalbumin; DCs, Deiters' cells; IP, inner pillar cell; OP, outer pillar cell; IHC, inner hair cell; OHCs, outer hair cells. Scale bar, shown in G: C-F', 20 µm; G, 180 µm.

    Techniques Used: Infection, Labeling, Transduction, Staining, Immunohistochemistry

    31) Product Images from "Distinct gene-selective roles for a network of core promoter factors in Drosophila neural stem cell identity"

    Article Title: Distinct gene-selective roles for a network of core promoter factors in Drosophila neural stem cell identity

    Journal: Biology Open

    doi: 10.1242/bio.042168

    NSC-TAF or TRF2-depleted NSCs exhibit defective cell polarity but do not express differentiation markers. (A) Taf4 LL07382 NSCs or (B) Taf7 LL07382 NSCs fail to express normal levels of Insc. Mitotic cells are labeled with phospho Histone H3 (pH3), CD8:GFP (a membrane-tethered GFP) labels the clones and Insc is shown in red. (C) Knockdown of TBP, TRF2 or TAF9 does not result in nuclear accumulation of Prospero. (D) Knockdown of TBP, TRF2 or TAF9 does not result in nuclear accumulation of Elav. The white dotted line demarcates the optic lobe (left)/central brain (right) boundary. In both C and D, transgenes were expressed using a UAS-Dcr2; inscGAL4, UAS-CD8:GFP, tubGAL80 ts driver and transgenes induced for 72 h. Scale bars: 10 µm (A–C); 20 µm (D).
    Figure Legend Snippet: NSC-TAF or TRF2-depleted NSCs exhibit defective cell polarity but do not express differentiation markers. (A) Taf4 LL07382 NSCs or (B) Taf7 LL07382 NSCs fail to express normal levels of Insc. Mitotic cells are labeled with phospho Histone H3 (pH3), CD8:GFP (a membrane-tethered GFP) labels the clones and Insc is shown in red. (C) Knockdown of TBP, TRF2 or TAF9 does not result in nuclear accumulation of Prospero. (D) Knockdown of TBP, TRF2 or TAF9 does not result in nuclear accumulation of Elav. The white dotted line demarcates the optic lobe (left)/central brain (right) boundary. In both C and D, transgenes were expressed using a UAS-Dcr2; inscGAL4, UAS-CD8:GFP, tubGAL80 ts driver and transgenes induced for 72 h. Scale bars: 10 µm (A–C); 20 µm (D).

    Techniques Used: Labeling, Clone Assay

    A unique subset of TAFs (NSC-TAFs) required for NSC homeostasis. (A) Marker gene expression and cell division patterns of Type I (left) and Type II (right) NSCs. (B) Genetic schema of in vivo transgenic RNAi screen. (C) Subunit composition of Transcription Factor IID (TFIID); the red outline indicates subunits implicated in human neurological disorders. (D) Quantification of total central brain NSCs (large Dpn + cells) in nervous systems expressing the listed RNAi transgenes using a pan-NSC driver (worniuGAL4, UAS-Dcr-2, UAS-GFP). P -values were RpII33 (0.6655), MED12 (0.9376), TBP (0.3182), TRF2 (
    Figure Legend Snippet: A unique subset of TAFs (NSC-TAFs) required for NSC homeostasis. (A) Marker gene expression and cell division patterns of Type I (left) and Type II (right) NSCs. (B) Genetic schema of in vivo transgenic RNAi screen. (C) Subunit composition of Transcription Factor IID (TFIID); the red outline indicates subunits implicated in human neurological disorders. (D) Quantification of total central brain NSCs (large Dpn + cells) in nervous systems expressing the listed RNAi transgenes using a pan-NSC driver (worniuGAL4, UAS-Dcr-2, UAS-GFP). P -values were RpII33 (0.6655), MED12 (0.9376), TBP (0.3182), TRF2 (

    Techniques Used: Marker, Expressing, In Vivo, Transgenic Assay

    TBP, TRF2 and NSC-TAFs are required for normal NSC output but not for NSC survival. (A) Blocking apoptosis does not rescue NSC loss observed upon knockdown of TRF2, TAF1 or TAF9. The listed RNAi transgenes were co-expressed with UAS-miRHG, which blocks apoptosis, using a pan-NSC driver (worniuGAL4, UAS-GFP). P -values were RpII33 (0.4216), MED12 (0.9893), TBP (0.4710), TRF2 (
    Figure Legend Snippet: TBP, TRF2 and NSC-TAFs are required for normal NSC output but not for NSC survival. (A) Blocking apoptosis does not rescue NSC loss observed upon knockdown of TRF2, TAF1 or TAF9. The listed RNAi transgenes were co-expressed with UAS-miRHG, which blocks apoptosis, using a pan-NSC driver (worniuGAL4, UAS-GFP). P -values were RpII33 (0.4216), MED12 (0.9893), TBP (0.4710), TRF2 (

    Techniques Used: Blocking Assay

    32) Product Images from "Neuronal Chloride Regulation via KCC2 Is Modulated through a GABAB Receptor Protein Complex"

    Article Title: Neuronal Chloride Regulation via KCC2 Is Modulated through a GABAB Receptor Protein Complex

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2164-16.2017

    The GABA B R associates with the transmembrane domain of KCC2. A , The GABA B R1 monoclonal antibody 2D7 used for immunoblotting is specific for GABA B R1. In Western blots of total rat brain and rat hippocampus homogenate, two bands corresponding to GABA B R1a and GABA B R1b were detected. Blots of lysates from CHO cells stably expressing either GABA B R1a/R2 or GABA B R1b/R2 revealed multiple bands, consistent with differential glycosylation of GABA B R1 proteins in this system. B , The KCC2-GABA B R association can be reconstituted in a heterologous cell system. CHO cells stably expressing GABA B R1b/R2 were transiently transfected with FL-KCC2-GFP and used for coimmunoprecipitation experiments. Western blots of resulting complexes showed that KCC2 can be coimmunoprecipitated with GABA B R1b (left), and the reciprocal coimmunoprecipitation confirms the association (right). IgG controls were included in each experiment. Sh, Sheep; Rab, rabbit. C , Schematic diagram of KCC2 showing the intracellular NTD, CTD, and TMD with its 12 predicted transmembrane helices. D , GFP fusion proteins of FL-KCC2 and different KCC2 deletion constructs containing the TMD are expressed and transported to the plasma membrane. Biotinylation experiment comparing total (T) and cell surface (S) protein levels in CHO GABA B R1b/R2 cells, transiently transfected with different GFP fusion constructs (left). FL-KCC2 (predicted molecular mass 150 kDa, although the TM region is glycosylated), TMD + CTD (predicted 140 kDa), NTD + TMD (predicted 96 kDa), and TMD (predicted 86 kDa) are all detected on the cell surface, whereas GFP (27 kDa) alone is not. Additional bands detected likely represent alternatively glycosylated, degraded, or aggregated proteins. The blot was reprobed with GABA B R1 antibody to confirm surface expression of the receptor (bottom). E , The transmembrane domain of KCC2 is required for the association with the GABA B R. Coimmunoprecipitation experiments on CHO GABA B R1b/R2 cells transiently expressing KCC2-GFP deletion constructs were performed using anti-GFP as the precipitating antibody. When the resulting complexes were probed for GABA B R1 (top), all KCC2-GFP fusion proteins containing the TMD successfully coimmunoprecipitated GABA B R1b. However, GFP fusion proteins containing only the NTD (predicted 38 kDa) or the CTD (predicted 82 kDa) did not capture GABA B R1b. Under these conditions, and compared with FL-KCC2 (100%), the relative amounts of GABA B R detected after isolation with the GFP antibody was 68 ± 16% for NTD + TMD, 36 ± 2% for TMD + CTD, 76 ± 19% for TMD, 3 ± 1% for NTD, 3 ± 2% for CTD, and 3 ± 1% for GFP ( n = 2–4 in each case). These quantifications will be influenced by the level of expression of each of the different constructs. Cell lysates expressing the GFP fusion proteins are shown (bottom). Additional bands detected with the GFP antibody are likely to be alternatively glycosylated, degraded, or higher-order aggregates of the expressed fusion proteins. F , Control experiments show that the endogenous 100 kDa transferrin receptor (TfR) is not coimmunoprecipitated with KCC2-GFP proteins in CHO GABA B R cells (top; IgG bands are shown for clarity). Lysates are also shown (bottom).
    Figure Legend Snippet: The GABA B R associates with the transmembrane domain of KCC2. A , The GABA B R1 monoclonal antibody 2D7 used for immunoblotting is specific for GABA B R1. In Western blots of total rat brain and rat hippocampus homogenate, two bands corresponding to GABA B R1a and GABA B R1b were detected. Blots of lysates from CHO cells stably expressing either GABA B R1a/R2 or GABA B R1b/R2 revealed multiple bands, consistent with differential glycosylation of GABA B R1 proteins in this system. B , The KCC2-GABA B R association can be reconstituted in a heterologous cell system. CHO cells stably expressing GABA B R1b/R2 were transiently transfected with FL-KCC2-GFP and used for coimmunoprecipitation experiments. Western blots of resulting complexes showed that KCC2 can be coimmunoprecipitated with GABA B R1b (left), and the reciprocal coimmunoprecipitation confirms the association (right). IgG controls were included in each experiment. Sh, Sheep; Rab, rabbit. C , Schematic diagram of KCC2 showing the intracellular NTD, CTD, and TMD with its 12 predicted transmembrane helices. D , GFP fusion proteins of FL-KCC2 and different KCC2 deletion constructs containing the TMD are expressed and transported to the plasma membrane. Biotinylation experiment comparing total (T) and cell surface (S) protein levels in CHO GABA B R1b/R2 cells, transiently transfected with different GFP fusion constructs (left). FL-KCC2 (predicted molecular mass 150 kDa, although the TM region is glycosylated), TMD + CTD (predicted 140 kDa), NTD + TMD (predicted 96 kDa), and TMD (predicted 86 kDa) are all detected on the cell surface, whereas GFP (27 kDa) alone is not. Additional bands detected likely represent alternatively glycosylated, degraded, or aggregated proteins. The blot was reprobed with GABA B R1 antibody to confirm surface expression of the receptor (bottom). E , The transmembrane domain of KCC2 is required for the association with the GABA B R. Coimmunoprecipitation experiments on CHO GABA B R1b/R2 cells transiently expressing KCC2-GFP deletion constructs were performed using anti-GFP as the precipitating antibody. When the resulting complexes were probed for GABA B R1 (top), all KCC2-GFP fusion proteins containing the TMD successfully coimmunoprecipitated GABA B R1b. However, GFP fusion proteins containing only the NTD (predicted 38 kDa) or the CTD (predicted 82 kDa) did not capture GABA B R1b. Under these conditions, and compared with FL-KCC2 (100%), the relative amounts of GABA B R detected after isolation with the GFP antibody was 68 ± 16% for NTD + TMD, 36 ± 2% for TMD + CTD, 76 ± 19% for TMD, 3 ± 1% for NTD, 3 ± 2% for CTD, and 3 ± 1% for GFP ( n = 2–4 in each case). These quantifications will be influenced by the level of expression of each of the different constructs. Cell lysates expressing the GFP fusion proteins are shown (bottom). Additional bands detected with the GFP antibody are likely to be alternatively glycosylated, degraded, or higher-order aggregates of the expressed fusion proteins. F , Control experiments show that the endogenous 100 kDa transferrin receptor (TfR) is not coimmunoprecipitated with KCC2-GFP proteins in CHO GABA B R cells (top; IgG bands are shown for clarity). Lysates are also shown (bottom).

    Techniques Used: Western Blot, Stable Transfection, Expressing, Transfection, Construct, Isolation

    GABA B Rs associate with KCC2 at the cell membrane in cortex and hippocampus. A , Affinity purification and mass spectrometry were used to identify proteins associated with the GABA B R in synaptic membrane preparations from adult rat cortex. The table shows the number of unique peptides for each protein, obtained across three independent isolates of the GABA B R1 protein. Where the identified proteins have previously been shown to associate with the GABA B R, references are provided. B , KCC2 coimmunoprecipitates with the GABA B R. Rat hippocampal lysates were immunoprecipitated with an anti-GABA B R1 antibody, and subsequent Western blot analysis for KCC2 revealed two distinct bands at ∼130 and 270 kDa, which correspond to the monomeric and dimeric forms of KCC2 (top blot, right lane). Probing for GABA B R1 confirmed successful immunoprecipitation of both the GABA B R1a and GABA B R1b isoforms (bottom blot). In contrast, controls using sheep anti-IgG antibody failed to pull down KCC2 or GABA B R1 (middle lanes). C , The GABA B R coimmunoprecipitates with KCC2. Immunoprecipitates from rat hippocampal lysates isolated with an anti-KCC2 antibody were positive for GABA B R1a, GABA B R1b, and KCC2 (top and bottom blots, right lanes). In contrast, control experiments using a rabbit anti-IgG failed to pull down KCC2 or GABA B R1 from the same lysate (middle lanes). D , The GABA B R (left) and KCC2 (right) are both localized at the plasma membrane of pyramidal neurons, as revealed by rabbit polyclonal antibodies against GABA B R2 (left) and KCC2 (right) in separate rat organotypic hippocampal slices (P7 + 7–14 DIV). Scale bars, 10 μm. E , Using a sequential double-labeling technique in dissociated neuronal cultures (see Materials and Methods), GABA B R2 (red) and KCC2 (green) were found to be colocalized (yellow) at somatic and dendritic membranes of hippocampal pyramidal neurons. Magnifications of the areas highlighted within the white boxes are provided in the panels below. Control staining (bottom) for GABA B R2 and β-tubulin revealed nonoverlapping signals. Scale bars, 20 and 5 μm.
    Figure Legend Snippet: GABA B Rs associate with KCC2 at the cell membrane in cortex and hippocampus. A , Affinity purification and mass spectrometry were used to identify proteins associated with the GABA B R in synaptic membrane preparations from adult rat cortex. The table shows the number of unique peptides for each protein, obtained across three independent isolates of the GABA B R1 protein. Where the identified proteins have previously been shown to associate with the GABA B R, references are provided. B , KCC2 coimmunoprecipitates with the GABA B R. Rat hippocampal lysates were immunoprecipitated with an anti-GABA B R1 antibody, and subsequent Western blot analysis for KCC2 revealed two distinct bands at ∼130 and 270 kDa, which correspond to the monomeric and dimeric forms of KCC2 (top blot, right lane). Probing for GABA B R1 confirmed successful immunoprecipitation of both the GABA B R1a and GABA B R1b isoforms (bottom blot). In contrast, controls using sheep anti-IgG antibody failed to pull down KCC2 or GABA B R1 (middle lanes). C , The GABA B R coimmunoprecipitates with KCC2. Immunoprecipitates from rat hippocampal lysates isolated with an anti-KCC2 antibody were positive for GABA B R1a, GABA B R1b, and KCC2 (top and bottom blots, right lanes). In contrast, control experiments using a rabbit anti-IgG failed to pull down KCC2 or GABA B R1 from the same lysate (middle lanes). D , The GABA B R (left) and KCC2 (right) are both localized at the plasma membrane of pyramidal neurons, as revealed by rabbit polyclonal antibodies against GABA B R2 (left) and KCC2 (right) in separate rat organotypic hippocampal slices (P7 + 7–14 DIV). Scale bars, 10 μm. E , Using a sequential double-labeling technique in dissociated neuronal cultures (see Materials and Methods), GABA B R2 (red) and KCC2 (green) were found to be colocalized (yellow) at somatic and dendritic membranes of hippocampal pyramidal neurons. Magnifications of the areas highlighted within the white boxes are provided in the panels below. Control staining (bottom) for GABA B R2 and β-tubulin revealed nonoverlapping signals. Scale bars, 20 and 5 μm.

    Techniques Used: Affinity Purification, Mass Spectrometry, Immunoprecipitation, Western Blot, Isolation, Labeling, Staining

    33) Product Images from "Astrocytes Modulate Somatostatin Interneuron Signaling in the Visual Cortex"

    Article Title: Astrocytes Modulate Somatostatin Interneuron Signaling in the Visual Cortex

    Journal: Cells

    doi: 10.3390/cells11091400

    Experimental approach for Channelrhodopsin-2 (ChR2)-mCherry specific expression in somatostatin (SST) interneurons and GCaMP6f specific expression in astrocytes from the visual cortex (VCx). ( A ) Schematic representation of adeno-associated virus (AAV) injections in the VCx of newborn SST Cre mice and brain slice preparations at least 2 weeks after injections. ( B ) Left upper panel, representative differential interference contrast (DIC) image of a coronal slice containing the VCx (square). Right upper panel, combined fluorescence and DIC images of the square shown in left panel, showing ChR2-mCherry expression in VCx SST interneurons, the optical fiber for optogenetic stimulation, and the patch pipette (arrow) for electrophysiological recordings. Scale bar, 50 µm. Bottom, fluorescence image of ChR2-mCherry-expressing SST interneurons (scale bar, 10 µm) and a representative action potential discharge, recorded in cell-attached mode, upon a single 150 ms blue light (470 nm) pulse stimulation. Scale bars, 20 ms, 20 pA. ( C ) Schematic of the experimental approach. Ca 2+ imaging in layer II/III astrocytes and whole-cell patch-clamp recordings from layer II/III pyramidal neurons (PNs) were performed before and after optogenetic stimulation of SST interneurons. In this figure and the other figures, violet-shaped cells represent astrocytes, red cells represent SST interneurons, and blue cells represent PNs. ( D ) Confocal microscope fluorescence images of the VCx from a mouse injected with AAV2/1.EF1.dflox.hChR2(H134R)-mCherry.WPRE.hGH, illustrating the specific localization of ChR2-mCherry in SST interneurons. Red fluorescence, ChR2-mCherry (α-RFP staining); blue fluorescence, nuclear TO—PRO-3; specific green staining for either SST interneurons (α-SST staining) or parvalbumin (PV) interneurons (α-PV staining). Scale bar, 25 μm. ( E ) Bar chart showing that cells expressing ChR2-mCherry are mainly SST interneurons. α-SST: 406 SST+ cells out of 432 ChR2-mCherry+ cells (3 mice, 8 slices); α-PV: 33 PV+ cells out of 461 ChR2-mCherry+ cells (3 mice, 6 slices). ( F ) Confocal microscope fluorescence images of the VCx from a mouse injected with AAV5.GfaABC1DcytoGCaMP6f.SV40, illustrating the specific localization of GCaMP6f in astrocytes. Green fluorescence, GCaMP6f (α-GFP staining); blue fluorescence, nuclear TO-PRO-3; specific red staining for either astrocytes (α-S100β staining) or neurons (α-NeuN staining). Scale bar, 25 μm. ( G ) Bar chart showing that cells expressing GCaMP6f are mainly astrocytes. α-S100β: 2528 S100β+ cells out of 2856 GCaMP6f-GFP+ cells (5 mice, 12 slices); α-NeuN: 2 NeuN+ cells out of 510 GCaMP6f-GFP+ cells (2 mice, 6 slices).
    Figure Legend Snippet: Experimental approach for Channelrhodopsin-2 (ChR2)-mCherry specific expression in somatostatin (SST) interneurons and GCaMP6f specific expression in astrocytes from the visual cortex (VCx). ( A ) Schematic representation of adeno-associated virus (AAV) injections in the VCx of newborn SST Cre mice and brain slice preparations at least 2 weeks after injections. ( B ) Left upper panel, representative differential interference contrast (DIC) image of a coronal slice containing the VCx (square). Right upper panel, combined fluorescence and DIC images of the square shown in left panel, showing ChR2-mCherry expression in VCx SST interneurons, the optical fiber for optogenetic stimulation, and the patch pipette (arrow) for electrophysiological recordings. Scale bar, 50 µm. Bottom, fluorescence image of ChR2-mCherry-expressing SST interneurons (scale bar, 10 µm) and a representative action potential discharge, recorded in cell-attached mode, upon a single 150 ms blue light (470 nm) pulse stimulation. Scale bars, 20 ms, 20 pA. ( C ) Schematic of the experimental approach. Ca 2+ imaging in layer II/III astrocytes and whole-cell patch-clamp recordings from layer II/III pyramidal neurons (PNs) were performed before and after optogenetic stimulation of SST interneurons. In this figure and the other figures, violet-shaped cells represent astrocytes, red cells represent SST interneurons, and blue cells represent PNs. ( D ) Confocal microscope fluorescence images of the VCx from a mouse injected with AAV2/1.EF1.dflox.hChR2(H134R)-mCherry.WPRE.hGH, illustrating the specific localization of ChR2-mCherry in SST interneurons. Red fluorescence, ChR2-mCherry (α-RFP staining); blue fluorescence, nuclear TO—PRO-3; specific green staining for either SST interneurons (α-SST staining) or parvalbumin (PV) interneurons (α-PV staining). Scale bar, 25 μm. ( E ) Bar chart showing that cells expressing ChR2-mCherry are mainly SST interneurons. α-SST: 406 SST+ cells out of 432 ChR2-mCherry+ cells (3 mice, 8 slices); α-PV: 33 PV+ cells out of 461 ChR2-mCherry+ cells (3 mice, 6 slices). ( F ) Confocal microscope fluorescence images of the VCx from a mouse injected with AAV5.GfaABC1DcytoGCaMP6f.SV40, illustrating the specific localization of GCaMP6f in astrocytes. Green fluorescence, GCaMP6f (α-GFP staining); blue fluorescence, nuclear TO-PRO-3; specific red staining for either astrocytes (α-S100β staining) or neurons (α-NeuN staining). Scale bar, 25 μm. ( G ) Bar chart showing that cells expressing GCaMP6f are mainly astrocytes. α-S100β: 2528 S100β+ cells out of 2856 GCaMP6f-GFP+ cells (5 mice, 12 slices); α-NeuN: 2 NeuN+ cells out of 510 GCaMP6f-GFP+ cells (2 mice, 6 slices).

    Techniques Used: Expressing, Mouse Assay, Slice Preparation, Fluorescence, Transferring, Imaging, Patch Clamp, Microscopy, Injection, Staining

    34) Product Images from "Cell–Fibronectin Interactions and Actomyosin Contractility Regulate the Segmentation Clock and Spatio-Temporal Somite Cleft Formation during Chick Embryo Somitogenesis"

    Article Title: Cell–Fibronectin Interactions and Actomyosin Contractility Regulate the Segmentation Clock and Spatio-Temporal Somite Cleft Formation during Chick Embryo Somitogenesis

    Journal: Cells

    doi: 10.3390/cells11132003

    Experimental approaches to challenge the fibronectin–integrin–actomyosin communication axis. ( A ) Schematic representation of the four experimental treatments used: (1) expression of the 70 kDa N-terminal fragment of fibronectin impairs fibronectin matrix assembly; (2) addition of the linear RGD peptide to the culture medium competes with native fibronectin for integrin α5β1 engagement, with effects on fibronectin matrix assembly and cell–fibronectin interactions; (3) addition of RockOut, a chemical inhibitor of ROCK I and II, enzymes involved in activating NM II downstream of integrins; and (4) addition of Blebbistatin, a chemical inhibitor that blocks NM II activation directly. ( B ) Electroporation strategy used for 70 kDa overexpression. PSM/ectoderm progenitors of primitive-streak stage embryos were electroporated on one side with either a pCAGGs GFP-expressing vector alone (pCAGGS), or co-electroporated with pCAGGs-GFP and a 70 kDa-expressing vector (70 kDa). Electroporated embryos were then cultured ex ovo for 26 h using the Early Chick culture method [ 53 ], after which they were fixed. ( C ) Incubation of tissue explants with RGD or chemical inhibitors. HH11-14 embryos were collected, and the posterior region was isolated and bisected into two embryo half explants. Control (right or left) explants were cultured in medium only or medium containing DMSO, while experimental (left or right) explants were cultured in the presence of RGD, RockOut or Blebbistatin for the designated time. Both explant halves were fixed at the end of the culture period.
    Figure Legend Snippet: Experimental approaches to challenge the fibronectin–integrin–actomyosin communication axis. ( A ) Schematic representation of the four experimental treatments used: (1) expression of the 70 kDa N-terminal fragment of fibronectin impairs fibronectin matrix assembly; (2) addition of the linear RGD peptide to the culture medium competes with native fibronectin for integrin α5β1 engagement, with effects on fibronectin matrix assembly and cell–fibronectin interactions; (3) addition of RockOut, a chemical inhibitor of ROCK I and II, enzymes involved in activating NM II downstream of integrins; and (4) addition of Blebbistatin, a chemical inhibitor that blocks NM II activation directly. ( B ) Electroporation strategy used for 70 kDa overexpression. PSM/ectoderm progenitors of primitive-streak stage embryos were electroporated on one side with either a pCAGGs GFP-expressing vector alone (pCAGGS), or co-electroporated with pCAGGs-GFP and a 70 kDa-expressing vector (70 kDa). Electroporated embryos were then cultured ex ovo for 26 h using the Early Chick culture method [ 53 ], after which they were fixed. ( C ) Incubation of tissue explants with RGD or chemical inhibitors. HH11-14 embryos were collected, and the posterior region was isolated and bisected into two embryo half explants. Control (right or left) explants were cultured in medium only or medium containing DMSO, while experimental (left or right) explants were cultured in the presence of RGD, RockOut or Blebbistatin for the designated time. Both explant halves were fixed at the end of the culture period.

    Techniques Used: Expressing, Activation Assay, Electroporation, Over Expression, Plasmid Preparation, Cell Culture, Incubation, Isolation

    Somite morphology and cleft formation is compromised in 70 kDa-electroporated embryos. ( A ) Number of somitic structures formed in pCAGGs- (mean = 14.5; standard error of the mean (SEM) = 0.27) and 70 kDa-electroporated (mean = 14.6; SEM = 0.29) embryos after 26 h. ( B , C ) Close up of embryos electroporated with 70 kDa showing examples of fewer somites ( B ), ill-defined somites (arrowheads in B ) and fused somitic structures (arrowheads in C ) on the electroporated side (GFP, left). ( D – F ) Sagittal views of somite SI in embryos electroporated with pCAGGs ( D , D’ ) and 70 kDa with either mild ( E , E’ ) or severe ( F , F’ ) phenotypes, immunostained for ZO-1 and fibronectin and stained for DNA. SI of pCAGGs- and 70 kDa-electroporated embryos polarize ZO-1 normally ( D – F ). A thick fibronectin matrix surrounds the somites of pCAGGs-electroporated embryos (white arrowheads in D ), while this matrix is disrupted in 70 kDa-electroporated embryos (open arrowheads in E , F ). Somitic clefts (*) form in pCAGGs embryos, whereas 70 kDa-electroporated embryos retain cells within one of the clefts (arrow in E’ ) or in both (arrows in F’ ) clefts, suggesting incomplete cleft formation. Somites of 70 kDa-electroporated embryos are also detached from either the endoderm or ectoderm (brackets in E’ ), or from both (brackets in F’ ). ( G , H ) Sagittal sections of embryos electroporated with pCAGGs and 70 kDa (severe phenotype). Asterisks mark complete clefts, filled with a fibronectin matrix, and arrowhead indicates forming cleft ( G ). Arrows point to incomplete somitic clefts ( H ). Rostral to the left and dorsal on top. Error bars indicate standard error of the mean. ns—not statistically significant (paired Student’s t -test). GFP—green fluorescent protein. FN—fibronectin. ZO-1—Zonula occludens protein 1. Scale bars: 200 µm ( B , C ), 50 µm ( D – F’ ), 100 µm ( G , H ).
    Figure Legend Snippet: Somite morphology and cleft formation is compromised in 70 kDa-electroporated embryos. ( A ) Number of somitic structures formed in pCAGGs- (mean = 14.5; standard error of the mean (SEM) = 0.27) and 70 kDa-electroporated (mean = 14.6; SEM = 0.29) embryos after 26 h. ( B , C ) Close up of embryos electroporated with 70 kDa showing examples of fewer somites ( B ), ill-defined somites (arrowheads in B ) and fused somitic structures (arrowheads in C ) on the electroporated side (GFP, left). ( D – F ) Sagittal views of somite SI in embryos electroporated with pCAGGs ( D , D’ ) and 70 kDa with either mild ( E , E’ ) or severe ( F , F’ ) phenotypes, immunostained for ZO-1 and fibronectin and stained for DNA. SI of pCAGGs- and 70 kDa-electroporated embryos polarize ZO-1 normally ( D – F ). A thick fibronectin matrix surrounds the somites of pCAGGs-electroporated embryos (white arrowheads in D ), while this matrix is disrupted in 70 kDa-electroporated embryos (open arrowheads in E , F ). Somitic clefts (*) form in pCAGGs embryos, whereas 70 kDa-electroporated embryos retain cells within one of the clefts (arrow in E’ ) or in both (arrows in F’ ) clefts, suggesting incomplete cleft formation. Somites of 70 kDa-electroporated embryos are also detached from either the endoderm or ectoderm (brackets in E’ ), or from both (brackets in F’ ). ( G , H ) Sagittal sections of embryos electroporated with pCAGGs and 70 kDa (severe phenotype). Asterisks mark complete clefts, filled with a fibronectin matrix, and arrowhead indicates forming cleft ( G ). Arrows point to incomplete somitic clefts ( H ). Rostral to the left and dorsal on top. Error bars indicate standard error of the mean. ns—not statistically significant (paired Student’s t -test). GFP—green fluorescent protein. FN—fibronectin. ZO-1—Zonula occludens protein 1. Scale bars: 200 µm ( B , C ), 50 µm ( D – F’ ), 100 µm ( G , H ).

    Techniques Used: Staining

    35) Product Images from "Endosomal TLR3, TLR7, and TLR8 control neuronal morphology through different transcriptional programs"

    Article Title: Endosomal TLR3, TLR7, and TLR8 control neuronal morphology through different transcriptional programs

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201712113

    P38 and TAK1 inhibitors neutralize the negative effect of TLR8 activation on dendritic growth. (A and D) WT neurons were treated with CL075/poly dT for 30 min at 18 DIV and subjected to immunostaining with phospho-P38, phospho-TAK1, and neuronal marker NeuN antibodies as indicated. Counterstaining with DAPI was performed to label nuclei. (B and E) GFP was transfected at 12 DIV. CL075/poly dT and P38 inhibitor SB203580 (B) and three TAK1 inhibitors, Takinib, 5ZO, and NG25 (E), were added at 17 DIV for 1 d in WT cultured neurons. (C) SB203580 treatment did not influence the effect of CL075 on younger WT neurons at 4 DIV. (B–E) Sample size ( n ) indicates the number of examined neurons, which were randomly collected blind from two independent experiments. Data are presented as the mean + SEM (error bars). Bars: (A) 25 µm; (B) 50 µm; (D) 20 µm. **, P
    Figure Legend Snippet: P38 and TAK1 inhibitors neutralize the negative effect of TLR8 activation on dendritic growth. (A and D) WT neurons were treated with CL075/poly dT for 30 min at 18 DIV and subjected to immunostaining with phospho-P38, phospho-TAK1, and neuronal marker NeuN antibodies as indicated. Counterstaining with DAPI was performed to label nuclei. (B and E) GFP was transfected at 12 DIV. CL075/poly dT and P38 inhibitor SB203580 (B) and three TAK1 inhibitors, Takinib, 5ZO, and NG25 (E), were added at 17 DIV for 1 d in WT cultured neurons. (C) SB203580 treatment did not influence the effect of CL075 on younger WT neurons at 4 DIV. (B–E) Sample size ( n ) indicates the number of examined neurons, which were randomly collected blind from two independent experiments. Data are presented as the mean + SEM (error bars). Bars: (A) 25 µm; (B) 50 µm; (D) 20 µm. **, P

    Techniques Used: Activation Assay, Immunostaining, Marker, Transfection, Cell Culture

    Tlr8 knockdown promotes dendritic growth in vitro and in vivo. (A) Immunoblotting using HA tag antibody to examine the effect of TLR8 knockdown constructs on TLR8 expression in HEK293T cells. Two expression vectors were used to express miR-Ctrl and miR-Tlr8 as indicated. (B) Tlr7 −/− -cultured neurons were transfected with miR-Ctrl and miR-Tlr8 at 2 DIV, and then treated with CL075/poly dT at 4 DIV. Dendritic morphology was analyzed at 5 DIV based on GFP signals. The sample sizes ( n ) of the examined neurons are indicated. Data were collected from two independent experiments. (C and D) IUE with miR-Ctrl or miR-Tlr8 was performed at E15.5 with WT (C) and Tlr7 −/− (D) mice. Layer 2/3 cortical neurons were outlined by GFP signal at P7, P14, and P21 as indicated. N, number of examined mice; n , number of examined neurons. Bars: (B) 20 µm; (C and D) 50 µm. The data are presented as mean + SEM (error bars). *, P
    Figure Legend Snippet: Tlr8 knockdown promotes dendritic growth in vitro and in vivo. (A) Immunoblotting using HA tag antibody to examine the effect of TLR8 knockdown constructs on TLR8 expression in HEK293T cells. Two expression vectors were used to express miR-Ctrl and miR-Tlr8 as indicated. (B) Tlr7 −/− -cultured neurons were transfected with miR-Ctrl and miR-Tlr8 at 2 DIV, and then treated with CL075/poly dT at 4 DIV. Dendritic morphology was analyzed at 5 DIV based on GFP signals. The sample sizes ( n ) of the examined neurons are indicated. Data were collected from two independent experiments. (C and D) IUE with miR-Ctrl or miR-Tlr8 was performed at E15.5 with WT (C) and Tlr7 −/− (D) mice. Layer 2/3 cortical neurons were outlined by GFP signal at P7, P14, and P21 as indicated. N, number of examined mice; n , number of examined neurons. Bars: (B) 20 µm; (C and D) 50 µm. The data are presented as mean + SEM (error bars). *, P

    Techniques Used: In Vitro, In Vivo, Construct, Expressing, Cell Culture, Transfection, Mouse Assay

    36) Product Images from "Astrocytic ApoE underlies maturation of hippocampal neurons and cognitive recovery after traumatic brain injury in mice"

    Article Title: Astrocytic ApoE underlies maturation of hippocampal neurons and cognitive recovery after traumatic brain injury in mice

    Journal: Communications Biology

    doi: 10.1038/s42003-021-02841-4

    The reduction in astrocytic ApoE impairs dendritic complexity in newborn neurons in the injured dentate gyrus. a – d Reconstructed dendritic trees of newborn neurons 4 weeks after being injected with a GFP-expressing retrovirus in the dentate gyrus. e Neither the reduction of astrocytic ApoE nor CCI results in deficits in dendrites of newborn neurons in the dentate gyrus. f , g Similar observations were seen in the total length and nodes in the newborn neurons. Scale bar in ( d ) = 10 µm.
    Figure Legend Snippet: The reduction in astrocytic ApoE impairs dendritic complexity in newborn neurons in the injured dentate gyrus. a – d Reconstructed dendritic trees of newborn neurons 4 weeks after being injected with a GFP-expressing retrovirus in the dentate gyrus. e Neither the reduction of astrocytic ApoE nor CCI results in deficits in dendrites of newborn neurons in the dentate gyrus. f , g Similar observations were seen in the total length and nodes in the newborn neurons. Scale bar in ( d ) = 10 µm.

    Techniques Used: Injection, Expressing

    37) Product Images from "Neurotransmitter phenotype switching by spinal excitatory interneurons regulates locomotor recovery after spinal cord injury"

    Article Title: Neurotransmitter phenotype switching by spinal excitatory interneurons regulates locomotor recovery after spinal cord injury

    Journal: Nature Neuroscience

    doi: 10.1038/s41593-022-01067-9

    The approach to suppress vGAT gene expression after adult injury. a . Representative image of an injection site with GFP ON neurons representing AAV-floxed-shRNA-vGAT-GFP infected vGlut2 ON neurons from vGlut2 cre mouse. Scale bar: 100 µm. b . Representative image of synaptic apposition of SynGFP ON terminals to a ChAT ON motor neuron and colocalization of vGAT antibody, or lack thereof, in intact vGAT cre :: Tau LSL-SynGFP mouse after shRNA-vGAT injection. Scale bar: 2 µm. c . Quantification of average % SynGFP ON /vGAT + to MNs with or without the sh RNA-vGAT virus injection in intact vGAT cre :: Tau LSL-SynGFP mice (n=5 for each group, control 91 ± 0.52 %, sh RNA-vGAT 46 ± 2.5 %). Each data point represents 1 mouse, with the error bar indicating ± 2 SD of SynGFP ON /vGAT + input density per MN (n=10 MN per mouse).
    Figure Legend Snippet: The approach to suppress vGAT gene expression after adult injury. a . Representative image of an injection site with GFP ON neurons representing AAV-floxed-shRNA-vGAT-GFP infected vGlut2 ON neurons from vGlut2 cre mouse. Scale bar: 100 µm. b . Representative image of synaptic apposition of SynGFP ON terminals to a ChAT ON motor neuron and colocalization of vGAT antibody, or lack thereof, in intact vGAT cre :: Tau LSL-SynGFP mouse after shRNA-vGAT injection. Scale bar: 2 µm. c . Quantification of average % SynGFP ON /vGAT + to MNs with or without the sh RNA-vGAT virus injection in intact vGAT cre :: Tau LSL-SynGFP mice (n=5 for each group, control 91 ± 0.52 %, sh RNA-vGAT 46 ± 2.5 %). Each data point represents 1 mouse, with the error bar indicating ± 2 SD of SynGFP ON /vGAT + input density per MN (n=10 MN per mouse).

    Techniques Used: Expressing, Injection, shRNA, Infection, Mouse Assay

    38) Product Images from "Developmental independence of median fins from the larval fin fold revises their evolutionary origin"

    Article Title: Developmental independence of median fins from the larval fin fold revises their evolutionary origin

    Journal: Scientific Reports

    doi: 10.1038/s41598-022-11180-1

    Cell-tracking analysis of the epithelial cells in the reducing LMFF area. ( a ) Schematic of the plasmid DNA construct used to generate the Tg. ( b ) Scheme of the Tg observation. ( c – f’ ) GFP-positive labelled cells in the reducing LMFF area at 6.1 mm SL ( c – d’ ), 6.6 mm SL ( e – e’ ), and 7.0 mm SL ( f – f’ ). The right panels ( d’ , e’ , f’ ) are magnified views of the dashed rectangles in the left panes ( d , e , f ), respectively. White dashed lines in ( d- f’ ) indicate outlines of the LMFFs. Yellow dashed lines indicate outlines of the EGFP-positive populations of epidermal cells. Magenta brackets in ( d’ , e’ , f’ ) indicate EGFP-positive populations of epidermal cells experiencing proximo-distal shrinking. Magenta arrowheads in ( d’ , e’ , f’ ) indicate EGFP-positive populations of epidermal cells migrating down to the trunk. ( g – h’ ) Cell morphology and distribution in the reducing LMFF area at 6.0–6.5 mm SL ( g – g’ ) and 6.5–7.0 mm SL ( h – h’ ). Cell membrane visualized by CellMask. The right panels ( g’ , h’ ) are magnified views of the dashed rectangles in the left panels ( g,h ), respectively. Yellow dashed lines indicate outlines of the epidermal cells. ( i,j ) Boxplots of cell length along the AP and PD axis in the reducing LMFF area. Whiskers in ( i ) and ( j ) show maximum and minimum values within 1.5 times the interquartile range. Boxes show the median and 25th and 75th percentiles. The P value in ( i ) and ( j ) is the result of Brunner-Munzel test ( P = 0.4407 and P = 8.34e-10). Scale bars in ( c,d,d’,g ) and that in ( g’ ) indicate 200 μm and 100 μm, respectively.
    Figure Legend Snippet: Cell-tracking analysis of the epithelial cells in the reducing LMFF area. ( a ) Schematic of the plasmid DNA construct used to generate the Tg. ( b ) Scheme of the Tg observation. ( c – f’ ) GFP-positive labelled cells in the reducing LMFF area at 6.1 mm SL ( c – d’ ), 6.6 mm SL ( e – e’ ), and 7.0 mm SL ( f – f’ ). The right panels ( d’ , e’ , f’ ) are magnified views of the dashed rectangles in the left panes ( d , e , f ), respectively. White dashed lines in ( d- f’ ) indicate outlines of the LMFFs. Yellow dashed lines indicate outlines of the EGFP-positive populations of epidermal cells. Magenta brackets in ( d’ , e’ , f’ ) indicate EGFP-positive populations of epidermal cells experiencing proximo-distal shrinking. Magenta arrowheads in ( d’ , e’ , f’ ) indicate EGFP-positive populations of epidermal cells migrating down to the trunk. ( g – h’ ) Cell morphology and distribution in the reducing LMFF area at 6.0–6.5 mm SL ( g – g’ ) and 6.5–7.0 mm SL ( h – h’ ). Cell membrane visualized by CellMask. The right panels ( g’ , h’ ) are magnified views of the dashed rectangles in the left panels ( g,h ), respectively. Yellow dashed lines indicate outlines of the epidermal cells. ( i,j ) Boxplots of cell length along the AP and PD axis in the reducing LMFF area. Whiskers in ( i ) and ( j ) show maximum and minimum values within 1.5 times the interquartile range. Boxes show the median and 25th and 75th percentiles. The P value in ( i ) and ( j ) is the result of Brunner-Munzel test ( P = 0.4407 and P = 8.34e-10). Scale bars in ( c,d,d’,g ) and that in ( g’ ) indicate 200 μm and 100 μm, respectively.

    Techniques Used: Cell Tracking Assay, Plasmid Preparation, Construct

    39) Product Images from "Deciphering the Molecular Interaction Between the Adhesion G Protein-Coupled Receptor ADGRV1 and its PDZ-Containing Regulator PDZD7"

    Article Title: Deciphering the Molecular Interaction Between the Adhesion G Protein-Coupled Receptor ADGRV1 and its PDZ-Containing Regulator PDZD7

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2022.923740

    Western blot detection of ADGRV1 β subunit (prey) pulled down by PDZD7-GFP (bait) constructs from HeLa cell lysates. PDZD7 constructs are detected with an antibody directed against the fused GFP. ADGRV1 is detected using an antibody directed against its cytoplasmic domain. For each condition, fractions corresponding to the input (I), unbound fraction (U), wash (W) and elution (E) are loaded. Red squares indicate the expected position for ADGRV1 β in the elution fraction.
    Figure Legend Snippet: Western blot detection of ADGRV1 β subunit (prey) pulled down by PDZD7-GFP (bait) constructs from HeLa cell lysates. PDZD7 constructs are detected with an antibody directed against the fused GFP. ADGRV1 is detected using an antibody directed against its cytoplasmic domain. For each condition, fractions corresponding to the input (I), unbound fraction (U), wash (W) and elution (E) are loaded. Red squares indicate the expected position for ADGRV1 β in the elution fraction.

    Techniques Used: Western Blot, Construct

    40) Product Images from "Human Immunodeficiency Virus Type 1 Protease Regulation of Tat Activity Is Essential for Efficient Reverse Transcription and Replication"

    Article Title: Human Immunodeficiency Virus Type 1 Protease Regulation of Tat Activity Is Essential for Efficient Reverse Transcription and Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.18.9912-9921.2003

    Cleavage of wild-type (WT) and mutant HIV-1 Tat-GFP proteins by PR. (A) For each PR cleavage assay, 35 S-labeled wild-type and mutant Tat-GFP proteins were synthesized in RRL translation reaction mixtures, and equivalent amounts of the Tat-GFP proteins were mixed as indicated with unlabeled wild-type (plus-strand) or mutant (minus-strand) HIV-1 PR made in separate RRL reaction mixtures. Each experiment was repeated three to six times, and the mean result with standard deviation (error bar) is shown. The level of proteolysis was calculated by comparing the ratio of full-length to cleaved wild-type Tat-GFP protein to the ratio of full-length to cleaved mutant protein. The results were analyzed on a Molecular Dynamics PhosphorImager. (B) Virus stocks were prepared from two independent cell lines making HIV-1Δtat virus and stably transcomplemented with wild-type (WT) or mutant Tat-GFP; the parent cell line is shown as Δtat. The efficiency of minus-strand SS DNA synthesis in HIV-1 made in stably transfected cell lines expressing wild-type or mutated tat was determined by NERT-PCR assays. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. At least three independent virus stocks were collected and assayed, and a representative experiment is shown. The relative fluorescence level of Tat-GFP made by each cell line is shown below the graph. NA, not applicable. (C) Virus stocks collected after transient expression of different Tat-GFP plasmids in 293HIVΔtat cells were assayed by NERT-PCR. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. These experiments were performed three times, and a representative experiment is shown. (D) Western blot analysis of infected 293 cells stably expressing Tat-GFP using either anti-GFP monoclonal antibody or a purified pooled human anti-HIV-1 polyclonal antibody as indicated.
    Figure Legend Snippet: Cleavage of wild-type (WT) and mutant HIV-1 Tat-GFP proteins by PR. (A) For each PR cleavage assay, 35 S-labeled wild-type and mutant Tat-GFP proteins were synthesized in RRL translation reaction mixtures, and equivalent amounts of the Tat-GFP proteins were mixed as indicated with unlabeled wild-type (plus-strand) or mutant (minus-strand) HIV-1 PR made in separate RRL reaction mixtures. Each experiment was repeated three to six times, and the mean result with standard deviation (error bar) is shown. The level of proteolysis was calculated by comparing the ratio of full-length to cleaved wild-type Tat-GFP protein to the ratio of full-length to cleaved mutant protein. The results were analyzed on a Molecular Dynamics PhosphorImager. (B) Virus stocks were prepared from two independent cell lines making HIV-1Δtat virus and stably transcomplemented with wild-type (WT) or mutant Tat-GFP; the parent cell line is shown as Δtat. The efficiency of minus-strand SS DNA synthesis in HIV-1 made in stably transfected cell lines expressing wild-type or mutated tat was determined by NERT-PCR assays. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. At least three independent virus stocks were collected and assayed, and a representative experiment is shown. The relative fluorescence level of Tat-GFP made by each cell line is shown below the graph. NA, not applicable. (C) Virus stocks collected after transient expression of different Tat-GFP plasmids in 293HIVΔtat cells were assayed by NERT-PCR. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. These experiments were performed three times, and a representative experiment is shown. (D) Western blot analysis of infected 293 cells stably expressing Tat-GFP using either anti-GFP monoclonal antibody or a purified pooled human anti-HIV-1 polyclonal antibody as indicated.

    Techniques Used: Mutagenesis, Cleavage Assay, Labeling, Synthesized, Standard Deviation, Stable Transfection, DNA Synthesis, Transfection, Expressing, Polymerase Chain Reaction, Fluorescence, Western Blot, Infection, Purification

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    Thermo Fisher rabbit anti gfp
    Immunohistochemistry and indirect immunofluorescence on bronchial cells infected with HRSV. Bronchial cells were infected with either rHRSV A2 EGFP(5), rHRSV A11 EGFP(5), or rHRSV B05 EGFP(5) at a standardized low viral inoculum. Samples were fixed in formalin at 3 days postinfection (DPI) and embedded in paraffin. (A and B) Paraffin-embedded slides were used for H E staining and indirect immunofluorescence using antibodies against green fluorescent protein (HRSV, green), acetylated <t>α-tubulin</t> (cilia, red), and Hoechst (nuclei, blue). Arrows indicate putative mucus channels in the cultures. (C and D) Transwell filters were additionally stained with antibodies against zona-occludens 1 (tight junctions, magenta) and acetylated α-tubulin (cilia, orange). Representative images are shown of rHRSV A11 EGFP(5).
    Rabbit Anti Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Immunohistochemistry and indirect immunofluorescence on bronchial cells infected with HRSV. Bronchial cells were infected with either rHRSV A2 EGFP(5), rHRSV A11 EGFP(5), or rHRSV B05 EGFP(5) at a standardized low viral inoculum. Samples were fixed in formalin at 3 days postinfection (DPI) and embedded in paraffin. (A and B) Paraffin-embedded slides were used for H E staining and indirect immunofluorescence using antibodies against green fluorescent protein (HRSV, green), acetylated α-tubulin (cilia, red), and Hoechst (nuclei, blue). Arrows indicate putative mucus channels in the cultures. (C and D) Transwell filters were additionally stained with antibodies against zona-occludens 1 (tight junctions, magenta) and acetylated α-tubulin (cilia, orange). Representative images are shown of rHRSV A11 EGFP(5).

    Journal: mSphere

    Article Title: Human Respiratory Syncytial Virus Subgroup A and B Infections in Nasal, Bronchial, Small-Airway, and Organoid-Derived Respiratory Cultures

    doi: 10.1128/mSphere.00237-21

    Figure Lengend Snippet: Immunohistochemistry and indirect immunofluorescence on bronchial cells infected with HRSV. Bronchial cells were infected with either rHRSV A2 EGFP(5), rHRSV A11 EGFP(5), or rHRSV B05 EGFP(5) at a standardized low viral inoculum. Samples were fixed in formalin at 3 days postinfection (DPI) and embedded in paraffin. (A and B) Paraffin-embedded slides were used for H E staining and indirect immunofluorescence using antibodies against green fluorescent protein (HRSV, green), acetylated α-tubulin (cilia, red), and Hoechst (nuclei, blue). Arrows indicate putative mucus channels in the cultures. (C and D) Transwell filters were additionally stained with antibodies against zona-occludens 1 (tight junctions, magenta) and acetylated α-tubulin (cilia, orange). Representative images are shown of rHRSV A11 EGFP(5).

    Article Snippet: Primary antibodies (acetylated α-tubulin [Santa Cruz Biotechnology, catalog no. sc-23950 AF488] and rabbit anti-GFP [Invitrogen, catalog no. A11122]) were added for 1 h of incubation at RT and after washing secondary antibodies (acetylated α-tubulin and goat-anti-rabbit [AF594; Invitrogen, catalog no. A11012) were added for 1 h of incubation at RT.

    Techniques: Immunohistochemistry, Immunofluorescence, Infection, Staining

    Immunohistochemistry and indirect immunofluorescence on primary well-differentiated airway organoid cultures at ALI infected with HRSV. Well-differentiated airway organoid cultures at ALI were infected with either rHRSV A2 EGFP(5), rHRSV A11 EGFP(5), or rHRSV B05 EGFP(5) at a standardized low viral inoculum. Samples were fixed in formalin at 3 days postinfection (DPI) and embedded in paraffin. (A and B) Paraffin-embedded slides were used for H E staining and indirect immunofluorescence using antibodies against green fluorescent protein (HRSV, green), acetylated α-tubulin (cilia, red), and Hoechst (nuclei, blue). Arrows indicate putative mucus channels in the cultures. (C and D) Transwell filters were additionally stained with antibodies against zona-occludens 1 (tight junctions, magenta) and acetylated α-tubulin (cilia, orange). Representative images are shown of rRHRSV A11 .

    Journal: mSphere

    Article Title: Human Respiratory Syncytial Virus Subgroup A and B Infections in Nasal, Bronchial, Small-Airway, and Organoid-Derived Respiratory Cultures

    doi: 10.1128/mSphere.00237-21

    Figure Lengend Snippet: Immunohistochemistry and indirect immunofluorescence on primary well-differentiated airway organoid cultures at ALI infected with HRSV. Well-differentiated airway organoid cultures at ALI were infected with either rHRSV A2 EGFP(5), rHRSV A11 EGFP(5), or rHRSV B05 EGFP(5) at a standardized low viral inoculum. Samples were fixed in formalin at 3 days postinfection (DPI) and embedded in paraffin. (A and B) Paraffin-embedded slides were used for H E staining and indirect immunofluorescence using antibodies against green fluorescent protein (HRSV, green), acetylated α-tubulin (cilia, red), and Hoechst (nuclei, blue). Arrows indicate putative mucus channels in the cultures. (C and D) Transwell filters were additionally stained with antibodies against zona-occludens 1 (tight junctions, magenta) and acetylated α-tubulin (cilia, orange). Representative images are shown of rRHRSV A11 .

    Article Snippet: Primary antibodies (acetylated α-tubulin [Santa Cruz Biotechnology, catalog no. sc-23950 AF488] and rabbit anti-GFP [Invitrogen, catalog no. A11122]) were added for 1 h of incubation at RT and after washing secondary antibodies (acetylated α-tubulin and goat-anti-rabbit [AF594; Invitrogen, catalog no. A11012) were added for 1 h of incubation at RT.

    Techniques: Immunohistochemistry, Immunofluorescence, Infection, Staining

    VPS35 colocalises with Tollip and maintains lysosomal integrity, while its loss of function has no effect on Tollip–Parkin interactions SH‐SY5Y cells were transfected with GFP‐Tollip and then treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A (BfnA1) for 2 h prior to fixation and immunostaining. Antibodies specific to GFP (green) and VPS35 (red) were used. Zoom insets represent 4× magnification. Quantification of GFP‐Tollip/VPS35 colocalisation was assessed by Pearson's correlation from two independent experiments. Each data point represents 1 cell, and bar indicates mean value (7 cells/condition from two independent experiments). Statistical significance was determined using a two‐way ANOVA followed by Dunnett's multiple comparison test. Error bars represent SEM. Tollip siRNA knockdown in SH‐SY5Y cells was performed over 72 h, then cells were treated with AO or 25 μM antimycin A (AA) for 2 h, and fixed and immunostained with antibodies specific to VPS35 (green) and TOM20 (red). Colocalisation between VPS35 and TOM20 was not observed. Images were captured at 100× magnification. Zoom insets represent 3.5× magnification. HeLa wild type (WT) or VPS35 KO cells transfected with GFP‐Tollip were treated with 5 μM antimycin A/10 μM oligomycin (AO) for 2 h, then fixed and immunostained with antibodies specific to GFP (green) and LAMP1 (red). HeLa WT or VPS35 KO cells were treated with AO for 2 h and then fixed and immunostained with antibodies specific to Rab7a (green) and LAMP1 (red). Images were captured at 63× magnification. Rab7a/LAMP1 colocalisation in HeLa WT and VPS35 KO was assessed by Pearson's correlation. Each data point represents 1 cell, and bar indicates mean value (6–7 cells/condition from two independent experiments). Statistical significance was determined using a two‐way ANOVA (Sidak). Error bars represent SEM. HeLa VPS35 KO cells stably expressing myc BioID–Tollip (WT) and HA‐Parkin were left untreated or treated with AO or AO/100 nM bafilomycin A (BfnA1) for 6 h in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole‐cell extracts (lysate) and pulldowns (SA pulldown) from each condition were then separated by SDS–PAGE and membranes probed with antibodies specific to the indicated proteins or epitope tags. Data information: Scale bars represent 10 μM. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Tollip coordinates Parkin‐dependent trafficking of mitochondrial‐derived vesicles

    doi: 10.15252/embj.2019102539

    Figure Lengend Snippet: VPS35 colocalises with Tollip and maintains lysosomal integrity, while its loss of function has no effect on Tollip–Parkin interactions SH‐SY5Y cells were transfected with GFP‐Tollip and then treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A (BfnA1) for 2 h prior to fixation and immunostaining. Antibodies specific to GFP (green) and VPS35 (red) were used. Zoom insets represent 4× magnification. Quantification of GFP‐Tollip/VPS35 colocalisation was assessed by Pearson's correlation from two independent experiments. Each data point represents 1 cell, and bar indicates mean value (7 cells/condition from two independent experiments). Statistical significance was determined using a two‐way ANOVA followed by Dunnett's multiple comparison test. Error bars represent SEM. Tollip siRNA knockdown in SH‐SY5Y cells was performed over 72 h, then cells were treated with AO or 25 μM antimycin A (AA) for 2 h, and fixed and immunostained with antibodies specific to VPS35 (green) and TOM20 (red). Colocalisation between VPS35 and TOM20 was not observed. Images were captured at 100× magnification. Zoom insets represent 3.5× magnification. HeLa wild type (WT) or VPS35 KO cells transfected with GFP‐Tollip were treated with 5 μM antimycin A/10 μM oligomycin (AO) for 2 h, then fixed and immunostained with antibodies specific to GFP (green) and LAMP1 (red). HeLa WT or VPS35 KO cells were treated with AO for 2 h and then fixed and immunostained with antibodies specific to Rab7a (green) and LAMP1 (red). Images were captured at 63× magnification. Rab7a/LAMP1 colocalisation in HeLa WT and VPS35 KO was assessed by Pearson's correlation. Each data point represents 1 cell, and bar indicates mean value (6–7 cells/condition from two independent experiments). Statistical significance was determined using a two‐way ANOVA (Sidak). Error bars represent SEM. HeLa VPS35 KO cells stably expressing myc BioID–Tollip (WT) and HA‐Parkin were left untreated or treated with AO or AO/100 nM bafilomycin A (BfnA1) for 6 h in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole‐cell extracts (lysate) and pulldowns (SA pulldown) from each condition were then separated by SDS–PAGE and membranes probed with antibodies specific to the indicated proteins or epitope tags. Data information: Scale bars represent 10 μM. Source data are available online for this figure.

    Article Snippet: Primary antibodies used from immunofluorescence were specific against Tollip (GTX116566, 1:100) from GeneTex or (ATO0918, 1:100) from Insight Biotechnology Ltd; Tom1 (ab99356, 1:100), Parkin (ab77924, 1:250), PDH E2/E3bp (ab110333, 1:1,000), Rab7a (ab137029, 1:100) and GFP (A11122, 1:1,000) from Abcam; LAMP1 (555798, 1:250), Rab5 (610282, 1:200), EEA1 (610456, 1:250) and GM130 (610823, 1:1,000) from BD Biosciences; HA (901501, 1:500) and Cytochrome c (612302, 1:1,000) from BioLegend; MFN2 (7581, 1:500) and HA (37245, 1:1,000) from Cell Signalling; TOM20 (SC‐11415, 1:1,000) and VPS35 (SC‐374372, 1:1,000) from Santa Cruz; GFP (A11122, 1:2,000) from Invitrogen; Myc (9E10, MAB3696‐SP, 1:1,000) from R & D Systems; ubiquitin (FK2, BML‐PW8810‐0100, 1:1,000) from Enzo; and Cathepsin D (IM16, 1:100) from Oncogene.

    Techniques: Transfection, Immunostaining, Stable Transfection, Expressing, SDS Page

    Parkin and Tollip interact on a vesicular compartment TOM20‐positive MDV formation was induced in SH‐SY5Y by siRNA knockdown of Tollip. MDVs were defined as TOM20 +ve /Cytochrome c (Cyt c) −ve via immunofluorescence microscopy, which indicated some colocalisation with Parkin (arrowheads). SH‐SY5Y cells were transfected with GFP‐Tollip for 24 h and then treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A1 (BfnA1) for 2 h prior to fixation and immunostaining. Antibodies specific to GFP (green), Parkin (red) and ubiquitin (blue) were used. GFP‐Tollip/Parkin colocalisation was quantified from cells under steady‐state conditions or treated with AO and AO/BfnA1 by counting overall GFP puncta and GFP puncta positive for Parkin per cell, across 3–6 GFP‐transfected cells/condition, per experiment ( n = 3). Results are represented as a percentage of Tollip puncta positive for Parkin. SH‐SY5Y cells expressing GFP‐Tollip were immunostained for GFP (green), Parkin (red) and Rab7a (blue). Arrowheads indicate areas of colocalisation between all three markers. SH‐SY5Y cells were transfected with GFP‐Tollip R78A or CUE mutant for 24 h, and then treated with AO or AO/BfnA1 for 2 h prior to fixation and immunostaining, as described above for (B). The percentage of GFP‐Tollip puncta positive for Parkin was quantified from immunofluorescence images immunostained for GFP and Parkin from WT, R78A and CUE mutant expressing cells across 3–6 GFP‐transfected cells/condition, per experiment ( n = 3; * P = 0.0162, ** P = 0.0034, **** P

    Journal: The EMBO Journal

    Article Title: Tollip coordinates Parkin‐dependent trafficking of mitochondrial‐derived vesicles

    doi: 10.15252/embj.2019102539

    Figure Lengend Snippet: Parkin and Tollip interact on a vesicular compartment TOM20‐positive MDV formation was induced in SH‐SY5Y by siRNA knockdown of Tollip. MDVs were defined as TOM20 +ve /Cytochrome c (Cyt c) −ve via immunofluorescence microscopy, which indicated some colocalisation with Parkin (arrowheads). SH‐SY5Y cells were transfected with GFP‐Tollip for 24 h and then treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A1 (BfnA1) for 2 h prior to fixation and immunostaining. Antibodies specific to GFP (green), Parkin (red) and ubiquitin (blue) were used. GFP‐Tollip/Parkin colocalisation was quantified from cells under steady‐state conditions or treated with AO and AO/BfnA1 by counting overall GFP puncta and GFP puncta positive for Parkin per cell, across 3–6 GFP‐transfected cells/condition, per experiment ( n = 3). Results are represented as a percentage of Tollip puncta positive for Parkin. SH‐SY5Y cells expressing GFP‐Tollip were immunostained for GFP (green), Parkin (red) and Rab7a (blue). Arrowheads indicate areas of colocalisation between all three markers. SH‐SY5Y cells were transfected with GFP‐Tollip R78A or CUE mutant for 24 h, and then treated with AO or AO/BfnA1 for 2 h prior to fixation and immunostaining, as described above for (B). The percentage of GFP‐Tollip puncta positive for Parkin was quantified from immunofluorescence images immunostained for GFP and Parkin from WT, R78A and CUE mutant expressing cells across 3–6 GFP‐transfected cells/condition, per experiment ( n = 3; * P = 0.0162, ** P = 0.0034, **** P

    Article Snippet: Primary antibodies used from immunofluorescence were specific against Tollip (GTX116566, 1:100) from GeneTex or (ATO0918, 1:100) from Insight Biotechnology Ltd; Tom1 (ab99356, 1:100), Parkin (ab77924, 1:250), PDH E2/E3bp (ab110333, 1:1,000), Rab7a (ab137029, 1:100) and GFP (A11122, 1:1,000) from Abcam; LAMP1 (555798, 1:250), Rab5 (610282, 1:200), EEA1 (610456, 1:250) and GM130 (610823, 1:1,000) from BD Biosciences; HA (901501, 1:500) and Cytochrome c (612302, 1:1,000) from BioLegend; MFN2 (7581, 1:500) and HA (37245, 1:1,000) from Cell Signalling; TOM20 (SC‐11415, 1:1,000) and VPS35 (SC‐374372, 1:1,000) from Santa Cruz; GFP (A11122, 1:2,000) from Invitrogen; Myc (9E10, MAB3696‐SP, 1:1,000) from R & D Systems; ubiquitin (FK2, BML‐PW8810‐0100, 1:1,000) from Enzo; and Cathepsin D (IM16, 1:100) from Oncogene.

    Techniques: Immunofluorescence, Microscopy, Transfection, Immunostaining, Expressing, Mutagenesis

    Tollip translocates to a LAMP 1 compartment during mitochondrial stress Quantitation of Tollip vesicular clusters in SH‐SY5Ys treated with 5 μM antimycin A/10 μM oligomycin (AO) for 2 h. The percentage of cells with vesicular clusters was quantified from 7 to 23 cells for each condition from four independent experiments. Statistical significance was assessed using an unpaired t ‐test. Bars indicate the mean value, and error bars represent SEM. Quantitation of the prevalence of Tollip clusters in HEK293 WT or Tom1 KO cells treated with AO for 2 h. The percentage of cells with vesicular clusters was quantified from an average of 50 cells for each condition from five independent experiments. Statistical significance was determined using a two‐way ANOVA (Sidak). Bars indicate the mean value, and error bars represent SEM. Endogenous Tollip and LAMP1 colocalise in SH‐SY5Y cells treated with AO for 2 h. Antibodies specific to LAMP1 (green) and Tollip (red) were used. Zoom insets represent 2.5× magnification. SH‐SY5Y cells were transfected with either wild‐type GFP‐Tollip (WT), GFP‐Tollip lacking its N‐terminus (▵Nterm) or GFP‐Tollip containing a CUE domain mutation (CUEmut) and then treated with AO for 2 h. Cells were fixed and immunostained with antibodies specific to GFP (green) and LAMP1 (red). Zoom insets represent 2.5× magnification. The extent of colocalisation between GFP‐Tollip and LAMP1 from untreated and AO‐treated SH‐SY5Y cells was quantitated from immunofluorescence images and represented as Pearson's correlation (2–3 cells per experiment, from two independent experiments). Statistical significance was determined using an unpaired t ‐test. Bars indicate the mean, and error bars represent the SEM. The extent of colocalisation between GFP‐Tollip and LAMP1 in Tom1 KO (G), ATG5 KO (H) and VPS35 KO (I) compared with wild‐type (WT) HeLa cells was quantified from immunofluorescence images immunostained for GFP and LAMP1 and represented as Pearson's correlation (2–3 cells per experiment, from three independent experiments). Statistical significance was determined using a two‐way ANOVA (Sidak). Bars indicate the mean, and error bars represent SEM. Western blot analysis of lysates harvested from wild type (WT) alongside either Tom1 KO (G) or VPS35 KO (I) HeLa cells was performed using antibodies specific to the indicated proteins. Data information: Images were captured at 100× magnification. Scale bars represent 10 μm. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Tollip coordinates Parkin‐dependent trafficking of mitochondrial‐derived vesicles

    doi: 10.15252/embj.2019102539

    Figure Lengend Snippet: Tollip translocates to a LAMP 1 compartment during mitochondrial stress Quantitation of Tollip vesicular clusters in SH‐SY5Ys treated with 5 μM antimycin A/10 μM oligomycin (AO) for 2 h. The percentage of cells with vesicular clusters was quantified from 7 to 23 cells for each condition from four independent experiments. Statistical significance was assessed using an unpaired t ‐test. Bars indicate the mean value, and error bars represent SEM. Quantitation of the prevalence of Tollip clusters in HEK293 WT or Tom1 KO cells treated with AO for 2 h. The percentage of cells with vesicular clusters was quantified from an average of 50 cells for each condition from five independent experiments. Statistical significance was determined using a two‐way ANOVA (Sidak). Bars indicate the mean value, and error bars represent SEM. Endogenous Tollip and LAMP1 colocalise in SH‐SY5Y cells treated with AO for 2 h. Antibodies specific to LAMP1 (green) and Tollip (red) were used. Zoom insets represent 2.5× magnification. SH‐SY5Y cells were transfected with either wild‐type GFP‐Tollip (WT), GFP‐Tollip lacking its N‐terminus (▵Nterm) or GFP‐Tollip containing a CUE domain mutation (CUEmut) and then treated with AO for 2 h. Cells were fixed and immunostained with antibodies specific to GFP (green) and LAMP1 (red). Zoom insets represent 2.5× magnification. The extent of colocalisation between GFP‐Tollip and LAMP1 from untreated and AO‐treated SH‐SY5Y cells was quantitated from immunofluorescence images and represented as Pearson's correlation (2–3 cells per experiment, from two independent experiments). Statistical significance was determined using an unpaired t ‐test. Bars indicate the mean, and error bars represent the SEM. The extent of colocalisation between GFP‐Tollip and LAMP1 in Tom1 KO (G), ATG5 KO (H) and VPS35 KO (I) compared with wild‐type (WT) HeLa cells was quantified from immunofluorescence images immunostained for GFP and LAMP1 and represented as Pearson's correlation (2–3 cells per experiment, from three independent experiments). Statistical significance was determined using a two‐way ANOVA (Sidak). Bars indicate the mean, and error bars represent SEM. Western blot analysis of lysates harvested from wild type (WT) alongside either Tom1 KO (G) or VPS35 KO (I) HeLa cells was performed using antibodies specific to the indicated proteins. Data information: Images were captured at 100× magnification. Scale bars represent 10 μm. Source data are available online for this figure.

    Article Snippet: Primary antibodies used from immunofluorescence were specific against Tollip (GTX116566, 1:100) from GeneTex or (ATO0918, 1:100) from Insight Biotechnology Ltd; Tom1 (ab99356, 1:100), Parkin (ab77924, 1:250), PDH E2/E3bp (ab110333, 1:1,000), Rab7a (ab137029, 1:100) and GFP (A11122, 1:1,000) from Abcam; LAMP1 (555798, 1:250), Rab5 (610282, 1:200), EEA1 (610456, 1:250) and GM130 (610823, 1:1,000) from BD Biosciences; HA (901501, 1:500) and Cytochrome c (612302, 1:1,000) from BioLegend; MFN2 (7581, 1:500) and HA (37245, 1:1,000) from Cell Signalling; TOM20 (SC‐11415, 1:1,000) and VPS35 (SC‐374372, 1:1,000) from Santa Cruz; GFP (A11122, 1:2,000) from Invitrogen; Myc (9E10, MAB3696‐SP, 1:1,000) from R & D Systems; ubiquitin (FK2, BML‐PW8810‐0100, 1:1,000) from Enzo; and Cathepsin D (IM16, 1:100) from Oncogene.

    Techniques: Quantitation Assay, Transfection, Mutagenesis, Immunofluorescence, Western Blot

    Parkin and Tollip are both associated with mitochondrial quality control SH‐SY5Y cells were seeded on glass coverslips for 24 h and then treated with 5 μM antimycin/10 μM oligomycin for 2 or 6 h prior to fixation. Cells were immunostained with antibodies specific to LAMP1 (green) to define late endosomes/lysosomes, Parkin (red) and TOM20 (blue) to define mitochondria. Images were captured by widefield immunofluorescence microscopy at 63× magnification. Zoom insets represent 3.5× magnification. HEK293 WT and Tollip KO cells were immunostained for PDH (green) and TOM20 (red). Nuclei were labelled with Hoechst (blue). A number of TOM20 +ve /PDH −ve MDVs were observed in Tollip KO cells (arrowheads). Images were captured at 100× magnification. Zoom insets represent 5× magnification. Western blot analysis of lysates harvested from HEK293 parental and Tollip KO cells, immunoblotted with antibodies specific to Tollip and actin. HeLa cells expressing HA‐Parkin were transfected with GFP‐Tollip 24 h prior to treatment with 5 μM antimycin/10 μM oligomycin (AO) or 25 μM antimycin A (AA) for 2 h. Cells were fixed and stained for GFP (green), PDH (red) and TOM20 (blue). GFP‐Tollip was observed to localise to a small subset of TOM20‐positive MDVs (arrowheads). Images were captured at 100× magnification. Zoom insets represent 4× magnification. Data information: Scale bars represent 10 μm. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Tollip coordinates Parkin‐dependent trafficking of mitochondrial‐derived vesicles

    doi: 10.15252/embj.2019102539

    Figure Lengend Snippet: Parkin and Tollip are both associated with mitochondrial quality control SH‐SY5Y cells were seeded on glass coverslips for 24 h and then treated with 5 μM antimycin/10 μM oligomycin for 2 or 6 h prior to fixation. Cells were immunostained with antibodies specific to LAMP1 (green) to define late endosomes/lysosomes, Parkin (red) and TOM20 (blue) to define mitochondria. Images were captured by widefield immunofluorescence microscopy at 63× magnification. Zoom insets represent 3.5× magnification. HEK293 WT and Tollip KO cells were immunostained for PDH (green) and TOM20 (red). Nuclei were labelled with Hoechst (blue). A number of TOM20 +ve /PDH −ve MDVs were observed in Tollip KO cells (arrowheads). Images were captured at 100× magnification. Zoom insets represent 5× magnification. Western blot analysis of lysates harvested from HEK293 parental and Tollip KO cells, immunoblotted with antibodies specific to Tollip and actin. HeLa cells expressing HA‐Parkin were transfected with GFP‐Tollip 24 h prior to treatment with 5 μM antimycin/10 μM oligomycin (AO) or 25 μM antimycin A (AA) for 2 h. Cells were fixed and stained for GFP (green), PDH (red) and TOM20 (blue). GFP‐Tollip was observed to localise to a small subset of TOM20‐positive MDVs (arrowheads). Images were captured at 100× magnification. Zoom insets represent 4× magnification. Data information: Scale bars represent 10 μm. Source data are available online for this figure.

    Article Snippet: Primary antibodies used from immunofluorescence were specific against Tollip (GTX116566, 1:100) from GeneTex or (ATO0918, 1:100) from Insight Biotechnology Ltd; Tom1 (ab99356, 1:100), Parkin (ab77924, 1:250), PDH E2/E3bp (ab110333, 1:1,000), Rab7a (ab137029, 1:100) and GFP (A11122, 1:1,000) from Abcam; LAMP1 (555798, 1:250), Rab5 (610282, 1:200), EEA1 (610456, 1:250) and GM130 (610823, 1:1,000) from BD Biosciences; HA (901501, 1:500) and Cytochrome c (612302, 1:1,000) from BioLegend; MFN2 (7581, 1:500) and HA (37245, 1:1,000) from Cell Signalling; TOM20 (SC‐11415, 1:1,000) and VPS35 (SC‐374372, 1:1,000) from Santa Cruz; GFP (A11122, 1:2,000) from Invitrogen; Myc (9E10, MAB3696‐SP, 1:1,000) from R & D Systems; ubiquitin (FK2, BML‐PW8810‐0100, 1:1,000) from Enzo; and Cathepsin D (IM16, 1:100) from Oncogene.

    Techniques: Immunofluorescence, Microscopy, Western Blot, Expressing, Transfection, Staining

    Tollip mediates the trafficking of TOM 20 +ve MDV s siRNA knockdown of Tollip in SH‐SY5Y cells was performed over 72 h, with cells reverse‐transfected with siRNA oligos specific for Tollip (or mock) for 24 h before a second transfection was performed for a further 48 h. Knockdown was confirmed by Western blotting. After knockdown of Tollip, SH‐SY5Y cells were treated with 5 μM antimycin A/10 μM oligomycin (AO) or 25 μM antimycin A (AA) alone for 2 h, then fixed and stained for PDH E2/E3 bp (green), TOM20 (red) and nuclei (blue). Images were captured using widefield immunofluorescence microscopy. Zoom insets represent 4× magnification, and arrowheads indicate TOM20‐positive MDVs. The number of TOM20 +ve /PDH −ve MDVs (C) or PDH +ve /TOM20 −ve MDVs (D) per cell was quantified from immunofluorescence images captured ( n = 30 cells per condition from two independent experiments). Statistical significance was determined using a two‐way ANOVA (Tukey). Centre line indicates the mean, and error bars represent SEM. SH‐SY5Y cells were transfected with GFP‐labelled Tollip for 24 h and then treated with AO, AO/100 μM bafilomycin A1 (AO/BfnA1) or AA alone for 2 h prior to fixation. Cells were then immunostained for GFP (green), PDH E2/E3 bp (red) and TOM20 (blue) and imaged by widefield immunofluorescence microscopy. GFP‐Tollip was observed on TOM20 +ve /PDH −ve MDVs (arrowheads). Zoom insets represent 7× magnification. Images captured using super‐resolution radial fluctuation (SRRF) microscopy showing both TOM20 (arrowheads) and PDH (arrows) MDVs in SH‐SY5Y cells after Tollip siRNA knockdown. Left side images are the first in each of the original widefield image stacks. Scale bar for zoom images is 0.5 μm. Data information: Scale bars are 10 μm, unless otherwise stated. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Tollip coordinates Parkin‐dependent trafficking of mitochondrial‐derived vesicles

    doi: 10.15252/embj.2019102539

    Figure Lengend Snippet: Tollip mediates the trafficking of TOM 20 +ve MDV s siRNA knockdown of Tollip in SH‐SY5Y cells was performed over 72 h, with cells reverse‐transfected with siRNA oligos specific for Tollip (or mock) for 24 h before a second transfection was performed for a further 48 h. Knockdown was confirmed by Western blotting. After knockdown of Tollip, SH‐SY5Y cells were treated with 5 μM antimycin A/10 μM oligomycin (AO) or 25 μM antimycin A (AA) alone for 2 h, then fixed and stained for PDH E2/E3 bp (green), TOM20 (red) and nuclei (blue). Images were captured using widefield immunofluorescence microscopy. Zoom insets represent 4× magnification, and arrowheads indicate TOM20‐positive MDVs. The number of TOM20 +ve /PDH −ve MDVs (C) or PDH +ve /TOM20 −ve MDVs (D) per cell was quantified from immunofluorescence images captured ( n = 30 cells per condition from two independent experiments). Statistical significance was determined using a two‐way ANOVA (Tukey). Centre line indicates the mean, and error bars represent SEM. SH‐SY5Y cells were transfected with GFP‐labelled Tollip for 24 h and then treated with AO, AO/100 μM bafilomycin A1 (AO/BfnA1) or AA alone for 2 h prior to fixation. Cells were then immunostained for GFP (green), PDH E2/E3 bp (red) and TOM20 (blue) and imaged by widefield immunofluorescence microscopy. GFP‐Tollip was observed on TOM20 +ve /PDH −ve MDVs (arrowheads). Zoom insets represent 7× magnification. Images captured using super‐resolution radial fluctuation (SRRF) microscopy showing both TOM20 (arrowheads) and PDH (arrows) MDVs in SH‐SY5Y cells after Tollip siRNA knockdown. Left side images are the first in each of the original widefield image stacks. Scale bar for zoom images is 0.5 μm. Data information: Scale bars are 10 μm, unless otherwise stated. Source data are available online for this figure.

    Article Snippet: Primary antibodies used from immunofluorescence were specific against Tollip (GTX116566, 1:100) from GeneTex or (ATO0918, 1:100) from Insight Biotechnology Ltd; Tom1 (ab99356, 1:100), Parkin (ab77924, 1:250), PDH E2/E3bp (ab110333, 1:1,000), Rab7a (ab137029, 1:100) and GFP (A11122, 1:1,000) from Abcam; LAMP1 (555798, 1:250), Rab5 (610282, 1:200), EEA1 (610456, 1:250) and GM130 (610823, 1:1,000) from BD Biosciences; HA (901501, 1:500) and Cytochrome c (612302, 1:1,000) from BioLegend; MFN2 (7581, 1:500) and HA (37245, 1:1,000) from Cell Signalling; TOM20 (SC‐11415, 1:1,000) and VPS35 (SC‐374372, 1:1,000) from Santa Cruz; GFP (A11122, 1:2,000) from Invitrogen; Myc (9E10, MAB3696‐SP, 1:1,000) from R & D Systems; ubiquitin (FK2, BML‐PW8810‐0100, 1:1,000) from Enzo; and Cathepsin D (IM16, 1:100) from Oncogene.

    Techniques: Transfection, Western Blot, Staining, Immunofluorescence, Microscopy

    Trafficking of MDV s to the lysosome is dependent on Tollip A subset of TOM20 +ve /PDH −ve MDVs are trafficked to the lysosome. siRNA knockdown of Tollip was conducted in SH‐SY5Y cells over a 72‐h period prior to treatment with 5 μM antimycin A/10 μM oligomycin (AO) or 25 μM antimycin A (AA) for 2 h. Cells were then fixed and stained with antibodies specific to LAMP1 (green), PDH E2/E3 bp (red) and TOM20 (blue). Arrows denote TOM20 +ve /PDH −ve MDVs colocalising with LAMP1, whereas arrowheads denote MDVs that do not colocalise with LAMP1. Zoom insets represent 4× magnification. From immunofluorescence images, the total number of TOM20 +ve /PDH −ve MDVs was counted as well as the number of TOM20 MDVs that colocalised with LAMP1 to calculate the % of TOM20‐positive MDVs colocalising with LAMP1 per cell ( n = 25–32 cells per condition from three independent experiments). Each data point represents 1 cell, and centre line indicates the mean. Statistical significance was determined using a two‐way ANOVA (Dunnett). Error bars represent the SEM. siRNA knockdown was performed in SH‐SY5Y cells, and then 18 h prior to fixation, cells were transfected with GFP‐Tollip wild type (WT), ▵Nterm, CUEmut or R78A. Cells were fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). Arrowheads denote TOM20 +ve /PDH −ve MDVs. Zoom insets represent 3.5× magnification. Quantification of TOM20 MDVs per cell was performed by eye ( n = 11–14 cells per mutant from two independent experiments). Cells expressing a GFP‐Tollip construct were identified using the GFP channel. Surrounding cells not expressing GFP were counted as the Tollip siRNA only population. Statistical significance was determined using a one‐way ANOVA (Tukey). Each data point represents 1 cell, centre line indicates the mean, and error bars represent SEM. Data information: Images were captured at 100× magnification. Scale bars represent 10 μm.

    Journal: The EMBO Journal

    Article Title: Tollip coordinates Parkin‐dependent trafficking of mitochondrial‐derived vesicles

    doi: 10.15252/embj.2019102539

    Figure Lengend Snippet: Trafficking of MDV s to the lysosome is dependent on Tollip A subset of TOM20 +ve /PDH −ve MDVs are trafficked to the lysosome. siRNA knockdown of Tollip was conducted in SH‐SY5Y cells over a 72‐h period prior to treatment with 5 μM antimycin A/10 μM oligomycin (AO) or 25 μM antimycin A (AA) for 2 h. Cells were then fixed and stained with antibodies specific to LAMP1 (green), PDH E2/E3 bp (red) and TOM20 (blue). Arrows denote TOM20 +ve /PDH −ve MDVs colocalising with LAMP1, whereas arrowheads denote MDVs that do not colocalise with LAMP1. Zoom insets represent 4× magnification. From immunofluorescence images, the total number of TOM20 +ve /PDH −ve MDVs was counted as well as the number of TOM20 MDVs that colocalised with LAMP1 to calculate the % of TOM20‐positive MDVs colocalising with LAMP1 per cell ( n = 25–32 cells per condition from three independent experiments). Each data point represents 1 cell, and centre line indicates the mean. Statistical significance was determined using a two‐way ANOVA (Dunnett). Error bars represent the SEM. siRNA knockdown was performed in SH‐SY5Y cells, and then 18 h prior to fixation, cells were transfected with GFP‐Tollip wild type (WT), ▵Nterm, CUEmut or R78A. Cells were fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). Arrowheads denote TOM20 +ve /PDH −ve MDVs. Zoom insets represent 3.5× magnification. Quantification of TOM20 MDVs per cell was performed by eye ( n = 11–14 cells per mutant from two independent experiments). Cells expressing a GFP‐Tollip construct were identified using the GFP channel. Surrounding cells not expressing GFP were counted as the Tollip siRNA only population. Statistical significance was determined using a one‐way ANOVA (Tukey). Each data point represents 1 cell, centre line indicates the mean, and error bars represent SEM. Data information: Images were captured at 100× magnification. Scale bars represent 10 μm.

    Article Snippet: Primary antibodies used from immunofluorescence were specific against Tollip (GTX116566, 1:100) from GeneTex or (ATO0918, 1:100) from Insight Biotechnology Ltd; Tom1 (ab99356, 1:100), Parkin (ab77924, 1:250), PDH E2/E3bp (ab110333, 1:1,000), Rab7a (ab137029, 1:100) and GFP (A11122, 1:1,000) from Abcam; LAMP1 (555798, 1:250), Rab5 (610282, 1:200), EEA1 (610456, 1:250) and GM130 (610823, 1:1,000) from BD Biosciences; HA (901501, 1:500) and Cytochrome c (612302, 1:1,000) from BioLegend; MFN2 (7581, 1:500) and HA (37245, 1:1,000) from Cell Signalling; TOM20 (SC‐11415, 1:1,000) and VPS35 (SC‐374372, 1:1,000) from Santa Cruz; GFP (A11122, 1:2,000) from Invitrogen; Myc (9E10, MAB3696‐SP, 1:1,000) from R & D Systems; ubiquitin (FK2, BML‐PW8810‐0100, 1:1,000) from Enzo; and Cathepsin D (IM16, 1:100) from Oncogene.

    Techniques: Staining, Immunofluorescence, Transfection, Mutagenesis, Expressing, Construct

    Tollip facilitates endosomal “capture” of MDV s siRNA knockdown of Tollip or Parkin in SH‐SY5Y cells was performed over a 72‐h period, and then 18 h prior to treatment, cells were transfected with GFP‐2xFYVE to label endosomal membranes. Cells were treated with 25 μM antimycin A (AA) for 2 h, then fixed and immunostained with antibodies specific to PDH E2/E3 bp (red) and TOM20 (blue). Arrowheads denote TOM20 +ve /PDH −ve MDVs that colocalise with GFP‐2xFYVE, and arrows denote TOM20 +ve /PDH −ve MDVs that do not. Zoom insets represent 4.5× magnification. Quantification of TOM20 +ve /PDH −ve MDVs that are positive for GFP‐2xFYVE is shown as a % of TOM20 MDVs positive for GFP, with each data point indicating 1 cell. MDV colocalisation was quantified by eye from images captured ( n = 20–22 cells per condition from three independent experiments). Statistical significance was determined using a two‐way ANOVA (Dunnett). Centre line indicates the mean, and error bars represent SEM. SH‐SY5Y cells were left untreated or treated with 25 μM AA for 2 h prior to fixation and immunostaining for EEA1 (green), PDH E2/E3 bp (red) and TOM20 (blue). Arrowheads indicate TOM20 +ve /PDH −ve MDVs positive for the early endosome marker EEA1. Zoom insets represent 5× magnification. siRNA knockdown of Parkin was performed in SH‐SY5Y cells followed by 25 μM AA for 2 h prior to fixation and immunostaining with antibodies specific to TOM20 (green), Tollip (red) and Cytochrome c (blue). We observed Tollip colocalisation with TOM20 +ve /PDH −ve MDVs (denoted by arrowheads), which was still maintained following Parkin siRNA knockdown. Zoom insets represent 5× magnification. SH‐SY5Y cells were transfected with GFP‐Tollip and treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A (BfnA1) for 2 h prior to fixation and immunostaining with antibodies specific to GFP (green), Rab7a (red) and PDH E2/E3 bp (blue). Zoom insets represent 4× magnification. Quantification of GFP‐Tollip/Rab7a colocalisation using Pearson's correlation. Each data point represents 1 cell (12–14 cells/condition from three independent experiments), and bar indicates the mean. Statistical significance was assessed using a one‐way ANOVA (Dunnett). Error bars represent SEM. Data information: Scale bars represent 10 μm.

    Journal: The EMBO Journal

    Article Title: Tollip coordinates Parkin‐dependent trafficking of mitochondrial‐derived vesicles

    doi: 10.15252/embj.2019102539

    Figure Lengend Snippet: Tollip facilitates endosomal “capture” of MDV s siRNA knockdown of Tollip or Parkin in SH‐SY5Y cells was performed over a 72‐h period, and then 18 h prior to treatment, cells were transfected with GFP‐2xFYVE to label endosomal membranes. Cells were treated with 25 μM antimycin A (AA) for 2 h, then fixed and immunostained with antibodies specific to PDH E2/E3 bp (red) and TOM20 (blue). Arrowheads denote TOM20 +ve /PDH −ve MDVs that colocalise with GFP‐2xFYVE, and arrows denote TOM20 +ve /PDH −ve MDVs that do not. Zoom insets represent 4.5× magnification. Quantification of TOM20 +ve /PDH −ve MDVs that are positive for GFP‐2xFYVE is shown as a % of TOM20 MDVs positive for GFP, with each data point indicating 1 cell. MDV colocalisation was quantified by eye from images captured ( n = 20–22 cells per condition from three independent experiments). Statistical significance was determined using a two‐way ANOVA (Dunnett). Centre line indicates the mean, and error bars represent SEM. SH‐SY5Y cells were left untreated or treated with 25 μM AA for 2 h prior to fixation and immunostaining for EEA1 (green), PDH E2/E3 bp (red) and TOM20 (blue). Arrowheads indicate TOM20 +ve /PDH −ve MDVs positive for the early endosome marker EEA1. Zoom insets represent 5× magnification. siRNA knockdown of Parkin was performed in SH‐SY5Y cells followed by 25 μM AA for 2 h prior to fixation and immunostaining with antibodies specific to TOM20 (green), Tollip (red) and Cytochrome c (blue). We observed Tollip colocalisation with TOM20 +ve /PDH −ve MDVs (denoted by arrowheads), which was still maintained following Parkin siRNA knockdown. Zoom insets represent 5× magnification. SH‐SY5Y cells were transfected with GFP‐Tollip and treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A (BfnA1) for 2 h prior to fixation and immunostaining with antibodies specific to GFP (green), Rab7a (red) and PDH E2/E3 bp (blue). Zoom insets represent 4× magnification. Quantification of GFP‐Tollip/Rab7a colocalisation using Pearson's correlation. Each data point represents 1 cell (12–14 cells/condition from three independent experiments), and bar indicates the mean. Statistical significance was assessed using a one‐way ANOVA (Dunnett). Error bars represent SEM. Data information: Scale bars represent 10 μm.

    Article Snippet: Primary antibodies used from immunofluorescence were specific against Tollip (GTX116566, 1:100) from GeneTex or (ATO0918, 1:100) from Insight Biotechnology Ltd; Tom1 (ab99356, 1:100), Parkin (ab77924, 1:250), PDH E2/E3bp (ab110333, 1:1,000), Rab7a (ab137029, 1:100) and GFP (A11122, 1:1,000) from Abcam; LAMP1 (555798, 1:250), Rab5 (610282, 1:200), EEA1 (610456, 1:250) and GM130 (610823, 1:1,000) from BD Biosciences; HA (901501, 1:500) and Cytochrome c (612302, 1:1,000) from BioLegend; MFN2 (7581, 1:500) and HA (37245, 1:1,000) from Cell Signalling; TOM20 (SC‐11415, 1:1,000) and VPS35 (SC‐374372, 1:1,000) from Santa Cruz; GFP (A11122, 1:2,000) from Invitrogen; Myc (9E10, MAB3696‐SP, 1:1,000) from R & D Systems; ubiquitin (FK2, BML‐PW8810‐0100, 1:1,000) from Enzo; and Cathepsin D (IM16, 1:100) from Oncogene.

    Techniques: Transfection, Immunostaining, Marker

    Ndfip1 and Ndfip2 Expression in the Developing Spinal Cord (A and B) mRNA in situ hybridization reveals clear expression of Ndfip1(A) and Ndfip2 (B) in E10.5 and E11.5 mouse spinal cord. mRNA probes to the sense strand serve as controls for the specificity of Ndfip1 (A) and Ndfip2 (B) expression. Yellow arrows in the E11.5 images show expression in regions of dorsal commissural axon cell bodies. (C) Representative confocal images of transverse sections of wild-type mouse spinal cord from E10.5 to E12.5 labeled with anti-Ndfip1 antibody. Ndfip1 is expressed at the floor plate, in the motor column, and in DRGs. (D) Anti-GFP immunostaining of E10, E10.5, and E11.5 of embryos reveals the pattern of Ndfip2 expression. Embryos are heterozygous for an allele of Ndfip2 where the coding sequence has been replaced by a GFP reporter. Commissural axons are clearly labeled by E11.5. (E–G) Higher magnification images of E10.5 and E11.5 spinal cord sections illustrate co-labeling of Ndfip1 and TAG1 (E and F) or Robo1 (G) in the ventral commissure. Co-localization of Ndfip1 with TAG1-positive commissural axons demonstrates the commissural axonal expression of Ndfip1. (H–J) Higher magnification of anti-GFP immunostaining of E10.5 and E11.5 of Ndfip2-GFP heterozygous embryos reveals co-labeling of Ndfip2 and TAG1 (H and I) or DCC (J) in the ventral commissure. (K and L) Double immunostaining of Ndfip1 (K and L, green) and DCC (K, red) or TAG1 (L, red) in dissociated commissural neurons showing the expression of Ndfip1 in the cell body, axon and growth cone of commissural neurons. Scale bars represent 50 μm in (A)–(D), 20 μm in (E)–(J), and 10 μm in (K) and (L).

    Journal: Cell reports

    Article Title: Ndfip Proteins Target Robo Receptors for Degradation and Allow Commissural Axons to Cross the Midline in the Developing Spinal Cord

    doi: 10.1016/j.celrep.2019.02.080

    Figure Lengend Snippet: Ndfip1 and Ndfip2 Expression in the Developing Spinal Cord (A and B) mRNA in situ hybridization reveals clear expression of Ndfip1(A) and Ndfip2 (B) in E10.5 and E11.5 mouse spinal cord. mRNA probes to the sense strand serve as controls for the specificity of Ndfip1 (A) and Ndfip2 (B) expression. Yellow arrows in the E11.5 images show expression in regions of dorsal commissural axon cell bodies. (C) Representative confocal images of transverse sections of wild-type mouse spinal cord from E10.5 to E12.5 labeled with anti-Ndfip1 antibody. Ndfip1 is expressed at the floor plate, in the motor column, and in DRGs. (D) Anti-GFP immunostaining of E10, E10.5, and E11.5 of embryos reveals the pattern of Ndfip2 expression. Embryos are heterozygous for an allele of Ndfip2 where the coding sequence has been replaced by a GFP reporter. Commissural axons are clearly labeled by E11.5. (E–G) Higher magnification images of E10.5 and E11.5 spinal cord sections illustrate co-labeling of Ndfip1 and TAG1 (E and F) or Robo1 (G) in the ventral commissure. Co-localization of Ndfip1 with TAG1-positive commissural axons demonstrates the commissural axonal expression of Ndfip1. (H–J) Higher magnification of anti-GFP immunostaining of E10.5 and E11.5 of Ndfip2-GFP heterozygous embryos reveals co-labeling of Ndfip2 and TAG1 (H and I) or DCC (J) in the ventral commissure. (K and L) Double immunostaining of Ndfip1 (K and L, green) and DCC (K, red) or TAG1 (L, red) in dissociated commissural neurons showing the expression of Ndfip1 in the cell body, axon and growth cone of commissural neurons. Scale bars represent 50 μm in (A)–(D), 20 μm in (E)–(J), and 10 μm in (K) and (L).

    Article Snippet: Antibodies used: rabbit anti-Ndfip1 (1:100, Sigma #HPA009682), mouse anti-TAG1 (1:100, DSHB #4D7), goat anti-Robo3 (1:200, R & D systems #AF3076), goat anti-Robo1 (1:200, R & D systems #AF1749), rabbit anti-GFP (1:1000, Invitrogen #A11122), rat anti-L1CAM (1:300, Millipore #MAB5272), Alexa488 goat anti-rabbit (Invitrogen, 1:500 #A11034), Cy3 goat anti-mouse (1:1000, Jackson Immunoresearch # 115-165-003), Cy3 donkey anti-goat (1:400, Jackson Immunoresearch #705-165-003), and Alexa633 goat anti-Rat (1:500, Invitrogen #A-21094).

    Techniques: Expressing, In Situ Hybridization, Labeling, Immunostaining, Sequencing, Droplet Countercurrent Chromatography, Double Immunostaining

    Integrin β3 modulates SMC transdifferentiation. a – c Mice were fed a HFD for 6 or 16 weeks as indicated, and then transverse aortic root sections were stained. In a , b sections from ApoE (−/−) , SMMHC - CreER T2 , ROSA26R ( mTmG /+) mice were stained for SMA, GFP (fate marker), nuclei (DAPI), and either integrin β3 ( a ) or CD36 ( b ). Dashed yellow lines separate cap from core ( a , b ) and core from media ( b ). n = 5. In c sections from ApoE (−/−) mice that were also wild type or null for Itgb3 were stained for SMMHC, CD68, and nuclei (DAPI). n = 3. Boxed regions ( a , c ) are shown as close-ups on right; in c CD68 + SMMHC + cells in the media (arrowheads) and plaque (arrows) of the Itgb3 null atherosclerotic aorta are indicated. Med, tunica media; Lu, lumen; Pl, plaque. Scale bars, 25 μm. d – h Aortic SMCs were isolated from ApoE (−/−) mice and then subjected to siRNA-mediated knockdown with si-Itgb3 vs. scrambled (Scr; d – f ) or with si-Itgb3, si-Tlr4 vs. si-Itgb3 ( g , h ). Levels of indicated transcripts from qRT-PCR are relative to Gapdh and normalized to either Scr in d , f or to si-Itgb3 in g , h . For d , g , n = 4–5 in duplicate. In e silenced SMCs were cultured with DiI-conjugated ox-LDL for 10 h and stained with DAPI; n = 5. In f , h silenced SMCs were exposed to soluble cholesterol:methyl-β-cyclodextrin complexes for 3 days, and then mRNA levels were assessed; n = 4–7 in duplicate. * , ** , *** , ^ , **** vs. control (Scr in d , f and si-Itgb3 in g , h ), p

    Journal: Nature Communications

    Article Title: Integrin beta3 regulates clonality and fate of smooth muscle-derived atherosclerotic plaque cells

    doi: 10.1038/s41467-018-04447-7

    Figure Lengend Snippet: Integrin β3 modulates SMC transdifferentiation. a – c Mice were fed a HFD for 6 or 16 weeks as indicated, and then transverse aortic root sections were stained. In a , b sections from ApoE (−/−) , SMMHC - CreER T2 , ROSA26R ( mTmG /+) mice were stained for SMA, GFP (fate marker), nuclei (DAPI), and either integrin β3 ( a ) or CD36 ( b ). Dashed yellow lines separate cap from core ( a , b ) and core from media ( b ). n = 5. In c sections from ApoE (−/−) mice that were also wild type or null for Itgb3 were stained for SMMHC, CD68, and nuclei (DAPI). n = 3. Boxed regions ( a , c ) are shown as close-ups on right; in c CD68 + SMMHC + cells in the media (arrowheads) and plaque (arrows) of the Itgb3 null atherosclerotic aorta are indicated. Med, tunica media; Lu, lumen; Pl, plaque. Scale bars, 25 μm. d – h Aortic SMCs were isolated from ApoE (−/−) mice and then subjected to siRNA-mediated knockdown with si-Itgb3 vs. scrambled (Scr; d – f ) or with si-Itgb3, si-Tlr4 vs. si-Itgb3 ( g , h ). Levels of indicated transcripts from qRT-PCR are relative to Gapdh and normalized to either Scr in d , f or to si-Itgb3 in g , h . For d , g , n = 4–5 in duplicate. In e silenced SMCs were cultured with DiI-conjugated ox-LDL for 10 h and stained with DAPI; n = 5. In f , h silenced SMCs were exposed to soluble cholesterol:methyl-β-cyclodextrin complexes for 3 days, and then mRNA levels were assessed; n = 4–7 in duplicate. * , ** , *** , ^ , **** vs. control (Scr in d , f and si-Itgb3 in g , h ), p

    Article Snippet: On the next day, sections were washed with PBS-T and then incubated with secondary antibodies for 1 h. Primary antibodies used were anti-CD31 (1:500, BD Pharmingen, 553370), anti-SMMHC (1:100, Biomedical Technologies, J64817), anti-GFP (1:500, Invitrogen, A11122; 1:100, Abcam, ab13970), anti-pH3 (1:200, Millipore, 06–570), anti-Ki67 (1:100, Vector Labs, VP-RM04), anti-integrin β3 (1:200, Abcam, ab75872 and ab197662), anti-CD68 (1:200, Bio-Rad, MCA1957), anti-CD36 (1:100, Novus Biologicals, NB400-144), directly conjugated Cy3 anti-SMA (1:500, Sigma-Aldrich, C6198), anti-PDGFR-β (1:100, Abcam, ab32570), and biotinylated anti-PDGFR-β (1:50, R & D, AF1042).

    Techniques: Mouse Assay, Staining, Marker, Isolation, Quantitative RT-PCR, Cell Culture

    Aortic SMCs are polyclonal and highly migratory in development. a Cells of ROSA26R ( CreER / Rb ) embryo induced with high dose 4-OH-T at E5.25 are expected to be of multiple colors at E5.5–7.25. If the aortic wall derives from a single cell (or instead from multiple polyclonal cells) present at ~E7.25 or thereafter, wall cells will be a single color (or multiple colors). b – g Sections of ROSA26R ( CreER / Rb ) mice in transverse ( b – e , g ) or longitudinal axis ( f ) imaged with direct fluorescence of Rb channels. At E5.25, dams were induced with a single high 4-OH-T dose in b , c (150 µg) or, to label clonally, at a limiting dose in d , d′ (100 µg; Supplementary Table 1 , clone ID1-1), e (20 µg; ID1-14), f (20 µg; ID1-18), and g (50 µg; ID1-10). Arrowheads indicate mOrange + cells ( d , e ), with marked cells in d suggesting ventral migration of aortic progenitors. By the time of recombination, progenitors of aorta and lung mesenchyme ( d , d ′ ) or epithelium ( g , g′ ) are differentially specified ( h ) (Supplementary Table 1 ). i , j Transverse sections of X GFP X + aorta stained for GFP at E14.5 and in the adult (5 weeks), representative of n = 4 embryos and 6 adults. k , l SMMHC - CreER T2 embryos also carrying ROSA26R ( mTmG /+) or ROSA26R ( Rb /+) induced with tamoxifen 0.5 mg at E9.5 (when there is one SMC layer; Supplementary Fig. 1b, c ) and analyzed at E16.5. In k transverse aortic section (left panel, section #30) of a 21-cell GFP-marked clone (Supplementary Table 2 , clone ID2-12; Supplementary Fig. 3 ) after GFP staining. The middle panel shows positions of two marked cells in this section, and columnar clone schematic (right panel) is a compilation of transverse sections, among 60 consecutive sections analyzed, that contain marked cells. The first and 60th sections are included for reference. A 15-cell clone (ID2-10) is shown in l with the section stained for DAPI and imaged for Rb colors. The clones in k , l expanded radially, circumferentially, and longitudinally. Lu, aortic lumen; E, endothelial layer; 1–5, smooth muscle layers; A, adventitial layer. Scale bars, 100 µm ( b , d , e , g , i ) and 10 µm ( c , f , j – l )

    Journal: Nature Communications

    Article Title: Integrin beta3 regulates clonality and fate of smooth muscle-derived atherosclerotic plaque cells

    doi: 10.1038/s41467-018-04447-7

    Figure Lengend Snippet: Aortic SMCs are polyclonal and highly migratory in development. a Cells of ROSA26R ( CreER / Rb ) embryo induced with high dose 4-OH-T at E5.25 are expected to be of multiple colors at E5.5–7.25. If the aortic wall derives from a single cell (or instead from multiple polyclonal cells) present at ~E7.25 or thereafter, wall cells will be a single color (or multiple colors). b – g Sections of ROSA26R ( CreER / Rb ) mice in transverse ( b – e , g ) or longitudinal axis ( f ) imaged with direct fluorescence of Rb channels. At E5.25, dams were induced with a single high 4-OH-T dose in b , c (150 µg) or, to label clonally, at a limiting dose in d , d′ (100 µg; Supplementary Table 1 , clone ID1-1), e (20 µg; ID1-14), f (20 µg; ID1-18), and g (50 µg; ID1-10). Arrowheads indicate mOrange + cells ( d , e ), with marked cells in d suggesting ventral migration of aortic progenitors. By the time of recombination, progenitors of aorta and lung mesenchyme ( d , d ′ ) or epithelium ( g , g′ ) are differentially specified ( h ) (Supplementary Table 1 ). i , j Transverse sections of X GFP X + aorta stained for GFP at E14.5 and in the adult (5 weeks), representative of n = 4 embryos and 6 adults. k , l SMMHC - CreER T2 embryos also carrying ROSA26R ( mTmG /+) or ROSA26R ( Rb /+) induced with tamoxifen 0.5 mg at E9.5 (when there is one SMC layer; Supplementary Fig. 1b, c ) and analyzed at E16.5. In k transverse aortic section (left panel, section #30) of a 21-cell GFP-marked clone (Supplementary Table 2 , clone ID2-12; Supplementary Fig. 3 ) after GFP staining. The middle panel shows positions of two marked cells in this section, and columnar clone schematic (right panel) is a compilation of transverse sections, among 60 consecutive sections analyzed, that contain marked cells. The first and 60th sections are included for reference. A 15-cell clone (ID2-10) is shown in l with the section stained for DAPI and imaged for Rb colors. The clones in k , l expanded radially, circumferentially, and longitudinally. Lu, aortic lumen; E, endothelial layer; 1–5, smooth muscle layers; A, adventitial layer. Scale bars, 100 µm ( b , d , e , g , i ) and 10 µm ( c , f , j – l )

    Article Snippet: On the next day, sections were washed with PBS-T and then incubated with secondary antibodies for 1 h. Primary antibodies used were anti-CD31 (1:500, BD Pharmingen, 553370), anti-SMMHC (1:100, Biomedical Technologies, J64817), anti-GFP (1:500, Invitrogen, A11122; 1:100, Abcam, ab13970), anti-pH3 (1:200, Millipore, 06–570), anti-Ki67 (1:100, Vector Labs, VP-RM04), anti-integrin β3 (1:200, Abcam, ab75872 and ab197662), anti-CD68 (1:200, Bio-Rad, MCA1957), anti-CD36 (1:100, Novus Biologicals, NB400-144), directly conjugated Cy3 anti-SMA (1:500, Sigma-Aldrich, C6198), anti-PDGFR-β (1:100, Abcam, ab32570), and biotinylated anti-PDGFR-β (1:50, R & D, AF1042).

    Techniques: Mouse Assay, Fluorescence, Migration, Staining, Clone Assay

    Cap cells and smooth muscle-derived cells in the plaque are highly proliferative. a – c ApoE (−/−) , SMMHC - CreER T2 , ROSA26R ( mTmG /+) were induced with tamoxifen and fed a HFD for 6, 12, or 16 weeks. In a transverse aortic root sections were stained for phosphohistoneH3 (pH3; mitotic marker), GFP (fate marker), SMA, and nuclei (DAPI). In b percentage of total cap or core cells that are pH3 + is shown. *vs. 6, 12 weeks, p

    Journal: Nature Communications

    Article Title: Integrin beta3 regulates clonality and fate of smooth muscle-derived atherosclerotic plaque cells

    doi: 10.1038/s41467-018-04447-7

    Figure Lengend Snippet: Cap cells and smooth muscle-derived cells in the plaque are highly proliferative. a – c ApoE (−/−) , SMMHC - CreER T2 , ROSA26R ( mTmG /+) were induced with tamoxifen and fed a HFD for 6, 12, or 16 weeks. In a transverse aortic root sections were stained for phosphohistoneH3 (pH3; mitotic marker), GFP (fate marker), SMA, and nuclei (DAPI). In b percentage of total cap or core cells that are pH3 + is shown. *vs. 6, 12 weeks, p

    Article Snippet: On the next day, sections were washed with PBS-T and then incubated with secondary antibodies for 1 h. Primary antibodies used were anti-CD31 (1:500, BD Pharmingen, 553370), anti-SMMHC (1:100, Biomedical Technologies, J64817), anti-GFP (1:500, Invitrogen, A11122; 1:100, Abcam, ab13970), anti-pH3 (1:200, Millipore, 06–570), anti-Ki67 (1:100, Vector Labs, VP-RM04), anti-integrin β3 (1:200, Abcam, ab75872 and ab197662), anti-CD68 (1:200, Bio-Rad, MCA1957), anti-CD36 (1:100, Novus Biologicals, NB400-144), directly conjugated Cy3 anti-SMA (1:500, Sigma-Aldrich, C6198), anti-PDGFR-β (1:100, Abcam, ab32570), and biotinylated anti-PDGFR-β (1:50, R & D, AF1042).

    Techniques: Derivative Assay, Staining, Marker

    Smooth muscle-derived cells initially coat the cap of the plaque and then invade the core and dedifferentiate. Transverse aortic root sections of mice fed a HFD for 5–12 weeks as indicated were immunostained. a , d Sections from ApoE (−/−) mice were stained for SMA, nuclei (DAPI), and either CD68 ( a ) or PDGFR-β ( d ). b , c Prior to high fat feeding, ApoE (−/−) , SMMHC - CreER T2 , ROSA26R ( mTmG /+) were induced with tamoxifen, and sections were stained for GFP (fate marker), nuclei (DAPI), and either SMA ( b ) or SMMHC ( c ). Results are representative of n = 5 mice. Lu, aortic lumen; Med, tunica media; Pl, plaque. Scale bars, 25 μm

    Journal: Nature Communications

    Article Title: Integrin beta3 regulates clonality and fate of smooth muscle-derived atherosclerotic plaque cells

    doi: 10.1038/s41467-018-04447-7

    Figure Lengend Snippet: Smooth muscle-derived cells initially coat the cap of the plaque and then invade the core and dedifferentiate. Transverse aortic root sections of mice fed a HFD for 5–12 weeks as indicated were immunostained. a , d Sections from ApoE (−/−) mice were stained for SMA, nuclei (DAPI), and either CD68 ( a ) or PDGFR-β ( d ). b , c Prior to high fat feeding, ApoE (−/−) , SMMHC - CreER T2 , ROSA26R ( mTmG /+) were induced with tamoxifen, and sections were stained for GFP (fate marker), nuclei (DAPI), and either SMA ( b ) or SMMHC ( c ). Results are representative of n = 5 mice. Lu, aortic lumen; Med, tunica media; Pl, plaque. Scale bars, 25 μm

    Article Snippet: On the next day, sections were washed with PBS-T and then incubated with secondary antibodies for 1 h. Primary antibodies used were anti-CD31 (1:500, BD Pharmingen, 553370), anti-SMMHC (1:100, Biomedical Technologies, J64817), anti-GFP (1:500, Invitrogen, A11122; 1:100, Abcam, ab13970), anti-pH3 (1:200, Millipore, 06–570), anti-Ki67 (1:100, Vector Labs, VP-RM04), anti-integrin β3 (1:200, Abcam, ab75872 and ab197662), anti-CD68 (1:200, Bio-Rad, MCA1957), anti-CD36 (1:100, Novus Biologicals, NB400-144), directly conjugated Cy3 anti-SMA (1:500, Sigma-Aldrich, C6198), anti-PDGFR-β (1:100, Abcam, ab32570), and biotinylated anti-PDGFR-β (1:50, R & D, AF1042).

    Techniques: Derivative Assay, Mouse Assay, Staining, Marker