Journal: PLoS ONE

Article Title: BAAV Transcytosis Requires an Interaction with β-1-4 Linked- Glucosamine and gp96

doi: 10.1371/journal.pone.0009336

Figure Lengend Snippet: (A) gp96 western blot from whole cell lysates. GAPDH detection was used as a loading control. (B) Non-permeabilized cells were stained with anti-gp96 followed by Alexafluor 488 secondary antibody and analyzed by confocal immunofluorescence laser microscopy. (C) Fluorescence quantification of 10 random fields. * P<0.05.

Article Snippet: MDCKI or Caco-2 cells grown on Transwell were fixed in 2% formaldehyde/PBS for 10–12 min. Non-permeabilized cells were probed with anti-gp96 (Santa Cruz) in 10% FBS in PBS/0.02% sodium azide for 1 h followed by a goat anti-rabbit Alexafluor 488 (Molecular Probes, Invitrogen) secondary antibody in 10% FBS in PBS/0.02% sodium azide for 1 h and analyzed by confocal immunofluorescence laser microscopy.

Techniques: Western Blot, Control, Staining, Immunofluorescence, Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: BAAV Transcytosis Requires an Interaction with β-1-4 Linked- Glucosamine and gp96

doi: 10.1371/journal.pone.0009336

Figure Lengend Snippet: Anti gp96, anti PDGFRα or soluble gp96 extra cellular domain, 25µg/ml, were placed on the apical surface of MDCKI cells grown on Transwell, prior to the addition of BAAV or AAV4. Transcytosis activity was measured by QPCR after 4 h. Values are expressed relative to the control (BAAV or AAV4 alone). * P<0.05.

Article Snippet: MDCKI or Caco-2 cells grown on Transwell were fixed in 2% formaldehyde/PBS for 10–12 min. Non-permeabilized cells were probed with anti-gp96 (Santa Cruz) in 10% FBS in PBS/0.02% sodium azide for 1 h followed by a goat anti-rabbit Alexafluor 488 (Molecular Probes, Invitrogen) secondary antibody in 10% FBS in PBS/0.02% sodium azide for 1 h and analyzed by confocal immunofluorescence laser microscopy.

Techniques: Activity Assay, Control

Journal: PLoS ONE

Article Title: BAAV Transcytosis Requires an Interaction with β-1-4 Linked- Glucosamine and gp96

doi: 10.1371/journal.pone.0009336

Figure Lengend Snippet: Anti-myc sepharose beads previously incubated with cell lysates either of GFP, or myc-tagged TRPC1 or gp96 transfected cells were then added with BAAV or AAV4 vector respectively containing the CMV-GFP expression cassette. After incubation at 4C° for 1h, beads were extensively washed and bound virus was quantified by QPCR. Bound vector was calculated as fold change in bound vector relative to vector bound to GFP beads alone. n = 3. * P<0.05.

Article Snippet: MDCKI or Caco-2 cells grown on Transwell were fixed in 2% formaldehyde/PBS for 10–12 min. Non-permeabilized cells were probed with anti-gp96 (Santa Cruz) in 10% FBS in PBS/0.02% sodium azide for 1 h followed by a goat anti-rabbit Alexafluor 488 (Molecular Probes, Invitrogen) secondary antibody in 10% FBS in PBS/0.02% sodium azide for 1 h and analyzed by confocal immunofluorescence laser microscopy.

Techniques: Incubation, Transfection, Plasmid Preparation, Expressing, Virus

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: RT-qPCR primer sequences

Article Snippet: The beads were then resuspended in 2 × Loading Buffer and boiled at 95 °C for 10 min. After washing, protein complexes were resolved in Sodium Dodecyl Sulfate (SDS) loading buffer and analyzed by Western blot using antibodies against HSP90B1 (ab3674, Abcam), EGFR (ab52894, Abcam), and IgG (ab37355, Abcam).

Techniques:

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: HSP90B1 is overexpressed in NPC. A : Bioinformatics website http://gepia.cancer-pku.cn/index.html analyzed the expression parttern of HSP90B1 in HNSC; B : RT-qPCR detection of HSP90B1 in NPC tissues and normal nasopharyngeal epithelial samples; C : Western blot detection of HSP90B1 expression pattern in NPC tissues and normal nasopharyngeal epithelial samples; D : IHC staining to assess the expression pattern of HSP90B1 in NPC tissues and normal nasopharyngeal epithelial samples; E : RT-qPCR to assess the expression pattern of HSP90B1 in the NPC cell lines (HNE1, HNE2, CNE1, CNE2, HONE1) and human immortalized nasopharyngeal epithelial cell line (NP69); F : Western blot assessment of HSP90B1 expression in NPC cell lines (HNE1, HNE2, CNE1, CNE2, HONE1) and NP69; G : Prognostic chart of 5 year survival of 42 NPC patients. Data are expressed as mean ± SD (N = 3). p < 0.05 was considered statistically significant

Article Snippet: The beads were then resuspended in 2 × Loading Buffer and boiled at 95 °C for 10 min. After washing, protein complexes were resolved in Sodium Dodecyl Sulfate (SDS) loading buffer and analyzed by Western blot using antibodies against HSP90B1 (ab3674, Abcam), EGFR (ab52894, Abcam), and IgG (ab37355, Abcam).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: Relationship between HSP90B1 and clinicopathological features of NPC patients

Article Snippet: The beads were then resuspended in 2 × Loading Buffer and boiled at 95 °C for 10 min. After washing, protein complexes were resolved in Sodium Dodecyl Sulfate (SDS) loading buffer and analyzed by Western blot using antibodies against HSP90B1 (ab3674, Abcam), EGFR (ab52894, Abcam), and IgG (ab37355, Abcam).

Techniques: Expressing

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: HSP90B1 affects NPC proliferation, glycolysis, and angiogenesis. The siRNA targeting HSP90B1 and pcDNA 3.1 overexpression vector were transfected into HNE1 and CNE2 cells. A : RT-qPCR to detect the expression of HSP90B1 in the cells; B : Western blot to detect the expression of HSP90B1 in the cells; C : Colony formation assay to detect the proliferation of HNE1 and CNE2 cells; D : EdU assay to detect proliferation of HNE1 and CNE2 cells; E : Flow cytometry to detect apoptosis in HNE1 and CNE2 cells; F : Commercial kits to detect ECAR in HNE1 and CNE2 cells; G : Commercial kits to detect glucose consumption in HNE1 and CNE2 cells; H : Commercial kit to detect lactate production in HNE1 and CNE2 cells; I : Western blot to assess GLUT1 and HK2 protein expression in HNE1 and CNE2 cells; J : Tube formation assay to detect angiogenic capacity of HUVECs; K : HUVEC migration capacity assay; L : Western blot to assess the expression of angiogenic proteins Ang-1 and VEGF in HUVECs. Data are expressed as mean ± SD (N = 3). p < 0.05 was considered statistically significant

Article Snippet: The beads were then resuspended in 2 × Loading Buffer and boiled at 95 °C for 10 min. After washing, protein complexes were resolved in Sodium Dodecyl Sulfate (SDS) loading buffer and analyzed by Western blot using antibodies against HSP90B1 (ab3674, Abcam), EGFR (ab52894, Abcam), and IgG (ab37355, Abcam).

Techniques: Over Expression, Plasmid Preparation, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Colony Assay, EdU Assay, Flow Cytometry, Tube Formation Assay, Migration

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: HSP90B1 targets EGFR expression. A : Western blot to detect the effect of knockdown or overexpression of HSP90B1 on the expression of phosphorylated EGFR; B : Biosignature website ( https://www.ebi.ac.uk ) to analyze the binding relationship between HSP90B1 and EGFR; C : Co-IP assay to detect the interaction between HSP90B1 and EGFR; D : RT-qPCR to detect EGFR expression pattern in NPC tissues and normal nasopharyngeal epithelial samples; E : Western blot to detect the expression pattern of EGFR in NPC tissues and normal nasopharyngeal epithelial samples; F : IHC staining to assess the expression pattern of EGFR in NPC tissues and normal nasopharyngeal epithelial samples; G : RT-qPCR to assess the expression pattern of EGFR in NPC cell lines (HNE1, HNE2, CNE1, CNE2, and HONE1) and human immortalized nasopharyngeal epithelial cell line (NP69); H : Western blot to assess EGFR expression in NPC cell lines (HNE1, HNE2, CNE1, CNE2, and HONE1) and NP69. Data are presented as mean ± SD (N = 3). P < 0.05 was considered statistically significant

Article Snippet: The beads were then resuspended in 2 × Loading Buffer and boiled at 95 °C for 10 min. After washing, protein complexes were resolved in Sodium Dodecyl Sulfate (SDS) loading buffer and analyzed by Western blot using antibodies against HSP90B1 (ab3674, Abcam), EGFR (ab52894, Abcam), and IgG (ab37355, Abcam).

Techniques: Expressing, Western Blot, Over Expression, Binding Assay, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Immunohistochemistry

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: HSP90B1 by regulating EGFR affects NPC malignancy. HNE1 and CNE2 cells transfected with pcDNA 3.1-HSP90B1 were treated with an EGFR inhibitor (cetuximab). A : Western blot to detect the phosphorylation level of EGFR; B : Colony formation assay to detect proliferation of HNE1 and CNE2 cells; C : EdU assay to detect proliferation of HNE1 and CNE2 cells; D : Flow cytometry assay to detect apoptosis in HNE1 and CNE2 cells; E : Commercial kits to detect ECAR in HNE1 and CNE2 cells; F : Commercial kits to detect glucose depletion in HNE1 and CNE2 cells; G : Commercial kits to detect lactate production in HNE1 and CNE2 cells; H : Western blot assessment of HNE1 and CNE2 cells GLUT1 and HK2 protein expression; I : Tube formation assay to detect angiogenic capacity of HUVECs; J : HUVEC migration capacity assay; K : Western blot to assess the expression of angiogenic proteins Ang-1 and VEGF in HUVECs. Data are presented as mean ± SD (N = 3). P < 0.05 was considered statistically significant

Article Snippet: The beads were then resuspended in 2 × Loading Buffer and boiled at 95 °C for 10 min. After washing, protein complexes were resolved in Sodium Dodecyl Sulfate (SDS) loading buffer and analyzed by Western blot using antibodies against HSP90B1 (ab3674, Abcam), EGFR (ab52894, Abcam), and IgG (ab37355, Abcam).

Techniques: Transfection, Western Blot, Colony Assay, EdU Assay, Flow Cytometry, Expressing, Tube Formation Assay, Migration

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: HSP90B1 activates the PI3K/Akt/mTOR pathway in NPC cells by regulating EGFR. HNE1 and CNE2 cells transfected with pcDNA 3.1-HSP90B1 were treated with an EGFR inhibitor (cetuximab). A – B : Western blot assessed the phosphorylation level of PI3K/Akt/mTOR. Data are presented as mean ± SD (N = 3). P < 0.05 was considered statistically significant

Article Snippet: The beads were then resuspended in 2 × Loading Buffer and boiled at 95 °C for 10 min. After washing, protein complexes were resolved in Sodium Dodecyl Sulfate (SDS) loading buffer and analyzed by Western blot using antibodies against HSP90B1 (ab3674, Abcam), EGFR (ab52894, Abcam), and IgG (ab37355, Abcam).

Techniques: Transfection, Western Blot

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: FKC by inhibiting HSP90B1 limits NPC proliferation, glycolysis, and angiogenesis. FKC-treated HNE1 and CNE2 cells were transfected with pcDNA 3.1-HSP90B1. A : CCK-8 assay to assess the effect of different concentrations of FKC treatment on NP69 cell viability; B : RT-qPCR to detect the expression of HSP90B1; C : Western blot to detect the expression of HSP90B1; D : Colony formation assay to detect HNE1 and CNE2 cell proliferation; E : EdU assay to detect HNE1 and CNE2 cell proliferation; F : Flow cytometry to detect HNE1 and CNE2 cell apoptosis; G : Commercial kits to detect ECAR in HNE1 and CNE2 cells; H : Commercial kits to detect glucose consumption in HNE1 and CNE2 cells; I : Commercial kits to detect HNE1 and lactate production in CNE2 cells; J : Western blot assessment of GLUT1 and HK2 protein expression in HNE1 and CNE2 cells; K : Tube formation assay to detect the angiogenic capacity of HUVECs; L : Migration capacity assay of HUVECs; M : Western blot assessment of angiogenic proteins Ang-1 and VEGF in HUVEC. Data are presented as mean ± SD (N = 3). P < 0.05 was considered statistically significant

Article Snippet: The beads were then resuspended in 2 × Loading Buffer and boiled at 95 °C for 10 min. After washing, protein complexes were resolved in Sodium Dodecyl Sulfate (SDS) loading buffer and analyzed by Western blot using antibodies against HSP90B1 (ab3674, Abcam), EGFR (ab52894, Abcam), and IgG (ab37355, Abcam).

Techniques: Transfection, CCK-8 Assay, Quantitative RT-PCR, Expressing, Western Blot, Colony Assay, EdU Assay, Flow Cytometry, Tube Formation Assay, Migration

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: FKC by inhibiting HSP90B1 affects the PI3K/Akt/mTOR pathway in NPC cells. pcDNA 3.1-HSP90B1 was transfected into FKC-treated HNE1 and CNE2 cells. A – B : Western blot detected phosphorylation levels of PI3K/Akt/mTOR. Data are presented as mean ± SD (N = 3). P < 0.05 was considered statistically significant

Article Snippet: The beads were then resuspended in 2 × Loading Buffer and boiled at 95 °C for 10 min. After washing, protein complexes were resolved in Sodium Dodecyl Sulfate (SDS) loading buffer and analyzed by Western blot using antibodies against HSP90B1 (ab3674, Abcam), EGFR (ab52894, Abcam), and IgG (ab37355, Abcam).

Techniques: Transfection, Western Blot

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: FKC by modulating the HSP90B1/EGFR axis affects NPC tumor growth and metastasis. A : Tumor images; B : Tumor volume; C : Tumor weight; D : Western blot assessment of relevant protein expression in tumors; E : Noninvasive bioluminescence imaging of luciferase-expressing intrahepatic HNE1 cell xenografts showing FKC inhibition of in situ liver metastatic tumor formation. Data are presented as mean ± SD (n = 5). P < 0.05 was considered statistically significant

Article Snippet: The beads were then resuspended in 2 × Loading Buffer and boiled at 95 °C for 10 min. After washing, protein complexes were resolved in Sodium Dodecyl Sulfate (SDS) loading buffer and analyzed by Western blot using antibodies against HSP90B1 (ab3674, Abcam), EGFR (ab52894, Abcam), and IgG (ab37355, Abcam).

Techniques: Western Blot, Expressing, Imaging, Luciferase, Inhibition, In Situ

Journal: The Journal of Cell Biology

Article Title: Cerebellar ataxia disease–associated Snx14 promotes lipid droplet growth at ER–droplet contacts

doi: 10.1083/jcb.201808133

Figure Lengend Snippet: Snx14 localizes at ER–LD contacts after OA treatment. (A) Localization of Snx14 in U2OS cells in the absence and presence of OA, respectively. CoIF staining of the cells transiently expressing an untagged Snx14 were performed with α-Snx14 antibody (green), and α-HSP90B1 (ER marker, red) antibodies and LDs were stained with MDH (blue) and imaged by confocal microscopy. Inset of OA-treated cells displays Snx14 accumulating around LDs. Scale bar = 20 µm. Scale bar of insets = 5 µm. (B) Schematic diagram showing Snx14 EGFP-APEX2 fusion at ER–LD contacts. (C) TEM micrographs showing untreated and DAB + H 2 O 2 –treated cells following OA treatment. The dark precipitate with DAB + H 2 O 2 treatment indicates presence of Snx14 EGFP-APEX2 . Scale bar = 0.5 µm. (D) TEM micrograph showing SNX14 EGFP-APEX2 -expressing cell with DAB precipitate at ER–LD contacts. (D′) Image from D showing pseudocolored ER (red), LD (yellow), and green arrows pointing at DAB precipitate specifically at the junction of ER and LD. The blue arrows indicate a region of LD surface that lacks ER wrapping and also lacks DAB precipitate. Purple stars indicate LDs without any detectable ER association (and no DAB precipitate). The orange arrow indicates an ER membrane itself. Scale bar = 0.5 µm. (E) Lower-magnification TEM micrographs of a Snx14 EGFP-APEX2 -expressing cell following OA treatment and stained with DAB, showing precipitate surrounding various LDs entangled with the ER network. Scale bar = 0.5 µm.

Article Snippet: The primary antibodies used are mouse anti-Hsp90B1 (1:300; Sigma-Aldrich; AMAb91019), rabbit anti-Snx14 (HPA017639), rabbit anti-EGFP (1:300), rabbit anti-Flag (1:200; Sigma-Aldrich; F7425), mouse anti-ACSL3 (1:200; Novus Biologicals; H00002181-B01P).

Techniques: Staining, Expressing, Marker, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: Cerebellar ataxia disease–associated Snx14 promotes lipid droplet growth at ER–droplet contacts

doi: 10.1083/jcb.201808133

Figure Lengend Snippet: Snx14 is topologically anchored in the ER and interacts with LDs in trans. (A) LD flotation assay of OA-treated U2OS cells expressing Snx14 EGFP . Lanes indicate postnuclear supernatant (PNS), cytosol, total membrane, and LD float fractions. (B) Schematic diagram of Snx14 fragment constructs tagged with EGFP. Snx14 FL depicts the full-length human Snx14. Snx14 N is the N-terminal fragment from the start that includes PXA and RGS domains. Snx14 PXCN includes the PX domain and C-Nexin domains. Snx14 PX consists of PX and Snx14 CN represents C-Nexin domain. An AH in the C-Nexin domain is identified as depicted in the schematic diagram. Snx14 PXCNΔH indicates the PX and C-Nexin domain from which the AH is deleted. Snx14 FLΔH depicts the full-length Snx14 with AH deletion. (C) Western blot showing distribution of Snx14 FL , Snx14 N , and Snx14 PXCN tagged with 3XFlag among total membrane and LD fractions following OA treatment. (D) U2OS cells transfected with Snx14 FL , Snx14 N , Snx14 PXCN , Snx14 PX , Snx14 CN , Snx14 PXCNΔH , and Snx14 FLΔH , respectively, and treated with OA for 16 h. CoIF staining with α−EGFP (green) and α-HSP90B1 (ER marker, red) and LDs stained with MDH (blue) and imaged by confocal microscopy. Scale bar = 20 µm. Inset scale bar = 5 µm. Cartoons represent the localization of the respective Snx14 fragments with respect to ER and LD.

Article Snippet: The primary antibodies used are mouse anti-Hsp90B1 (1:300; Sigma-Aldrich; AMAb91019), rabbit anti-Snx14 (HPA017639), rabbit anti-EGFP (1:300), rabbit anti-Flag (1:200; Sigma-Aldrich; F7425), mouse anti-ACSL3 (1:200; Novus Biologicals; H00002181-B01P).

Techniques: Expressing, Construct, Western Blot, Transfection, Staining, Marker, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: Cerebellar ataxia disease–associated Snx14 promotes lipid droplet growth at ER–droplet contacts

doi: 10.1083/jcb.201808133

Figure Lengend Snippet: Loss of Snx14 perturbs LD size and morphology but does not change neutral lipid levels. (A) Confocal micrographs of WT and SNX14 KO cells treated with OA overnight. LDs visualized by MDH (black) and nucleus stained with Syto 85 orange fluorescent stain (blue outline). Images were processed so that LDs were converted to grayscale and inverted. Scale bar = 25 µm. (B) Quantification of average area covered by LDs per cell of representative images from A. Total LD area was derived from more than five fields of view, each consisting of approximately five cells or more of two different sets of experiments (total no. of cells >75; ***, P < 0.0001 unpaired t test with α = 0.05). (C) TEM micrographs of WT and SNX14 KO cells treated with OA overnight to visualize LD distribution and morphology. The top panels are lower magnification (scale bar = 2 µm). The bottom panels are higher magnification (scale bar = 1 µm). (D) Scatter dot plot of cross-sectional areas of LDs in WT and SNX14 KO cells as in C. Total LDs = 896; ***, P < 0.0001, Kolmogorov–Smirnov D test with α = 0.05. (E) Rescue of LD morphology in SNX14 KO cells by readdition of empty vector (EV), Snx14 FL , Snx14 N , Snx14 PXCN , Snx14 PXCNΔH , and Snx14 FLΔH , respectively, all tagged with EGFP. Cells were coIF stained with α-EGFP (green), α-HSP90B1 (ER, red), and LDs stained with MDH (blue) and imaged with confocal microscope. Scale bar = 50 µm. (F) Area covered by LDs in each cell from E analyzed and plotted. Total no. of cells quantified are 45 from two different sets of experiments (***, P < 0.0001, one-way ANOVA with α = 0.05). Line bars indicate mean ± SD.

Article Snippet: The primary antibodies used are mouse anti-Hsp90B1 (1:300; Sigma-Aldrich; AMAb91019), rabbit anti-Snx14 (HPA017639), rabbit anti-EGFP (1:300), rabbit anti-Flag (1:200; Sigma-Aldrich; F7425), mouse anti-ACSL3 (1:200; Novus Biologicals; H00002181-B01P).

Techniques: Staining, Derivative Assay, Plasmid Preparation, Microscopy

Journal: The Journal of Cell Biology

Article Title: Cerebellar ataxia disease–associated Snx14 promotes lipid droplet growth at ER–droplet contacts

doi: 10.1083/jcb.201808133

Figure Lengend Snippet: Snx14 functions independently of ER–LD protein, Seipin. (A) Confocal micrographs showing localization of Snx14 in WT and SEIPIN KO SUM159 cells. The cells were transfected with Snx14 EGFP and treated with OA overnight. Labels were Snx14 EGFP (α-EGFP), ER (α-HSP90B1), and LDs (MDH). Scale bar = 20 µm. Inset scale bar = 5 µm. (B) Test of rescue of LD morphology in SEIPIN KO examined by ectopic expression of Snx14 EGFP into SEIPIN KO . Labels were Snx14 EGFP (α-EGFP), ER (α-HSP90B1), and LDs (MDH stained, converted to grayscale, and inverted by ImageJ). Scale bar = 20 µm. (C) Area covered by LDs in each cell from B analyzed and plotted. Total no. of cells quantified is 23 from two different sets of experiments (***, P < 0.0001, one-way ANOVA with α = 0.05). Line bars indicate mean ± SD. (D) Test of rescue of LD morphology in SNX14 KO examined by readdition of Seipin mCherry and comparing to that of WT and SNX14 KO . Blue line depicts cell boundary. Black speckles represent LDs that are MDH-stained, converted to grayscale, and inverted by ImageJ. Scale bar = 20 µm. (E) Area covered by LDs in each cell from D analyzed and plotted. Total no. of cells quantified is 45 from two different sets of experiments (***, P < 0.0001, one-way ANOVA with α = 0.05). Line bars indicate mean ± SD. (F) Confocal micrographs of U2OS cells to examine localization of Seipin mCherry in WT cells, those transfected with Snx14 EGFP , and in SNX14 KO cells. Cells were transfected with Seipin-mCherry and treated with OA overnight. Labels were Snx14 EGFP (α-EGFP), Seipin-mCherry (α-mCherry), and LDs (MDH). Scale bar = 20 µm.

Article Snippet: The primary antibodies used are mouse anti-Hsp90B1 (1:300; Sigma-Aldrich; AMAb91019), rabbit anti-Snx14 (HPA017639), rabbit anti-EGFP (1:300), rabbit anti-Flag (1:200; Sigma-Aldrich; F7425), mouse anti-ACSL3 (1:200; Novus Biologicals; H00002181-B01P).

Techniques: Transfection, Expressing, Staining

Journal: The Journal of Cell Biology

Article Title: Cerebellar ataxia disease–associated Snx14 promotes lipid droplet growth at ER–droplet contacts

doi: 10.1083/jcb.201808133

Figure Lengend Snippet: Snx14 localizes at ACSL3-positive preLDs following OA addition. (A) Confocal micrographs of immunofluorescently stained Snx14 EGFP -expressing cells following OA treatment for t = 0, 1, 2, 4, 8, and 16 h. Labels were Snx14 EGFP (α-EGFP), ER (α-HSP90B1), and LDs (MDH). Scale bar = 2 µm. Line scans show the spatial distribution of Snx14 (green) with respect to ER (red) and LDs (blue). (B) Confocal micrographs of immunofluorescently stained Snx14 EGFP -expressing cells following OA treatment for t = 0, 1, 2, 4, 8, and 16 h. Labels were Snx14 EGFP (α-EGFP), native ACSL3 (α-ACSL3), and LDs (MDH). Scale bar = 2 µm. Line scans show the spatial distribution of Snx14 (green) with respect to ACSL3 (red) and LDs (blue). Scale bar = 2 µm. (C) Quantification of Pearson’s coefficient between Snx14 and ER of n = ∼20 cells depicted in A. (D) Quantification of Pearson’s coefficient between ACSL3 and LDs, and Snx14 and LDs of n = ∼20 cells depicted in B. (E) Quantification of Pearson’s coefficient between Snx14 and ACSL3 of n = ∼20 cells depicted in B. (F) Confocal micrographs examining localization of ACSL3 in WT and SNX14 KO cells treated with OA overnight. Labels were native ACSL3 (α-ACSL3, green) and LDs (MDH, magenta). Scale bar = 20 µm. (G) Confocal micrographs to examine localization of Snx14 in WT cells treated with negative scrambled siRNA (neg Si) or ACSL3-targeted siRNA (ACSL3 Si), respectively. The cells were transfected with Snx14 EGFP along with the respective siRNAs and treated with OA overnight. Labels were Snx14 EGFP (α-EGFP), ER (α-HSP90B1), and LDs (MDH). Scale bar = 20 µm. Inset scale bar = 5 µm.

Article Snippet: The primary antibodies used are mouse anti-Hsp90B1 (1:300; Sigma-Aldrich; AMAb91019), rabbit anti-Snx14 (HPA017639), rabbit anti-EGFP (1:300), rabbit anti-Flag (1:200; Sigma-Aldrich; F7425), mouse anti-ACSL3 (1:200; Novus Biologicals; H00002181-B01P).

Techniques: Staining, Expressing, Transfection