Journal: PLOS ONE

Article Title: Rare CIDEC coding variants enriched in age-related macular degeneration patients with small low-luminance deficit cause lipid droplet and fat storage defects

doi: 10.1371/journal.pone.0280484

Figure Lengend Snippet: (A) 3xflag-tagged CIDEC wild-type (WT) was co-transfected with the indicated GFP-tagged CIDEC variants in HEK 293T cells. GFP alone was used as negative control. 3xflag-tagged CIDEC WT was immuno-precipitated (IP) using anti-Flag and pulled-down proteins were immuno-blotted (IB) with anti-GFP and anti-Flag. Total cell lysate was immunoblotted with anti-GFP to control for CIDEC-GFP expression levels. (B-E) HEK 293T cells were co-transfected with 3xFlag-CIDEC WT, E186X or AMD variants, and either PLIN1-mCherry (B), AS160-GFP (C) or RAB8A-mCherry (D). After immunoprecipitation (IP) of the 3xFlag-CIDEC, pulled-down proteins were probed with anti-mCherry or anti-GFP, and anti-Flag. Co-transfection with mCherry or GFP alone was used as negative controls. Total cell lysates were immunoblotted (IB) with anti-mCherry or anti-GFP to control for PLIN1, AS160 and RAB8A expression levels. (E) Representative fluorescence images of 3T3-L1 pre-adipocytes lipid droplets containing CIDEC-GFP wild-type (WT) or variants and RAB8A-mCherry. Scale bar: 2 μm.

Article Snippet: GFP- and mCherry-tagged plasmids were used to express human PLIN1, AS160, and RAB8A (Genecopoeia, Inc.).

Techniques: Transfection, Negative Control, Control, Expressing, Immunoprecipitation, Cotransfection, Fluorescence

Journal: EMBO Reports

Article Title: TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution

doi: 10.15252/embr.201948901

Figure Lengend Snippet: RPE1 cells were transfected with siRNAs as indicated, followed by transfection with wild‐type pGFP‐Rab8a (WT‐Rab8), constitutively active pGFP‐Rab8a (Q67L) (CA‐Rab8), or DN Rab8 dominant‐negative pGFP Rab8a (T22N), incubated in serum‐starved media for 12 h, and immunostained for ARL13B (red), GFP‐Rab8 (green), and DAPI (blue). Scale bar, 10 μm. Quantification of the percentage of ciliated cells shown in (A). Data represent mean ± SD ( n = 3 experiments), and 200 GFP‐positive cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. (Upper panel) Cells were transfected as indicated and the cell lysates were incubated with purified proteins, including GST or GST‐JCF1 (RBD). The amount of GTP‐Rab8 bound to GFT‐JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Intensity of the bands was quantified by ImageJ software. The amount of GTP‐Rab8 was normalized to the control level. Bar graph represents mean ± SD ( n = 3 experiments). * P < 0.05, Student's t ‐test. Cells were transfected as shown in (A), and cell lysates were incubated with purified GST‐JCF1 (RBD) fusion protein. The amount of GTP‐Rab8 bound to GFT‐JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Cells were transfected as shown in (A), and cell lysates were incubated with purified GST protein. The amount of GTP‐Rab8 bound to GFT‐JCF1(RBD) was analyzed by Western blot with Rab8 antibody. Source data are available online for this figure.

Article Snippet: Human wild‐type pGFP‐Rab8A (Plasmid #24898), human constitutively active (Q67L) pGFP‐Rab8A (Plasmid #24900), and human dominant‐negative (T22N) pGFP‐Rab8A (Plasmid #24899) were obtained from Addgene.

Techniques: Transfection, Dominant Negative Mutation, Incubation, Purification, Western Blot, Software, Control

Journal: EMBO Reports

Article Title: TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution

doi: 10.15252/embr.201948901

Figure Lengend Snippet: RPE1 cells were transfected with siRNAs as indicated, followed by 24‐h incubation in serum‐starved media. Cells were then and subjected to fractionation, and Western blot for IFT20, the Golgi marker GM130, membrane marker UBXD8, and nuclear marker CREB. Efficiency of IFT20 knockdown by Western blot. Cells were transfected with siRNAs as indicated, followed by incubation in serum‐starved media for 24 h, and immunostained for ARL13B (red). Scale bar, 10 μm. The bar graph represents the quantification of the percentage of ciliated cells. Data represent mean ± SD ( n = 3 experiments), and 250 cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. Cells were transfected as shown in (C), and immunostained for Rab8 and γ‐tubulin, followed by quantification of the percentage of cells with Rab8 localized to the centriole. Data represent average ( n = 2 experiments). Cells were transfected with siRNAs as indicated, followed by transfection with Flag‐IFT20, incubated in serum‐starvation media for 12 h, and immunostained for ARL13B. Representative fluorescent images of Flag‐IFT20 (green), ARL13B (red), and DAPI (blue) are shown. Scale bar, 10 μm. Quantification of the percentage of ciliated cells with both the Flag‐IFT20 and ARL13B localized in the cilium. Data represent mean ± SD ( n = 3 experiments), and 150 Flag‐positive cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. Cells were transfected with siRNAs as indicated, followed by further transfection with CA‐Rab8, incubated in serum‐starvation media for 12 h, and immunostained for acetylated tubulin. Representative fluorescent images of GFP‐Rab8 Q67L (green), acetylated tubulin (red), and DNA (blue) are shown. Scale bar, 10 μm. Quantification of the percentage of GFP‐positive ciliated cells (only those cilia having both GFP‐Rab8 and acetylated tubulin on cilium were considered for quantification). Data represent mean ± SD ( n = 3 experiments), and 200 GFP‐positive cells were scored per condition per experiment, * P < 0.05, Student's t ‐test. Source data are available online for this figure.

Article Snippet: Human wild‐type pGFP‐Rab8A (Plasmid #24898), human constitutively active (Q67L) pGFP‐Rab8A (Plasmid #24900), and human dominant‐negative (T22N) pGFP‐Rab8A (Plasmid #24899) were obtained from Addgene.

Techniques: Transfection, Incubation, Fractionation, Western Blot, Marker, Membrane, Knockdown

Journal: EMBO Reports

Article Title: TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution

doi: 10.15252/embr.201948901

Figure Lengend Snippet: A Efficiency of Rab8a depletion confirmed by Western blot in RPE1 cells. B RPE1 cells were transfected by siRNAs as indicated, followed by incubation in serum‐starvation media for 24 h, and immunostained for ARL13B (red) and γ‐tubulin (green). Scale bar, 10 μm. C Quantification of the percentage of ciliated cells shown in (B). Data represent mean ± SD ( n = 3 experiments), and 250 GFP‐positive cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. D Cells were transfected by siRNA as indicated followed by incubation in serum‐starvation media for 24 h, and immunostained for EHD1 (red) and γ‐tubulin (green). Scale bar, 10 μm. E Quantification of the percentage of cells with EHD1 in cilium or in the distal end of basal body as shown in (D). Data represent mean ± SD ( n = 3 experiments), and 150 cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. F Cells were transfected by siRNAs as indicated, followed by incubation in serum‐starvation media for 24 h, and immunostained for IFT20 (red) and γ‐tubulin (green). Scale bar, 10 μm. G Quantification of the percentage of IFT20 fluorescent intensity at the centriole shown in (F). Data represent mean ± SD ( n = 3 experiments), and 150 were scored per condition per experiment; * P < 0.05, Student's t ‐test. H–J Cells were transfected by siRNAs as indicated, followed by transfection with GFP‐Rab8 WT, GFP‐Rab8 Q67L, or GFP‐Rab8 T22N, and further incubated in serum‐starvation media for 12 h. Cell lysate was subjected to immunoprecipitation with anti‐GFP antibody, followed by Western blot with antibody against GFP. Source data are available online for this figure.

Article Snippet: Human wild‐type pGFP‐Rab8A (Plasmid #24898), human constitutively active (Q67L) pGFP‐Rab8A (Plasmid #24900), and human dominant‐negative (T22N) pGFP‐Rab8A (Plasmid #24899) were obtained from Addgene.

Techniques: Western Blot, Transfection, Incubation, Immunoprecipitation

Journal: Autophagy

Article Title: TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulators

doi: 10.1080/15548627.2017.1422850

Figure Lengend Snippet: Transport of latent TGFB1 to the cell surface requires RAB8A-mediated secretion. (A) Immunofluorescence analysis of human Wi26 fibroblasts expressing HA-LAP-TGFB1 (red), stained for endogenous RAB8A (green) and GOLGA2 (magenta) identified colocalization of HA-LAP with RAB8A at Golgi-derived autophagosomal intermediates (yellow in merged inset). Scale bar: 10 μm. (B) Immunofluorescence analysis of murine control and Ilk cKO fibroblasts, transfected with HA-tagged RAB8A (green; DAPI-stained nuclei appear blue) showed relocalization of RAB8A to perinuclear aggregates. Scale bar: 10 μm. (C and D) Immunoblots illustrating knockdown efficiency of RAB8A (C) and RAB8B (D) in comparison to control (si-Scr). ACTB levels were used to indicate comparable loading. (E and F) Secreted TGFB1 levels were significantly reduced upon knockdown of RAB8A (P<0.001) (E); whereas no change in TGFB1 secretion was detected upon knockdown of RAB8B (F). Each symbol represents one independent transfectant. (G and H) Autophagy was analyzed in human Wi26 fibroblasts following treatment either with DMSO, or with 100 nM BafA, or with 20 μg/ml rapamycin (Rapa) for 4 h. The upper and lower ‘<’ symbols in the immunoblots indicate the position of the MAP1LC3B-I and -II variants, respectively. Autophagy was visualized by the presence of MAP1LC3-II and SQSTM1 bands in immunoblots. Depletion of RAB8A (G) and of RAB8B (H) did not affect basal or stimulated autophagy. Data are representative of 3 experiments. Signal intensities were quantified densitometrically and the ratio of MAP1LC3-II to ACTB is presented below the blots. (I) Immunofluorescence analysis of MEFs derived from atg5 KO or Atg5 WT animals expressing HA-LAP-TGFB1 (red) and stained for endogenous RAB8A (green) identified partial colocalization of HA-LAP with RAB8A at Golgi-derived autophagosomal intermediates (yellow in merged inset), which is not observed in atg5 KO cells. Scale bar: 5 μm. (J) The images of 20 Atg5 WT and 26 atg5 KO cells were used to determine the Pearson correlation coefficient, which reflects the extent of colocalization of TGFB1 with RAB8A (LAP and RAB8A). Values of about 0.15 confirmed selective colocalization.

Article Snippet: Full-length human RAB8A , acc. no. {"type":"entrez-nucleotide","attrs":{"text":"NM_005370","term_id":"1519241776","term_text":"NM_005370"}} NM_005370 , was cloned into pCMV-HA (Clontech, 631604).

Techniques: Immunofluorescence, Expressing, Staining, Derivative Assay, Transfection, Western Blot

Journal: Autophagy

Article Title: TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulators

doi: 10.1080/15548627.2017.1422850

Figure Lengend Snippet: siRNA-mediated gene silencing

Article Snippet: Full-length human RAB8A , acc. no. {"type":"entrez-nucleotide","attrs":{"text":"NM_005370","term_id":"1519241776","term_text":"NM_005370"}} NM_005370 , was cloned into pCMV-HA (Clontech, 631604).

Techniques: