Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: N-glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting.

doi: 10.1210/mend.12.4.0085

Figure Lengend Snippet: Fig. 4. Perinuclear Localization of the Aglycosylated PRLR COS-7 cells transfected with WT (a and d), N80,108D (b and e), or N35,80,108D (c and f) PRLR cDNAs were fixed in 4% paraformaldehyde (a, b, and c: nonpermeabilized conditions) or in methanol, 220 C (d, e, and f: permeabilized conditions). Mouse monoclonal antibody U5 (160 mg/ml) followed by FITC goat anti-IgG (dilution 1:40) were used, as described in Materials and Methods. Note the immunostaining in the juxtanuclear area of the aglycosylated PRLR in permeabilized cells (f) and the absence of expression at the plasma membrane level in nonpermeabilized cells (c). 293 cells transfected with WT (g), N80,108D (h), or N35,80,108D (i) PRLR cDNAs were processed for fixation (4% paraformaldehyde) and immunofluorescence staining, as described for COS-7 cells. Note the absence of immunostaining at the cell surface and low perinuclear staining of the aglycosylated receptor in nonpermeabilized conditions (i). Magnification, 3400.

Article Snippet: The secondary antibodies used in these studies were the Texas-Red-conjugated and fluorescein-isothiocyanate-conjugated (FITC) goat anti-mouse IgG (dilution 1:40) (Vector Laboratories, Burlingame, CA; and Biosys, Compiègne, France), the biotinylated donkey anti-rabbit IgG and the Texas-Red-conjugated streptavidin (1:100; Amersham International, Aylesbury, UK).

Techniques: Transfection, Immunostaining, Expressing, Clinical Proteomics, Membrane, Immunofluorescence, Staining

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: N-glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting.

doi: 10.1210/mend.12.4.0085

Figure Lengend Snippet: Fig. 5. Deglycosylated PRLR Colocalizes with the Golgi rab6 in COS-7 Cells a, Labeling of fixed permeabilized COS-7 cells transfected with N35,80,108D receptor using U5 antibody. Panels b and c and panels d and e show a double- labeling experiment of fixed permeabilized COS-7 cells transfected with the N35,80,108D mutant, with anti-PRLR antibody [visualized with a secondary anti-mouse FITC secondary antibody (b and d)], and anti-Golgi antibody [anti-rab6 visualized with bioti- nylated secondary anti-rabbit IgG followed by Texas-Red- conjugated streptavidin (c and e)]. The choice of mouse anti- PRLR and rabbit anti-Golgi IgG as primary antibodies allows accurate double-labeling experiments. Panels b-c and d-e show two different COS-7 cells. f, Micrographs of COS-7 cells stained for anti-protein disulfide isomerase (endoplas- mic reticulum protein) antibody, visualized with Texas-Red- conjugated anti-mouse secondary antibody. Magnification, 3400.

Article Snippet: The secondary antibodies used in these studies were the Texas-Red-conjugated and fluorescein-isothiocyanate-conjugated (FITC) goat anti-mouse IgG (dilution 1:40) (Vector Laboratories, Burlingame, CA; and Biosys, Compiègne, France), the biotinylated donkey anti-rabbit IgG and the Texas-Red-conjugated streptavidin (1:100; Amersham International, Aylesbury, UK).

Techniques: Labeling, Transfection, Mutagenesis, Staining

Journal: Journal of cell science

Article Title: Tubular microdomains of Rab7-positive endosomes retrieve TrkA, a mechanism disrupted in Charcot-Marie-Tooth disease 2B.

doi: 10.1242/jcs.258559

Figure Lengend Snippet: Fig. 6. WASH1 is involved in late receptor tubulations. (A) Anti-GFP-conjugated beads were used to immunoprecipitate (IP) GFP, endophilinA1–GFP (A1–GFP), endophilinA2– GFP (A2–GFP) or endophilinA3–GFP (A3–GFP) with TrkA–RFP from co-transfected HEK293 cells (input on the left, IP on the right) in the presence or absence (NF) of 100 ng/ml NGF. (B) Anti-RFP-conjugated beads (or IgG control beads) were used to pull down EGFR–RFP with endophilinA1–GFP, endophilinA2–GFP or endophilinA3–GFP from co-transfected HEK293 cells in the presence or absence (NF) of 100 ng/ ml EGF (input on the left, IP on the right). Data in A and B are representative of three experiments. (C) Immunostaining of MEFs showing WASH1 on the rim of late Rab7 vacuoles when stimulated with NGF. Scale bar: 20 µm. (D) Immunostaining of MEFs showing WASH1 not localizing to late Rab7 vacuoles when stimulated with EGF. Scale bar: 20 µm. In C and D, boxes indicate regions shown as magnified images. Lines indicate transects shown in plot profiles. Nuclei are stained with DAPI. Images are representative of three experiments. (E) Colocalization of stained WASH1 and Rab7 increases in DRGs upon stimulation with NGF. Boxes indicate regions shown in magnified images on the right. Arrowheads indicate colocalization. Quantification shows mean±s.e.m. Pearson’s correlation coefficient. n=10–15 images per condition, the experiment was performed three times. **P=0.0046 (two-tailed, unpaired t-test). Scale bar: 10 µm. (F) EndophilinAs co- immunoprecipitate with WASH1 in lysates from HEK293 cells co-transfected with GFP-tagged endophilinAs and WASH1–mCherry. Anti-GFP- conjugated beads (or control IgG beads) were used for the IP [input and output (protein levels after incubation with conjugated beads) on the left, IP on the right]. Blots shown are representative of three experiments.

Article Snippet: Antibodies used for western blotting: Rab7a rabbit polyclonal antibody (1:1000, Synaptic Systems, 320 003), SNX1 mouse monoclonal antibody (51; Santa Cruz Biotechnology, sc-136247), SNX2 mouse monoclonal antibody (13; Santa Cruz Biotechnology, sc-136072), SNX5 mouse monoclonal antibody (F11; Santa Cruz Biotechnology, sc-515215), SNX6 mouse monoclonal antibody (D-1; Santa Cruz Biotechnology, sc-365795, all SNX antibodies were used 1:1000), endophilin I mouse monoclonal antibody (B-1; Santa Cruz Biotechnology, sc-374279), endophilin II mouse monoclonal antibody (A-11; Santa Cruz Biotechnology, sc-365704), endophilin III mouse monoclonal antibody (F-4; Santa Cruz Biotechnology, sc-376592, all endophilin antibodies were used at 1:1000), EGFR mouse monoclonal antibody (1:500, A-10; Santa Cruz Biotechnology, sc-373746), GFP rabbit polyclonal antibody (1:1000, pabg1-10; Chromotek, pabg1), RFP mouse monoclonal antibody (1:1000, Chromotek, 6G6), TrkA rabbit polyclonal antibody (1:1000,Millipore, 06-574), GAPDHmousemonoclonal antibody (1:1000, HyTest, 5G4).

Techniques: Transfection, Control, Immunostaining, Staining, Two Tailed Test, Incubation

Journal: Nutrients

Article Title: Pro-Osteogenic Properties of Violina pumpkin ( Cucurbita moschata ) Leaf Extracts: Data from In Vitro Human Primary Cell Cultures.

doi: 10.3390/nu13082633

Figure Lengend Snippet: Figure 3. Effects of organic extracts of Violina pumpkin leaves on osteogenic markers. (A) The expression of Runx2 early osteogenic differentiation marker was evaluated. hOBs were maintained in basal medium and treated with 5 µg/mL of hexane (Hex), acetone (Ac) and methanol (Me) extracts for 72 h. Representative optical photomicrographs of immunostaining are reported. Scale bars: 20 µm. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software and are expressed as means of pixels per one hundred cells ± SD (n = 3). * p < 0.01 vs. DMSO. (B) ALP activity was determined after 7 and 14 days of extract treatment in osteogenic medium. Data are presented as fold changes with respect to control group (CTR) (mean ± SD) (n = 3). * p < 0.05 vs. CTR, ** p < 0.01 vs. CTR, ◦p < 0.05 vs. OM and Hex-treated groups. (C) Representative images of mineralized nodule formation detected using Alizarin Red S staining. Cells were cultured in osteogenic medium in presence of extracts for 21 days. (D) Quantitative analysis of Alizarin Red S staining was performed after extraction with cethylpyridinium chloride. Data are presented as fold changes with respect to OM (mean ± SD) (n = 3). * p < 0.05 vs. OM, ◦p < 0.01 vs. Hex and Me-treated groups. CTR = cells maintained in basal medium containing 0.5% DMSO. OM = osteogenic medium containing 0.5% DMSO.

Article Snippet: Antibody against human Runx2 (#sc-10758) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Marker, Immunostaining, Software, Activity Assay, Control, Staining, Cell Culture, Extraction

Journal: Nutrients

Article Title: Pro-Osteogenic Properties of Violina pumpkin ( Cucurbita moschata ) Leaf Extracts: Data from In Vitro Human Primary Cell Cultures.

doi: 10.3390/nu13082633

Figure Lengend Snippet: Figure 4. Effects of TLC subfractions of acetone raw extract on cell viability and osteogenic markers. (A) hOBs were treated with 5–250 µg/mL of TLC subfractions 2, 6 and 7 (Fr2, Fr6, Fr7) for 72 h. The viability was monitored with MTT assays. Data represent mean ± SD (n = 3). * p < 0.05 vs. control cells (CTR), ** p < 0.001 vs. CTR, ◦p < 0.001 vs. DMSO-treated group. (B) The expression of Runx2 early osteogenic differentiation marker was evaluated. hOBs were maintained in basal medium and treated with 5 µg/mL of TLC subfractions or raw acetone extract for 72 h. Representative optical photomicrographs of immunostaining are reported. Scale bars: 20 µm. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software and are expressed as means of pixels per one hundred cells ± SD (n = 3). * p < 0.05 vs. DMSO, ** p < 0.001 vs. DMSO, ◦p < 0.001 vs. Ac, ◦◦p < 0.01 vs. Ac, # p < 0.001 vs. Fr2, ## p < 0.01 vs. Fr2. (C) ALP activity was determined after 7 and 14 days for TLC subfraction and raw acetone extract treatments in an osteogenic medium. Data are presented as fold changes with respect to the control group (CTR) (mean ± SD) (n = 3). * p < 0.05 vs. CTR, ** p < 0.01 vs. CTR, ◦p < 0.01 vs. OM, ◦◦p < 0.05 vs. OM, # p < 0.05 vs. Fr7-treated group. (D) Representative images of the formation of mineralized nodules detected using Alizarin Red S staining. Cells were cultured in osteogenic medium in the presence of TLC subfractions or raw acetone extract for 21 days. (E) Quantitative analysis of Alizarin Red S staining was performed after extraction with cethylpyridinium chloride. Data are presented as fold changes with respect to OM (mean ± SD) (n = 3). * p < 0.01 vs. OM, ◦p< 0.01 vs. Ac-treated group, # p < 0.01 vs. Fr2-treated group. CTR = cells maintained in basal medium containing 0.5% DMSO. OM = osteogenic medium containing 0.5% DMSO.

Article Snippet: Antibody against human Runx2 (#sc-10758) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Control, Expressing, Marker, Immunostaining, Software, Activity Assay, Staining, Cell Culture, Extraction

Journal: Nutrients

Article Title: Pro-Osteogenic Properties of Violina pumpkin ( Cucurbita moschata ) Leaf Extracts: Data from In Vitro Human Primary Cell Cultures.

doi: 10.3390/nu13082633

Figure Lengend Snippet: Figure 5. Effects of a pure compound (PU-13OH-FA) obtained from subfraction 2 on cell viability and osteogenic markers. (A) hOBs were treated with 5–250 µg/mL of compound PU-13OH-FA for 72 h. The viability was monitored with an MTT assay. Data represent mean ± SD (n = 3). * p < 0.05 vs. control cells (CTR), ** p < 0.001 vs. CTR, ◦p < 0.05 vs. DMSO-treated group, ◦◦p < 0.01 vs. DMSO-treated group. (B) The expression of Runx2 early osteogenic differentiation marker was evaluated. hOBs were maintained in basal medium and treated with 5 µg/mL of Fr2 and PU-13OH-FA for 72 h. Representative optical photomicrographs of immunostaining is reported. Scale bars: 20 µm. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software and expressed as means of pixels per one hundred cells ± SD (n = 3). * p < 0.001 vs. DMSO. (C) ALP activity was determined after 7 and 14 days of Fr2 and PU-13OH-FA treatment in osteogenic medium. Data are presented as fold changes with respect to the control group (CTR) (mean ± SD) (n = 3). * p < 0.05 vs. CTR, ** p < 0.01 vs. CTR, ◦p < 0.01 vs. OM. (D) Representative images of mineralized nodules formation detected using Alizarin Red S staining. Cells were cultured in osteogenic medium in the presence of Fr2 and PU-13OH-FA for 21 days. (E) Quantitative analysis of Alizarin Red S staining was performed after extraction with cethylpyridinium chloride. Data are presented as fold changes with respect to OM (mean ± SD) (n = 3). * p < 0.001 vs. OM, ◦p < 0.05 vs. Fr2-treated group. CTR = cells maintained in basal medium containing 0.5% DMSO. OM = osteogenic medium containing 0.5% DMSO.

Article Snippet: Antibody against human Runx2 (#sc-10758) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: MTT Assay, Control, Expressing, Marker, Immunostaining, Software, Activity Assay, Staining, Cell Culture, Extraction

Journal: Cell reports

Article Title: Cdc42 is required for male germline niche development in mice.

doi: 10.1016/j.celrep.2021.109550

Figure Lengend Snippet: Figure 6. Downregulation of DMRT1 and SOX8/9 in Cdc42-deficient testis (A) Western blot analysis of DMRT1 (n = 6), SOX8/9 (n = 5), and WT1 (n = 5) in busulfan-treated testes from Cdc42-deficient mice. (B) Immunostaining of Cdc42-deficient testes with GATA4 and DMRT1, SOX8/9, or WT1 antibodies. At least 12 tubules were counted. Arrows indicate Sertoli cells lacking candidate proteins. Arrowheads indicate GATA4+ Sertoli cells with candidate protein expression. (C) Western blot analysis of GDNF and candidate proteins following in vitro PD treatment (n = 7). (D) Immunostaining of DMRT1 and SOX8/9 in WT testes following in vitro PD treatment. At least 12 tubules were counted. Arrows indicate Sertoli cells lacking candidate proteins. Arrowheads indicate GATA4+ Sertoli cells with candidate protein expression. Bar, 20 mm (B and D). Stain, Hoechst 33342 (B and D). Asterisk indicates statistical significance (p < 0.05).

Article Snippet: Reagent or resource Source Identifier Antibodies Rat anti-mouse CD117 (KIT) eBioscience Cat#14-1171; RRID:AB_467434; Lot E03982-1632 Rabbit anti-mouse CDC42 Abcam Cat#64533; RRID:AB_1310067; Lot GR3200090-2 Rat anti-mouse CDH1 Gift from Dr. Masatoshi Takeichi, RIKEN CDB RRID: CVCL_A6WX Rabbit anti-mouse CLDN11 Gift from Dr. Sachiko Tsukita, Teikyo University N/A Rabbit anti-mouse non-phospho (active) CTNNB1 (Ser33/37/Thr41) Cell Signaling Technology Cat#8814 (clone: D13A1); Lot 3 Rabbit anti-mouse CXCL12 Torrey Pines Biolabs Cat#201; RRID:AB_2335896; Lot 010718 Goat anti-mouse DMRT1 Santa Cruz Cat#104885X; Lot J2610 Goat anti-mouse FGF2 Santa Cruz Cat#1390; RRID:AB_631496; Lot F1113 Goat anti-mouse GATA4 Santa Cruz Cat#1237; Lot K1611 Rabbit anti-mouse GATA4 Abcam Cat#84593; RRID:AB_10670538; Lot GR275402-1 Rabbit anti-mouse GDNF Santa Cruz Cat#328; RRID:AB_631570; Lot F2713 Rabbit anti-GFP Medical & Biological Laboratories Cat#598; RRID:AB_591819; Lot 081 Goat anti-mouse GFRA1 R&D Systems Cat#AF560; RRID:AB_2110307; Lot BQE0514081 Rabbit anti-human phospho-histone H2A.X (gH2AX) Cell Signaling Technology Cat#2577; RRID:AB_2118010; Lot 4 Rabbit anti-mouse LAMININ Sigma Cat#L9393; RRID:AB_477163; Lot 054K4780 Rat anti-Occludin Gift from Dr. Sachiko Tsukita, Teikyo University N/A Mouse anti-mouse PAFAH1B1 Santa Cruz, Dallas, TX Cat#374586 (clone: H-7); RRID:AB_11008596; Lot H0217 Mouse anti-mouse PEA15A Santa Cruz, Dallas, TX Cat#166678 (clone H-3); RRID:AB_2236964; Lot B1418 Rabbit anti-mouse phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Cell Signaling Technology Cat#9101S; RRID:AB_331646; Lot 26 Rabbit anti-mouse SOX8 Thermo Fisher Scientific Cat#PA1-28072; RRID:AB_2196251; Lot UE2770328F Goat anti-mouse SOX9 Santa Cruz Cat#17341; RRID:AB_661281; Lot F1311 Rabbit anti-mouse SOX9 Santa Cruz Cat#20095; RRID:AB_661282; Lot l1897 Rabbit anti-mouse SYCP1 Abcam Cat#15090; RRID:AB_371671; Lot GR3184119-1 Rabbit anti-mouse SYCP3 Thermo Fisher Scientific Cat#PA1-16764; RRID:AB_568727; Lot WD3244703A Mouse anti-mouse TUBB3 Sigma Cat#T5076; RRID:AB_532291; Lot 047K4852 Rabbit anti-mouse Vimentin Cell Signaling Technology Cat#5741 (clone: D21H3); RRID:AB_10695459; Lot 1 Rabbit anti-mouse WT1 Santa Cruz Cat#192; RRID:AB_632611; Lot K0811 Alexa Fluor 488-conjugated donkey anti-goat IgG Thermo Fisher Scientific Cat#A-11055; RRID:AB_2534102; Lot 2059218 (Continued on next page) e1 Cell Reports 36, 109550, August 17, 2021

Techniques: Western Blot, Immunostaining, Expressing, In Vitro, Staining

Journal: Biomolecules

Article Title: Fermented Oyster Extract Promotes Osteoblast Differentiation by Activating the Wnt/β-Catenin Signaling Pathway, Leading to Bone Formation.

doi: 10.3390/biom9110711

Figure Lengend Snippet: Figure 4. FO activates osteoblast-specific protein expression and mineralization/calcification in osteosarcoma MG-63 human osteoblast-like cells. (A) MG-63 cells (3 × 103 cells/mL) were seeded overnight and then treated with FO (50 µg/mL and 100 µg/mL) or DEX (100 nM) for 7 days. Protein was extracted and western blotting analysis was performed using each specific antibody. β-Actin was used as the internal control of protein expression. All protein expression was normalized by the density of β-actin. The nuclear localization of runt-related transcription factor 2 RUNX2 (B) and osterix OSX (C) was measured by immunostaining. (D) ALP activity (mineralization) was measured using a TRACP & ALP assay kit. (E) In vitro calcification was detected by alizarin red staining. Significant differences among the groups were determined using the one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (*** p < 0.001, ** p < 0.01, and * p < 0.05 versus untreated group). FO; fermented extract of C. gigas and DEX; dexamethasone. FO; fermented extract of C. gigas and DEX; dexamethasone.

Article Snippet: Specific antibodies against RUNX2, ALP, Col1α1, OCN, OSX, BMP4, β-actin, β-catenin, and nucleolin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Control, Immunostaining, Activity Assay, ALP Assay, In Vitro, Staining

Journal: Biomolecules

Article Title: Fermented Oyster Extract Promotes Osteoblast Differentiation by Activating the Wnt/β-Catenin Signaling Pathway, Leading to Bone Formation.

doi: 10.3390/biom9110711

Figure Lengend Snippet: Figure 4. FO activates osteoblast-specific protein expression and mineralization/calcification in osteosarcoma MG-63 human osteoblast-like cells. (A) MG-63 cells (3 × 103 cells/mL) were seeded overnight and then treated with FO (50 µg/mL and 100 µg/mL) or DEX (100 nM) for 7 days. Protein was extracted and western blotting analysis was performed using each specific antibody. β-Actin was used as the internal control of protein expression. All protein expression was normalized by the density of β-actin. The nuclear localization of runt-related transcription factor 2 RUNX2 (B) and osterix OSX (C) was measured by immunostaining. (D) ALP activity (mineralization) was measured using a TRACP & ALP assay kit. (E) In vitro calcification was detected by alizarin red staining. Significant differences among the groups were determined using the one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (*** p < 0.001, ** p < 0.01, and * p < 0.05 versus untreated group). FO; fermented extract of C. gigas and DEX; dexamethasone. FO; fermented extract of C. gigas and DEX; dexamethasone.

Article Snippet: Specific antibodies against RUNX2, ALP, Col1α1, OCN, OSX, BMP4, β-actin, β-catenin, and nucleolin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Control, Immunostaining, Activity Assay, ALP Assay, In Vitro, Staining

Journal: The Journal of cell biology

Article Title: ARFRP1 functions upstream of ARL1 and ARL5 to coordinate recruitment of distinct tethering factors to the trans-Golgi network.

doi: 10.1083/jcb.201905097

Figure Lengend Snippet: Figure 2. Both ARL5 and ARFRP1 are required for localization of GARP to the TGN. (A) KO of TGN-localized small GTPases in HeLa cells confirmed by immunoblot analysis with antibodies to the proteins indicated on the right. In this figure and subsequent figures, ARL5 KO represents KO of both ARL5A and ARL5B, and RAB6 KO represents KO of both RAB6A and RAB6B. α-Tubulin was used as a loading control. The positions of molecular mass markers are indicated on the left. (B) Immunofluorescence microscopy of WT and KO HeLa cells transfected with a plasmid encoding VPS54-13Myc and stained for the Myc epitope (red), giantin (green), and nuclei (DAPI; blue). Scale bars: 10 μm. Insets are magnified views of the boxed areas. Inset scale bars: 5 μm. (C) Quantification of the percentage of cells exhibiting VPS54-13Myc staining at the TGN. Values are the mean ± SEM from three independent experiments. More than 100 cells per sample were counted in each experiment. The statistical significance of the differences relative to WT cells was determined using Dunnett’s test. **, P < 0.01; ***, P < 0.001.

Article Snippet: The following antibodies were used for immunoblotting and/or immunofluorescence microscopy: rabbit anti-VPS51 (HPA039650; Atlas Antibodies), rabbit anti-VPS52 made in our laboratory (Pérez-Victoria et al., 2008), rabbit anti-VPS53 (HPA024446; Atlas Antibodies), mouse anti-VPS50 (FLJ20097, monoclonal antibody M01, 2D11; Abnova), mouse anti-Myc epitope (9E10; Santa Cruz Biotechnology), sheep anti-TGN46 (AHP500G; Bio-Rad), mouse anti–β-actin (G043; Applied Biological Materials), rabbit antigiantin (ab80864; Abcam), mouse HRP-conjugated anti-α-tubulin (DM1A; Santa Cruz Biotechnology), rabbit anti-ARL1 (16012-1-AP; Proteintech), rabbit anti-ARFRP1 (PA5-50606; Thermo Fisher Scientific), mouse anti-ARL5A (sc-514680; Santa Cruz Biotechnology), rabbit anti-RAB6A (GTX110646; GeneTex), rabbit anti-GCC88 (HPA021323; Sigma-Aldrich), rabbit anti-GCC185 (HPA035849; Sigma-Aldrich), mouse anti-Golgin-245 (611281; BD Biosciences), mouse anti-GM130 (610822; BD Biosciences), mouse anti–Golgin-97 (A-21270; Thermo Fisher Scientific), rabbit anti-TMF1 (HPA008729, Sigma-Aldrich), monoclonal HRP-conjugated anti-GFP (Miltenyi Biotec Inc.), rabbit anti-GFP (A-11122; Thermo Fisher Scientific), HRP-conjugated goat anti-rabbit and donkey anti-mouse antibodies (Jackson ImmunoResearch), HRP-conjugated donkey antisheep (R&D Systems), and Alexa Fluor–conjugated secondary antibodies for immunostaining (Thermo Fisher Scientific).

Techniques: Western Blot, Control, Immunofluorescence, Microscopy, Transfection, Plasmid Preparation, Staining

Journal: The Journal of cell biology

Article Title: ARFRP1 functions upstream of ARL1 and ARL5 to coordinate recruitment of distinct tethering factors to the trans-Golgi network.

doi: 10.1083/jcb.201905097

Figure Lengend Snippet: Figure 5. Small GTPases required for localization of golgins to the TGN. (A) Immunofluorescence microscopy of WT, ARL1-KO, ARFRP1-KO, ARL5-KO, and RAB6-KO cells immunostained for endogenous Golgin-245, Golgin-97, GCC88, GCC185, or TMF1 and counterstained with DAPI (blue). Scale bars: 10 μm. Insets are magnified views of the boxed areas. Inset scale bars: 5 μm. Notice that ARL1 KO or ARFRP1 KO caused complete disappearance of Golgin-245 and GCC88, and a partial decrease in the intensity of Golgin-97, at the TGN; quantification in 10 cells per sample in three independent experiment showed that Golgin-97 decrease was 75.6% ± 2.4% in ARL1-KO cells and 55.0% ± 1.9% in ARFRP1-KO cells. (B) SDS-PAGE and immunoblot analysis of endogenous golgins and α-tubulin (loading control) in WT and KO cells. The positions of molecular mass markers are indicated on the left. (C) Immunofluorescence microscopy of RAB6- KO cells transfected with a plasmid encoding GFP-tagged mouse Rab6A (green), immunostained for endogenous GCC185 and TMF1 (red), and counterstained with DAPI (blue). Cells were examined for GFP fluorescence by confocal microscopy. Scale bars: 10 μm.

Article Snippet: The following antibodies were used for immunoblotting and/or immunofluorescence microscopy: rabbit anti-VPS51 (HPA039650; Atlas Antibodies), rabbit anti-VPS52 made in our laboratory (Pérez-Victoria et al., 2008), rabbit anti-VPS53 (HPA024446; Atlas Antibodies), mouse anti-VPS50 (FLJ20097, monoclonal antibody M01, 2D11; Abnova), mouse anti-Myc epitope (9E10; Santa Cruz Biotechnology), sheep anti-TGN46 (AHP500G; Bio-Rad), mouse anti–β-actin (G043; Applied Biological Materials), rabbit antigiantin (ab80864; Abcam), mouse HRP-conjugated anti-α-tubulin (DM1A; Santa Cruz Biotechnology), rabbit anti-ARL1 (16012-1-AP; Proteintech), rabbit anti-ARFRP1 (PA5-50606; Thermo Fisher Scientific), mouse anti-ARL5A (sc-514680; Santa Cruz Biotechnology), rabbit anti-RAB6A (GTX110646; GeneTex), rabbit anti-GCC88 (HPA021323; Sigma-Aldrich), rabbit anti-GCC185 (HPA035849; Sigma-Aldrich), mouse anti-Golgin-245 (611281; BD Biosciences), mouse anti-GM130 (610822; BD Biosciences), mouse anti–Golgin-97 (A-21270; Thermo Fisher Scientific), rabbit anti-TMF1 (HPA008729, Sigma-Aldrich), monoclonal HRP-conjugated anti-GFP (Miltenyi Biotec Inc.), rabbit anti-GFP (A-11122; Thermo Fisher Scientific), HRP-conjugated goat anti-rabbit and donkey anti-mouse antibodies (Jackson ImmunoResearch), HRP-conjugated donkey antisheep (R&D Systems), and Alexa Fluor–conjugated secondary antibodies for immunostaining (Thermo Fisher Scientific).

Techniques: Immunofluorescence, Microscopy, SDS Page, Western Blot, Control, Transfection, Plasmid Preparation, Fluorescence, Confocal Microscopy

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 1. Protective effect of D4476 and possible association with autophagy in chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 (commercially available stable RPE cell line) and primary mouse RPE cells. Cytotoxicity of ARPE-19 cells treated with (A) varying concentrations of CQ (10–100 μM) alone for 24 h; (B) 100 μM CQ with vehicle or different D4476 concentrations (1–20 μM) for 24 h; (C) vehicle, 100 μM CQ, CQ+D4476 and 10 μM D4476 for different time-points. (D and E) Relative survival rate of ARPE-19 cells treated with casein kinase 1 (CK1) inhibitor, IC261, and selective activin receptor-like kinase (ALK)5 inhibitor, SB525334, instead of D4476 with the same combination of drugs for 24 h and (F) 100 μM CQ with vehicle and with 0.5 μM rapamycin (Rap) for 24 h. Cytotoxicity assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay unless otherwise specified. (G) Phase-contrast and fluorescence photo micrographs of primary mouse RPE cells. After 3 weeks of culture, cells were stained with RPE65, marker protein of RPE, and Na+/K+-ATPase, marker of plasma membrane. Scale bar, 20 μm. (H and I) Relative survival rate of primary mouse RPE cells. Cells were treated with vehicle, 100 μM CQ, CQ+D4476, 10 μM D4476, CQ+IC261, 1 μM IC261, CQ+SB525334, 1 μM SB525334, CQ+Rap, and 0.5 μM Rap for 24 h. Mean ± standard deviation (SD), n = 3; ##P < 0.01 and ###P < 0.001 compared to vehicle and *P < 0.05, **P < 0.01, ***P < 0.001 compared to CQ alone. ns, not significant, compared to vehicle or CQ-treated cells.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: MTT Assay, Fluorescence, Staining, Marker, Clinical Proteomics, Membrane, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 2. Attenuation of chloroquine (CQ)-induced inhibition of autophagy by D4476 in adult retinal pigment epithelial (ARPE)-19 cells. (A) Phase-contrast photo micrograph and (B) confocal images of green fluorescent protein-light chaing 3 (GFP-LC3, green)-transfected cells after immunostaining with anti-lysosomal asso ciated membrane protein (LAMP)-1 (red) antibody. Cells were treated with vehicle, 100 μM CQ alone, 10 μM D4476 alone, or 10 μM D4476 + 100 μM CQ for 6 h. 4ʹ,6-Diamidino-2-phenylindole (DAPI, blue) was used to counterstain nuclei. Scale bar, 10 μm. Quantification of LC3-positive vacuole (C) size, (D) number (green), and (E) number of LC3-positive (green) vacuoles colocalized (yellow) to LAMP-1 (red). One-hundred cells per experimental condition were analyzed using fluo rescence microscopy. (F) Western blot and quantitative analyses of Beclin 1, p62, and LC3 A/B expression in ARPE-19 cell lysates 6 h after treatment with vehicle, 100 μM CQ alone, 10 μM D4476 + 100 μM CQ, 10 μM D4476 alone, 0.5 μM rapamycin (Rap) plus 100 μM CQ, and 0.5 μM Rap alone. α-Tubulin was used as a loading control. Mean ± standard deviation (SD), n = 3; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to vehicle; *P < 0.05 and **P < 0.01 compared to CQ alone. ns, not significant, compared to vehicle or Bafilomycin A1 (BA1) alone-treated cells.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Transfection, Immunostaining, Membrane, Microscopy, Western Blot, Expressing, Control, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 5. Effect of D4476 on chloroquine (CQ)-induced interaction of Beclin 1 B-cell lymphoma 2 (Bcl-2) homology 3 (BH3) domain with Bcl-2 and cell proliferation- associated signaling in adult retinal pigment epithelial (ARPE)-19 cells. (A) immunoblotted or (B) immunoprecipitated ARPE-19 cells. Cells were immunoprecipitated with anti-mouse IgG, as an isotype control, or anti-Bcl-2 antibody, then immunoblotted with anti-Beclin 1 or anti-Bcl-2 antibody. (C and D) Densitometric analysis of Immunoblots with Beclin 1 and Bcl-2. Quantitative analysis performed using ImageJ software. (E) Western blot and (F) quantitative densitometric analyses of phosphorylated phosphoinositide 3-kinase (p-PI3K), PI3K, p-AKT, AKT, p-mechanistic target of rapamycin (Rap, mTOR), mTOR, p-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, p-c-Jun N terminal kinase (JNK), and JNK in APRE-19 cells 6 h after treatment with vehicle, 100 μM CQ alone, 10 μM D4476 + 100 μM CQ, 10 μM D4476 alone, 0.5 μM Rap+100 μM CQ, and 0.5 μM Rap alone. Data are means ± standard deviation (SD), n = 3; #P < 0.05 and ##P < 0.01 compared to vehicle and *P < 0.05 and **P < 0.01 compared to CQ alone. ns, not significant, compared to vehicle or CQ alone.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Control, Western Blot, Software, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 7. c-Jun N terminal kinase (JNK) inhibitor-induced reproduction of D4476 effects in chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells. (A) Relative survival percentage of APRE-19 cells treated with 100 μM CQ alone, 100 μM CQ+10 μM SP600125, and 10 μM SP600125 alone for 24 h. (B) immu noblotted or (C) immunoprecipitated ARPE-19 cells. Cell lysates immunoprecipitated with anti-mouse Ig G or anti-B-cell lymphoma 2 (Bcl-2) antibody were immunoblotted with anti-Beclin 1 or anti-Bcl-2 antibody. Bars denote densitometric analysis of Immunoblots. (D) Western blots and (E and F) quantitative densi tometric analyses for phosphorylated mechanistic target of rapamycin (p-mTOR), mTOR, p-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, p-JNK, JNK, light chain 3 (LC3) A/B, p62, and Bcl-xL in APRE-19 cells 6 h after treatment as in (A). Data are means ± standard deviation (SD), n = 3; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to vehicle and *P < 0.05, **P < 0.01, and ***P < 0.001 compared to CQ alone. ns, not significant, compared to vehicle or CQ alone.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 6. p38 Mitogen-activated protein kinase (MAPK) inhibitor-induced reproduction of D4476 effects in chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells. (A) Relative survival rate of APRE-19 cells 24 h after treatment with vehicle, 100 μM CQ alone, 100 μM CQ+20 μM SB203580, 20 μM SB203580 alone. (B) immunoblotted or (C) immunoprecipitated ARPE-19 cells. Cell lysates immunoprecipitated using anti-mouse Ig G or anti-B-cell lymphoma 2 (Bcl-2) antibody were immunoblotted with anti-Beclin 1 and anti-Bcl-2 antibodies. Bars denote densitometric analyses of Immunoblots. (D) Western blot and (E and F) quantitative densitometric analyses of phosphorylated mechanistic target of rapamycin (p-mTOR), mTOR, p-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, p-c-Jun N terminal kinase (JNK), JNK, light chain 3 (LC3) A/B, p62, and Bcl-2 extra-large (Bcl-xL) in APRE-19 cells 6 h after treatment as in (A). Data are means ± standard deviation (SD), n = 3; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to vehicle and *P < 0.05, **P < 0.01, and ***P < 0.001 compared to CQ alone. ns, not significant, compared to vehicle or CQ alone.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 8. Schematic representation of D4476 effects on crosstalk between autophagy and apoptosis in chlo roquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells. D4476 inhibits CQ-induced increase of mechanistic target of rapamycin (mTOR), c-Jun N terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) activities. This inhibitory ef fect alters CQ-mediated interaction between Beclin 1 and B-cell lymphoma 2 (Bcl-2) via the Bcl-2 homol ogy 3 (BH3) domain, resulting in release of Beclin 1 from Bcl-2 and activating autophagy. Beclin 1, light chain 3 (LC3) A/B, and p62 are all associated with autophagosome formation and autophagy flux. D4476 may play an important role in the gateway of intersection between autophagy and apoptosis during CQ-induced toxicity, mitigating damage through reinstating cellular homeostasis.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: