Journal: Communications Biology

Article Title: PACS1 syndrome mutation disrupts dynein-mediated cargo transport via HDAC6 and BICD2

doi: 10.1038/s42003-026-09924-0

Figure Lengend Snippet: a Silver-stained 6% SDS–PAGE of proteins co-immunoprecipitated with FLAG–PACS1 or FLAG–PACS1 R203W from HCT116 cells using M2 agarose. LC–MS/MS identified DHC1 and DNA-PKcs as the major high–molecular-weight species (Table ). b FLAG–PACS1 or FLAG–PACS1 R203W immunoprecipitated from HCT116 cells, with co-precipitating endogenous DHC1 detected by Western blot. Data are mean ± SEM, two-tailed t-test, n = 4 independently normalized experiments. c Endogenous DHC1 immunoprecipitated from mouse brain cytosol with co-precipitating PACS1 detected by Western blot (anti-PACS1 #703). IgG, negative control. Arrow, 530-kDa DHC1; lower band likely represents ~250-kDa brain-specific DHC1 isoforms. Experiments repeated three times with similar results. d Top : Control (160) fibroblasts nucleofected with PACS1, DHC1, or nonspecific siRNAs and analyzed by Western blot. Experiments repeated three times with similar results. Middle: Control (160) and patient (159) fibroblasts treated with the indicated siRNAs, stained for Giantin (Golgi, red), furin (TGN, green), and DAPI (blue), then imaged using confocal microscopy. Cell outlines, white. Scale bar, 20 µm. Bottom : Quantification of Giantin (Golgi) and furin dispersal (percentage of signal within or beyond 10 µm of the nuclear envelope). Data are mean ± SEM, ANOVA with Dunnett’s post hoc, n > 20 cells per group from three independent experiments. See Fig. for single-channel images.

Article Snippet: Samples were then incubated with primary antibodies for 2 h at RT or overnight at 4 °C, washed 3 times with PBS, and incubated with fluorophore-conjugated secondary antibodies at RT for 1 h. Finally, coverslips were mounted with DAPI-Fluoromount G (Southern Biotech, 0100-20) and incubated overnight in the dark.

Techniques: Staining, SDS Page, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, High Molecular Weight, Western Blot, Two Tailed Test, Negative Control, Control, Confocal Microscopy

Journal: Communications Biology

Article Title: PACS1 syndrome mutation disrupts dynein-mediated cargo transport via HDAC6 and BICD2

doi: 10.1038/s42003-026-09924-0

Figure Lengend Snippet: a FLAG-tagged PACS1 or PACS1 Dynmut (F121, S122, L128, V129, E168 → 5 A) immunoprecipitated from HCT116 cells (M2 agarose); co-precipitating endogenous DHC1 detected by Western blot (anti-DHC1). Data are mean ± SEM, two-tailed t-test, n = 3 independently normalized experiments. b FLAG–CD8-furin co-expressed with HA-tagged PACS1, PACS1 Dynmut , or PACS1 R203W was immunoprecipitated from HCT116 cells; co-precipitating HA-tagged PACS1 variants detected by Western blot (anti-HA). Data are mean ± SEM, ANOVA with Dunnett’s post hoc, n = 3 independently normalized experiments. c Top : U2OS cells expressing HA-tagged PACS1, PACS1 Dynmut , or PACS1 R203W stained for Giantin (Golgi, red), endogenous furin (green), HA (magenta), and DAPI (blue) and imaged by confocal microscopy. Cell outlines, white. Scale bar, 10 µm. Bottom : Quantification of Giantin and furin dispersal (percentage of signal within or beyond 5 µm of nuclear envelope). Data are mean ± SEM, ANOVA with Dunnett’s post hoc, n > 20 cells per group from three independent experiments. See Fig. for single-channel images.

Article Snippet: Samples were then incubated with primary antibodies for 2 h at RT or overnight at 4 °C, washed 3 times with PBS, and incubated with fluorophore-conjugated secondary antibodies at RT for 1 h. Finally, coverslips were mounted with DAPI-Fluoromount G (Southern Biotech, 0100-20) and incubated overnight in the dark.

Techniques: Immunoprecipitation, Western Blot, Two Tailed Test, Expressing, Staining, Confocal Microscopy

Journal: Communications Biology

Article Title: PACS1 syndrome mutation disrupts dynein-mediated cargo transport via HDAC6 and BICD2

doi: 10.1038/s42003-026-09924-0

Figure Lengend Snippet: a FLAG-HDAC6 co-expressed with BICD2–eGFP and HA-tagged PACS1 or PACS1 R203W was immunoprecipitated from HCT116 cells (M2 agarose); co-precipitating BICD2–eGFP and HA-tagged PACS1 variants detected by Western blot. Data are mean ± SEM, ANOVA with Dunnett’s post hoc, n = 3 independently normalized experiments. b Control (160) and patient (159) fibroblasts nucleofected with BICD2 or nonspecific (ns) siRNAs. After 48 h, cells were collected for Western blot ( bottom left ) or treated overnight with G1 inhibitor ribociclib (3 μM), G2/M inhibitor Ro-3306 (9 μM), or DMSO. Top: Cells were fixed, stained for Giantin (Golgi, red), α-tubulin (green), and DAPI (blue), then imaged by confocal microscopy. Scale bar, 20 μm. Bottom right : Quantification of Golgi dispersal (percentage of Giantin signal within or beyond 10 μm of nuclear envelope). Data are mean ± SEM, ANOVA with Dunnett’s post hoc, n > 50 cells per group from three independent experiments.

Article Snippet: Samples were then incubated with primary antibodies for 2 h at RT or overnight at 4 °C, washed 3 times with PBS, and incubated with fluorophore-conjugated secondary antibodies at RT for 1 h. Finally, coverslips were mounted with DAPI-Fluoromount G (Southern Biotech, 0100-20) and incubated overnight in the dark.

Techniques: Immunoprecipitation, Western Blot, Control, Staining, Confocal Microscopy

Journal: Communications Biology

Article Title: PACS1 syndrome mutation disrupts dynein-mediated cargo transport via HDAC6 and BICD2

doi: 10.1038/s42003-026-09924-0

Figure Lengend Snippet: a Left: Control (160) and patient (159) cells expressing Lis1-HA, fixed 48 h later, stained for Giantin (Golgi, red), HA (green; pseudocolored magenta), and DAPI (blue). Cell outlines, white. Scale bar, 20 µm. Lis1–HA⁺ cells (*). Scale bar, 20 µm. Right : Quantification of Golgi dispersal (percentage of Giantin signal located within or beyond 10 µm of nuclear envelope). Data are mean ± SEM, two-way ANOVA with Tukey’s post hoc, n > 40 cells per group from three independent experiments. b Top: Control (160) and patient (159) cells treated overnight with 5 µM tubacin or DMSO, fixed, stained for lysosomes (LAMTOR, red), α-tubulin (green), and DAPI (blue). Cell outlines, white. Scale bar, 20 µm. Bottom: Quantification of LAMTOR dispersal (percentage of signal located within or beyond 10 µm of nuclear envelope). Data are mean ± SEM, two-way ANOVA with Tukey’s post hoc, n > 100 cells per group from three independent experiments. c Left : Patient (159) cells expressing Lis1-HA stained as in ( b ); Cell outlines, white. Lis1–HA⁺ cells (*). Scale bar, 20 µm. Right : Quantification of LAMTOR dispersal (percentage of signal located within or beyond 10 µm of nuclear envelope). Data are mean ± SEM, two-way ANOVA with Tukey’s post hoc, n > 70 cells per group from three independent experiments. d U2OS cells expressing PEX3–eYFP–FKBP and BICD2 CC1/2–FRB with Lis1, plus either PACS1 or PACS1 R203W , treated with 1 µM rapalog to induce peroxisome transport. Live imaging (2 fps; six 3-min movies) was used to quantify peroxisome motility. Left: Representative kymographs. Arrowheads, rapalog-induced peroxisome motility in the presence of PACS1 R203W or PACS1 R203W +Lis1. y-axis, time (180 s); x-axis, distance. Scale bar, 2 µm. Top right : Mean peroxisome velocities per cell. Bottom right : Percentage of motile eYFP⁺ peroxisomes per cell over the 18-min interval. Data are mean ± SEM (two-way ANOVA with Tukey’s post hoc); n = 15 / 5547, 17 / 6777, 18 /7916, and 16 / 6438 (cells / total eYFP + peroxisomes) for PACS1, PACS1 + Lis1, PACS1 R203W , and PACS1 R203W + Lis1, respectively, from three independent experiments.

Article Snippet: Samples were then incubated with primary antibodies for 2 h at RT or overnight at 4 °C, washed 3 times with PBS, and incubated with fluorophore-conjugated secondary antibodies at RT for 1 h. Finally, coverslips were mounted with DAPI-Fluoromount G (Southern Biotech, 0100-20) and incubated overnight in the dark.

Techniques: Control, Expressing, Staining, Imaging