Fig 2: Pafah1b1 knockdown reverses the pro-growth of miR-181c/d inhibited murine Sertoli cells. The murine SCs were transfected with NC siRNA or Pafah1b1 siRNA. a Immunofluorescence staining of Ki67 (red) in Pafah1b1 siRNA treated murine SCs. Scale bar: 100 µm. b Quantification of Ki67-positive cells in Pafah1b1 siRNA treated murine SCs. c CCK-8 assay performed in Pafah1b1 siRNA treated murine SCs. d Western blot analysis of PAFAH1B1, PCNA, BAX, and BCL2 in Pafah1b1 siRNA treated murine SCs. The quantification of protein level is shown in the bar graph (e). f Annexin V-FITC/PI and flow cytometry analysis was used to examine cell apoptotic rate in Pafah1b1 siRNA treated murine SCs. g The quantification of cell apoptotic rate in Pafah1b1 siRNA treated murine SCs. Five co-transfection treatments were constructed in this experiment, including inhibitors NC + NC siRNA, miR-181c inhibitors + NC siRNA, miR-181d inhibitors + NC siRNA, miR-181c inhibitors + Pafah1b1 siRNA, and miR-181d inhibitors + Pafah1b1 siRNA. h-j Ki67 staining (h) and CCK-8 (j) assay were performed in murine SCs treated with co-transfections. Quantification of Ki67-positive murine SCs treated with co-transfections (i). Scale bar: 100 µm. k Western blot analysis of PAFAH1B1, PCNA, BAX, and BCL2 in murine SCs treated with co-transfections. The quantification of protein level is shown in the bar graph (l). m Annexin V-FITC/PI and flow cytometry analysis was used to examine cell apoptotic rate in murine SCs treated with co-transfections. n The quantification of cell apoptotic rate in murine SCs treated with co-transfections. Data are presented as mean ± SD of at least three independent experiments. *p < 0.05; **p < 0.01; ns, not significant

Fig 3: TRAK2 forms a complex with kinesin-1, dynein, and dynactin.a Representative kymographs show TRAK2-motor complexes switching direction during a run. b Lysates from COS-7 cells transfected with HA-TRAK2, FLAG-p150Glued, and Myc-KIF5B were immunoprecipitated with a FLAG antibody or negative control GFP antibody. Lysates from COS-7 cells expressing just FLAG-p150Glued and Myc-KIF5B were immunoprecipitated with a FLAG antibody in parallel. c Quantification of the difference in Myc-KIF5B co-immunoprecipitation with FLAG-p150Glued upon addition of HA-TRAK2. Data points are colored according to experimental replicate. Bars represent mean ± s.d. from three independent experiments. *p = 0.0405 (two-tailed t-test). d Lysates from COS-7 cells transfected with HA-TRAK2 and Myc-KIF5C-Halo were immunoprecipitated with a Halo antibody or negative control GFP antibody. Lysates from COS-7 cells expressing just Myc-KIF5C-Halo were immunoprecipitated with a Halo antibody in parallel. e Quantification of the difference in dynein heavy chain co-immunoprecipitation with Myc-KIF5C-Halo upon addition of HA-TRAK2. Data points are colored according to experimental replicate. Bars represent mean ± s.d. from three independent experiments. *p = 0.0410 (two-tailed t-test). f Schematic illustration for three-color single-molecule imaging of GFP-dynein heavy chain, Myc-KIF5C-Halo labeled with TMR, and SNAP-TRAK2 labeled with Janelia Fluor 646. HA-LIS1 is also present in this experiment. g Time series showing a processive complex containing KIF5C, TRAK2, and dynein heavy chain. The gray line indicates microtubule position, as inferred from the max projection of KIF5C. Right: corresponding kymographs.

Fig 4: The TRAK2 CC1-Box is important for processive motility and binding to dynein, but not kinesin-1.a Schematic overview of TRAK2 with sequence alignment showing conservation of the CC1-Box. The red stars and text indicate the mutations introduced to TRAK2. b Representative kymographs showing the effect of A/V and I/D mutations on TRAK2 motility along MTs when LIS1 is also expressed. Red, blue, and yellow arrows indicate minus-end, plus-end, and diffusive TRAK2 movement, respectively. c Normalized frequency of TRAK2 transport to the microtubule minus-end with exogenous LIS1. Data points are color-coded to experimental replicate, with smaller points representing TRAK2 frequency per video. The center line and bars represent the mean ± s.d. from independent experiments (n = 22 videos from four experiments for WT, 18 videos from three experiments for A/V, and 21 videos from four experiments for I/D). **p = 0.0047; ***p = 0.0009 (one-way ANOVA with the Dunnett’s multiple comparisons test). d, e Inverse cumulative distribution functions (CDF) of run length and histogram distributions of velocity for TRAK2 transport to the microtubule minus-end with exogenous LIS1 present (n = 92 events for TRAK2 WT, 30 events for TRAK2 A/V, and 17 events for TRAK2 I/D). The curves in (d) represent single exponential decay fits with decay constants indicated above. The values in (e) are mean ± s.d. f Same as c, but for TRAK2 transport to the microtubule plus-end. Exact p values from one-way ANOVA with the Dunnett’s multiple comparisons test are shown. g, h Same as d, e, but for TRAK2 transport to the microtubule plus-end (n = 693 events for TRAK2 WT, 369 events for TRAK2 A/V, and 391 events for TRAK2 I/D). i Immunoprecipitation using a Halo antibody of extracts from COS-7 cells transfected with Myc-KIF5B and Halo-tagged TRAK2, TRAK2 A/V, TRAK2 I/D, or negative control Optineurin (OPTN). j Quantification of co-immunoprecipitation of endogenous dynein heavy chain (DHC) with Halo-tagged constructs. Data points are color-coded according to experimental replicate. The center line and bars represent the mean ± s.d. from three independent experiments. *p < 0.05 (one-way ANOVA with the Dunnett’s multiple comparisons test). p values: WT vs. A/V, p = 0.0475; WT vs. I/D, p = 0.0416; WT vs. OPTN, p = 0.0346. k Same as j but for co-immunoprecipitation of Myc-KIF5B. ***p < 0.001; ns not significant (one-way ANOVA with the Dunnett’s multiple comparisons test). p values: WT vs. A/V, p = 0.8697; WT vs. I/D, p = 0.8513; WT vs. OPTN, p = 0.0004.

Fig 5: Inhibition of Pafah1b1 disturbs the Sertoli cell barrier in vitro. The murine SCs were transfected with NC siRNA or Pafah1b1 siRNA. NC siRNA and Pafah1b1 siRNA are abbreviated to si-NC and si-paf, respectively. a, b The permeability of the Sertoli cell barrier was assessed in vitro by quantifying TER (a) or measuring the permeability of Na-F (b) in Pafah1b1 siRNA treated murine SCs. c Western blot analysis of TJ proteins and basal ES proteins in Pafah1b1 siRNA treated murine SCs. The quantification of protein level is shown in the bar graph (d). e Immunofluorescence staining of TJ proteins (red) and basal ES proteins (red) in Pafah1b1 siRNA treated murine SCs. These proteins are tightly localized (white brackets) or diffusively localized (yellow brackets) at the Sertoli cell–cell interface. Scale bar: 5 µm. f Quantification of fluorescence signal distributed at the cell–cell interface. g TEM ultrastructural analysis in Pafah1b1 siRNA treated murine SCs. Intact (white arrowheads) or disrupted (yellow arrowheads) TJ structures between adjacent murine SC contact. Scale bar: 1 µm. Nu, nucleus; SC, Sertoli cell. h F-actin staining (green) in Pafah1b1 siRNA treated murine SCs. Ordered (white arrows) or disordered (yellow arrows) F-actin are indicated. Scale bar: 20 µm. Data are presented as mean ± SD of at least three independent experiments. *p < 0.05; **p < 0.01; ns, not significant