Fig 2: Expression of primed pluripotency cell-surface protein markers in bioreactor-cultured, naïve and primed hPSCs.a Average percentages of CD90-positive cells in bioreactor-cultured, naïve and primed H9 hPSCs. The data are represented as mean percentage of positive cells (n = 2). b Representative immunofluorescent images showing CD90 expression in bioreactor-cultured, naïve and primed H9 hPSC aggregates. CD90 antibody was conjugated with R-phycoerythrin (R-PE). Scale bars = 20 µm. c Histogram of flow cytometry analysis showing fluorescently labeled CD90, CD24, and CD317 in bioreactor-cultured, naïve and primed H9 hPSCs. Gates were drawn based on an isotype control. Aggregates of day 4 post-inoculation were used for flow cytometry analysis and for confocal imaging for both naïve and primed hPSCs. All the cultures underwent fed-batch condition (60% media change, 48-h post-inoculation) for bioreactor cultures. The data presented are generated from inoculation density of 50,000 cells/mL. d Expression dynamics of CD24 cell-surface protein during primed to naïve pluripotency conversion in the stirred suspension bioreactor within six days of culture. The media was changed gradually from mTESR1 to RSeT from day 2 to day 6 of culture in the bioreactor, and the cells were collected every day for analysis.

Fig 3: WDR81 forms a functional complex with WDR91. A) Mass spectrometry analysis of 2×HA-WDR81 immunoprecipitates from 2×HA-WDR81 expressing HeLa cells as compared with wild-type control HeLa cells identifies WDR91 as a WDR81 binding partner. B) WDR91 was depleted in HeLa cells by stable expression of Cas9 and three independent WDR91-specific sgRNAs. Tetherin trafficking in the resulting WDR91 KO cell lines and controls was analysed using confocal immunostaining. Scale bars represent 10 µm. C) WDR91 was depleted in WDR81KO clones and tetherin distribution was imaged using confocal microscopy. Scale bars represent 10 µm. D) Confocal microscopy images of early EEA1 positive and late LAMP1 positive endolysosomal compartments in control and WDR91 depleted HeLa cells. Scale bars represent 5 µm.

Fig 4: SIVagm constructs expressing functional Vpu and HIV-1 Nef. a Genomic organization of the WT SIVagm construct with the nef gene (yellow) and the GU1N derivative containing the SIVgsn vpu (blue) and the HIV-1 NA7 nef (orange). b HEK293T cells were cotransfected with a vpu-deleted HIV-1 NL4-3 construct, expression plasmids for HIV-1 or SIVgsn Vpu and a vector expressing AGM tetherin (BST-2). Infectious virus yield was measured by infection of TZM-bl cells. Curves represent the average values (n = 3) relative to those obtained in the absence of tetherin (100%). c Levels of AGM BST-2 and human CD4, NTB-A, and CD1d in HEK293T cells cotransfected with SIVgsn or HIV-1 Vpu constructs relative to those measured in cells transfected with the eGFP control vector (100%). Symbols represent the value obtained in one of three experiments. Mean ± SEM are indicated in panels c, d, e, and j. d Effect of Vpu on NF-?B activity. HEK293T cells were cotransfected with a firefly luciferase NF-?B reporter construct, a Gaussia luciferase construct for normalization, a vector for constitutively active IKKß and constructs expressing eGFP alone or together with Vpu. Luciferase activity was determined two dpi (n = 3 ± SEM). CTRL represents the vector control. e Receptor downmodulation in human PBMCs or CD4+ T cells (CD4) infected with HIV-1 constructs expressing the indicated Nefs relative the nef-defective virus (100%), (n = 3 ± SEM). f Effect of NA7 and SIVagm Nefs on virus release in the presence of AGM tetherin in transfected HEK293T cells. Curves represent averages (n = 3) relative to those obtained in the absence of tetherin (100%). g, h Infectivity and replication of SIVagm constructs in vitro. TZM-bl and MOLT-4 clone 8 cells (g) or AGM PBMCs (h) were infected with the indicated SIVagm constructs. Virus infectivity or production was monitored by ß-galactosidase assay or p27 antigen ELISA, respectively. Shown is g mean of triplicate infection or h one representative experiment. i Average infectious virus yield (n = 3) from HEK293T cells following co-transfection with SIVagm constructs and plasmids expressing AGM tetherin relative to that detected in the absence of tetherin (100%). j Effect of SIVagm constructs on receptor expression by PBMCs from three AGM relative to those infected with the nef-defective SIVagm clone (100%). p-values were calculated using the two-tailed unpaired Student's-t-test and significant differences are indicated as: *p < 0.05; **p < 0.01; ***p < 0.001

Fig 5: Depletion of WDR81 results in accumulation of tetherin in vesicular compartments in both the presence and absence of Vpu. A) WDR81 and tetherin expression was assessed by immunoblotting control and WDR81 depleted CEMT4 cells ± Vpu. B) Tetherin levels from three independent experiments using cells in ‘A’ were quantified with Image J and normalized by loading. Histograms represent mean normalized tetherin signal ±SEM. *p = 0.05, **p = 0.01, ***p = 0.001. C) WDR81 expression was assessed by immunoblot in HeLa cell clones ± Vpu stably expressing Cas9 and two independent WDR81 specific sgRNAs. D) Confocal microscopy images of endogenous tetherin using cells in ‘C’. Cell edges in Vpu expressing cells are outlined in red. E) HeLa cells were depleted of WDR81 by transient expression of Cas9 and WDR81 sgRNA#1 and single cell cloned. Exogenous expression of 2×HA-WDR81 in these WDR81KO clones was analysed by immunoblot. F) Immunostaining of endogenous tetherin in control, WDR81KO clones and WDR81KO clones genetically complemented with exogenous WDR81 or 2×HA-WDR81. G) Immunostaining of tetherin and 2HA-WDR81 in genetically complemented WDR81KO cells (prior to blasticidin selection). All scale bars represent 10 µm.