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. 2019 Jun 3;38(13):e100926. doi: 10.15252/embj.2018100926

Figure EV1. Quality control of GBP ablation in THP‐1 cells.

Figure EV1

  1. Fold‐change of mRNA of GBP1‐7 compared to hypoxanthine phosphoribosyltransferase 1 (HPRT1) in PMA‐differentiated THP‐1 cells (left) and primary MDMs (right) following treatment with IFNγ (50 IU/ml). Mean (red bar) ± SEM of n = 14 experiments (THP‐1) or n = 6 experiment (MDMs) shown; n.d. not detected.
  2. Immunoblots from differentiated THP‐1 WT or MDMs treated with IFNγ (50 IU/ml) or left untreated. Images represent n = 3 independent experiments.
  3. qRT–PCR measurement of silencing of expression of the indicated GBPs in IFNγ‐primed THP‐1 cells transfected with siRNA against GBP1‐5. mRNA fold‐change (mean ± SEM; n = 3 independent experiments) normalized to HPRT1 as percentage of cells transfected with non‐targeting control (CTRL) siRNA is indicated.
  4. Immunoblots from indicated THP‐1 cells treated with IFNγ. Images represent n = 3 independent experiments.
  5. qRT–PCR measurement of GBP1‐5 expression in THP‐1 and THP‐1 ΔGBP1 cells treated with IFNγ plotted as fold‐change to HPRT1. Mean ± SEM plotted from n = 3 experiments; n.d. not detected.
  6. RT–PCR amplification of GBP1‐5 coding sequences (CDS) from indicated THP‐1 cells treated with IFNγ. Yellow arrowhead indicates the truncated GBP1 CDS in the ΔGBP1 cells.
  7. Sequencing results showing loss of GBP1 coding region. Top: Needleman‐Wunsch alignment of GBP1 sequence from THP‐1 ΔGBP1 and GBP1 transcript sequence NM_002053.2 showing the deletion in knockout cells. Bottom left: GBP1 CDS with deletion highlighted in red. qRT–PCR primer binding sites marked in blue and bold letters and amplicon marked in blue. Bottom right: Summary table of sequencing result of GBP1‐5 in THP‐1 ΔGBP1 cells confirming loss of GBP1 and wild‐type coding sequences of GBP2‐5.