An Essential Role of UBXN3B in B Lymphopoiesis Tingting Geng Et Al. This File Contains 9 Supplemental Figures and Legends

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An Essential Role of UBXN3B in B Lymphopoiesis Tingting Geng et al.

This file contains 9 supplemental figures and legends. a Viral load(relative)

Serum TNF-α b +/+ Serum IL-6 Ubxn3b Ubxn3b-/- Ubxn3b+/+ 100 100 ** Ubxn3b-/- **

10 10 IL-6 (pg/ml)IL-6 GM-CSF (pg/ml)

1 1 0 3 8 14 35 0 3 8 14 35 Time post infection (Days) Time post infection (Days)

Serum IL-10 Serum CXCL10 Ubxn3b+/+ Ubxn3b-/- 10000 1000 +/+ -/- * Ubxn3b Ubxn3b

1000 100 IFN-γ (pg/ml) IFN-γ CXCL10 (pg/ml)

100 10 0 3 8 14 35 0 3 8 14 35 Time post infection (Days) Time post infection (Days) IL-1β (pg/ml)IL-1β

Supplemental Fig.s1 UBXN3B is essential for controlling SARS-CoV-2 pathogenesis. Sex- and-age matched littermates were administered 2x105 plaque forming units (PFU) of SARS-CoV-2 intranasally. a) Quantitative RT-PCR (qPCR) quantification of SARS-CoV-2 loads in the lung at days 3 and 10 post infection (p.i). Each symbol= one mouse, the small horizontal line: the median of the result. *, p<0.05; **, p<0.01, ***, p<0.001 (non-parametric Mann-Whitney test) between Ubxn3b+/+ and Ubxn3b-/- littermates at each time point.

Ubxn3b+/+ Ubxn3b-/-

Live Live 78.0 83.6 CD45+ CD45+ UV UV

CD45 94.1 CD45 90.9

FSC-A FSC-A FSC-A FSC-A

Mac Mac 23.0 38.1 MHC II MHC II MHC CD11b Eso CD11b Eso 5.81 7.44

CD19, MHCII subset F4_80, CD11b subset CD19, MHCII subset F4_80, CD11b subset

Myeloid panel 70.1 65.6 94.2 51.1

CD19 F4/80 CD19 F4/80

Neu DC Neu DC 4.87 1.02 16.2 2.24

CD11b, Ly-6G subset CD11b, Ly-6G subset 94.9 83.3 Ly-6G Ly-6G MHC II MHC II MHC

CD11b CD11c CD11b CD11c

Live Live 82.3 76.5 CD45+ CD45+ 91.1 91.3 UV UV CD45 CD45

FSC-A FSC-A FSC-A FSC-A

Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2 Lymphoid panel 20.1 0.29 54.1 1.17 4.27 0.060 39.2 2.55 CD4 CD4 CD19 CD19

Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3 47.0 32.6 7.21 37.5 67.7 28.0 7.27 51.0

CD3 CD8 CD3 CD8

Supplemental Fig.s2 Dysregulated immune compartmentalization in Ubxn3b-/- lung. The graphs illustrate the gating strategy of different immune populations in the lung at day 35 post SARS- CoV-2 infection (related to Fig.2f,g). Mac: macrophage, Neu: neutrophil, DC: dendritic cell, Eso: eosinophil. CD3+ T, CD8+ T, CD4+ T, CD19+ B. Ubxn3b+/+ Ubxn3b-/-

40x

WP RP

200x A MZ MS MS

Supplemental Fig.s3 Splenic atrophy in Ubxn3b-/- mice. The figures show H&E staining of spleens on day 35 after SARS-CoV-2 infection. The red arrow head indicates reduced cell density in the marginal zones of white pulps. The green circles likely show the myeloid clumps in red pulps. WP: white pulp, RP: red pulp, A: central arteriole, MS: marginal sinus, MZ: marginal zone, T: trabecula. Sting +/+ Sting-/- a Q1 Q2 Q1 Q2 28.1 0.38 29.4 0.62

Q4 Q3 Q4 Q3 Comp-APC-A :: CD19 :: Comp-APC-A CD19 :: Comp-APC-A 28.9 42.7 17.2 52.8

Comp-PE-Dazzle594-A :: CD3 Comp-PE-Dazzle594-A :: CD3

Neu Neu 61.5 46.2 Blood Blood cell

CD11b, Ly-6G subset CD11b, Ly-6G subset 37.6 52.5 Comp-Alexa Fluor 700-A :: Ly-6G :: 700-A Fluor Comp-Alexa Ly-6G :: 700-A Fluor Comp-Alexa Comp-APC-Fire750-A :: CD11b Comp-APC-Fire750-A :: CD11b

Mono Mono 15.9 13.0

CD11b, CD115 subset CD11b, CD115 subset 82.9 85.6 Comp-BVio711-A :: CD115 :: Comp-BVio711-A CD115 :: Comp-BVio711-A

Comp-APC-Fire750-A :: CD11b Comp-APC-Fire750-A :: CD11b b c + % ofCD45 % Cell counts / ml

Supplemental Fig.s4 STING is dispensable for steady-state hematopoietic homeostasis. a) The gating strategy, b) The percentage and c) counts of each blood cell type in Sting+/+ and Sting-/- littermates, quantitated by flow cytometry. Each symbol=one animal. The horizontal line indicate the mean of the results. RBC: red blood cell, WBC: white blood cell, CD19+: B cell, CD3+ : T cell, Neu: neutrophil, Mono: monocyte. a B cell T cell Neu Mono

Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2 0.015 4.51E-3 7.54 0.058 0 0 0 0 BM > BM> WT +/+

Comp-PE-A :: 45_1 Comp-PE-A Q4 Q3 :: 45_1 Comp-PE-A Q4 Q3 :: 45_1 Comp-PE-A Q4 Q3 :: 45_1 Comp-PE-A Q4 Q3 0 100.0 3.04E-3 92.4 0 100 0 100 Ubxn3b Comp-PE-Cy7-A :: 45_2 Comp-PE-Cy7-A :: 45_2 Comp-PE-Cy7-A :: 45_2 Comp-PE-Cy7-A :: 45_2

Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2 0.052 0.10 17.0 0.23 0 0 0 0.016 BM >WT -/- Ubxn3b Comp-PE-A :: 45_1 Comp-PE-A Q4 Q3 :: 45_1 Comp-PE-A Q4 Q3 :: 45_1 Comp-PE-A Q4 Q3 :: 45_1 Comp-PE-A Q4 Q3 0 99.8 0 82.8 0 100 0 100.0

Comp-PE-Cy7-A :: 45_2 Comp-PE-Cy7-A :: 45_2 Comp-PE-Cy7-A :: 45_2 Comp-PE-Cy7-A :: 45_2 b Q1 Q2 Neu Mono 57.0 1.17 48.3 53.6 BM > BM> WT +/+ Q4 Q3 CD11b, Ly-6G subset Comp-APC-A ::CD19 Comp-APC-A 23.3 18.6 51.0 Comp-BVio711-A :: CD115 :: Comp-BVio711-A Comp-Alexa Fluor 700-A :: Ly-6G :: 700-A Fluor Comp-Alexa Comp-PE-Dazzle594-A :: CD3 Comp-APC-Fire750-A :: CD11b Comp-APC-Fire750-A :: CD11b Ubxn3b

Q1 Q2 Neu Mono 6.26 0.17 52.7 67.2 BM >WT -/-

Q4 Q3 CD11b, Ly-6G subset Comp-APC-A :: CD19 :: Comp-APC-A 60.5 33.0 46.7 Comp-BVio711-A ::CD115 Comp-BVio711-A Comp-Alexa Fluor 700-A :: :: Ly-6G 700-A Fluor Comp-Alexa

Ubxn3b Comp-PE-Dazzle594-A :: CD3 Comp-APC-Fire750-A :: CD11b Comp-APC-Fire750-A :: CD11b

Supplemental Fig.s5. UBXN3B plays a cell-intrinsic role in B lymphopoiesis. Irradiated wild type (WT, CD45.1 ) mice were transplanted with Cre+Ubxn3bf/f (CD45.2) bone marrow . The mice were then treated with tamoxifen (TMX) to delete Ubxn3b (designated Ubxn3b−/− BM–WT) or not (designated Ubxn3b+/+ BM–WT) in hematopoietic cells. a) The percentage of blood CD45.1 and CD45.2 cells 1.5 month after bone marrow transplantation (BMT). Over 99% B/Neu/Mono are CD45.2+, over 82% T cell are CD45.2+, indicating successful irradiation and reconstitution. b) The gating strategy of blood immune cells in BMT mice (related to Fig.4). B: CD19 + B cell, T : CD3+ T cell, Neu: neutrophil, Mono: monocyte. a B cell T cell Neu Mono +/+ WT WT BM> Ubxn3b -/- WT WT BM >Ubxn3b b +/+ WT WT BM > Ubxn3b -/- WT WT BM >Ubxn3b

c WT BM→Ubxn3b−/− −/−

WT BM→Ubxn3b + % ofCD45 % Cell counts/ ml

Supplemental Fig.s6. UBXN3B plays a cell-intrinsic role in B lymphopoiesis. Irradiated Cre+Ubxn3bf/f and Ubxn3bf/f (CD45.2) mice were transplanted with wild type (WT, CD45.1) bone marrow . The mice were then treated with tamoxifen (TMX) to delete Ubxn3b (designated WT BM–Ubxn3b−/−) or not (designated WT BM–Ubxn3b+/+) in non-hematopoietic cells. a) The blood CD45.1 and CD45.2 cells 1.5 month after bone marrow transplantation (BMT). Over 99% B/Neu/Mono are CD45.1+, over 92% T cell are CD45.1+, indicating successful irradiation and reconstitution. b) The gating strategy of blood immune cells in BMT mice. c) The count and percentage (relative to CD45+) of each blood cell type in BMT mice at 15 days after induction of Ubxn3b deletion by tamoxifen (TMX), quantitated by flow cytometry. *, P<0.05; **, P<0.01, ***, P<0.001 (two-tailed Student’s t-test). Each symbol=one animal. The horizontal line indicate the mean of the results. RBC: red blood cell, WBC: white blood cell, B: B cell, T : T cell, Neu: neutrophil, Mono: monocyte. +/+ a Ubxn3b Ubxn3b-/-

CD19+ B CD19+ B 14.1 16.8 2.19 3.28 Comp-APC-A :: CD19 :: Comp-APC-A CD3, CD19 subset T CD19 :: Comp-APC-A CD3, CD19 subset T Comp-APC-A :: CD19 :: Comp-APC-A CD19 :: Comp-APC-A 83.6 1.65 96.4 0.78

Comp-PE-Alexa 594-A :: CD3 Comp-APC-Cy7-A :: IgD Comp-PE-Alexa 594-A :: CD3 Comp-APC-Cy7-A :: IgD

Neu Mono Neu Mono 56.3 24.1 53.8 35.2 BM BM Mature cell

CD11b, Ly6G subset CD11b, Ly6G subset Comp-BV711-A :: CD115 :: Comp-BV711-A 40.8 CD115 :: Comp-BV711-A 42.4 Comp-Alexa Fluor 700-A :: Ly6G :: 700-A Fluor Comp-Alexa Ly6G :: 700-A Fluor Comp-Alexa Comp-FITC-A :: CD11b Comp-FITC-A :: CD11b Comp-FITC-A :: CD11b Comp-FITC-A :: CD11b

b Ubxn3b+/+ Ubxn3b-/-

B220 B220 68.8 40.5

Dump Dump Comp-FITC-A :: dump :: Comp-FITC-A dump :: Comp-FITC-A 19.7 B220 :: Comp-BV650-A 10.2 B220 :: Comp-BV650-A

FSC-A FSC-A FSC-A FSC-A

D-F A-C' C C' D-F A-C' C C' 76.2 23.6 4.2917.7 36.4 61.7 6.444.81

Comp-PE-A ::BP-1 Comp-PE-A A B ::BP-1 Comp-PE-A A B Comp-BV650-A :: B220 :: Comp-BV650-A 44.5 31.9 B220 :: Comp-BV650-A 69.6 17.6 BM cell BM B linage

Comp-APC-A :: CD43 Comp-PerCP-Cy5-5-A :: CD24 Comp-APC-A :: CD43 Comp-PerCP-Cy5-5-A :: CD24

E F E F 24.1 19.2 Pre-Pro B 19.3 14.9 Pre-Pro B 25.6 40.1

D D Comp-BV421-A :: IgM :: Comp-BV421-A IgM :: Comp-BV421-A

51.3 CD93 :: Comp-PE-Cy7-A 60.3 CD93 :: Comp-PE-Cy7-A

Comp-APC-Cy7-A :: IgD Comp-PE-Alexa 594-A :: CD19 Comp-APC-Cy7-A :: IgD Comp-PE-Alexa 594-A :: CD19 Supplemental Fig.s7 UBXN3B is essential for early B lymphopoiesis in the bone marrow. The gating strategy for a) terminally differentiated (related to Fig.5a) and b) B lineage subsets in the bone marrow of Ubxn3b+/+ and Ubxn3b-/- littermates (related to Fig.5b). Fraction A: Pre-pro-B ( pre- progenitor B), Fraction B: pro-B (progenitor B), Fraction C: pre-BI, Fraction C’: large pre-BII, Fraction D: small pre-BII, Fraction E: immature B, Fraction F: recirculating mature B. T : T cell, Neu: neutrophil, Mono: monocyte. Ubxn3b+/+

Lin- GMC LSK 12.7 15.2 3.54

ST-HSC 59.2 cKit Flt3 FSC-A LT-HSC 39.7

Lin Sca-1 Sca-1

CD127, cKit subset 1.97 CLP 82.7 GMP 15.7 cKit

Count MEP CMP CD16/32 7.96 17.8

CD127 Sca-1 CD34

Ubxn3b-/-

Lin- GMC LSK 9.37 16.9 3.26

ST-HSC 32.2 cKit Flt3 FSC-A LT-HSC 66.6

Lin Sca-1 Sca-1

CD127, cKit subset 1.08 CLP 75.4 GMP 18.3 cKit

Count MEP CMP CD16/32 8.69 23.3

CD127 Sca-1 CD34

Supplemental Fig.s8 Bone marrow stem cells and CLPs are modestly reduced in Ubxn3b-/- mice. The gating strategy for hematopoietic stem cells (HSC, Lin-, Sca1+ Kit+) and lineage progenitors (related to Fig.5c) in the bone marrow of Ubxn3b+/+ and Ubxn3b-/- littermates. ST-HSC; short-term HSC, LT-HSC: long-term HSC, CLP: common lymphoid progenitor, CMP: common myeloid progenitor, GMP: granulocyte-macrophage progenitor, MEP: megakaryocyte-erythrocyte progenitors. a Pax5 +/+ 500 Ubxn3b Ubxn3b-/- 400

300

200

100 Relative mRNA expression Relative mRNA expression Relative mRNA expression 0 I I I B B B B T o u B B II T o I B T o e n e o-B -B n eu e n ro- ro- N r ro- N r Neu -P P Pre- tur Mo P Pre- Mo Pro-B Pre-BI tu Mo a e-P Pre-B Pre M Pr Mature B Pre-Pro-B e Ma Large Pre-BII Large Pre Larg Mef2c 3 Ubxn3b+/+ Ubxn3b-/- 2

1 Relative mRNA expression Relative mRNA expression 0 B B I o I o - - -BI T n eu BI B T eu -B -B B T -BI re B -BI e e on e e N e- N ro ro r Neu -Pro Pro Pr Mo Pro-B Pr tur Mono P Pre-BI re-BII tu M e atu Pre a e-P P M e Pr Pre-Pro-B g M Pr Ma r rge Large Pr La La Cebpa Runx1 3 Ubxn3b+/+ 8 Ubxn3b+/+ -/- Ubxn3b -/- 6 Ubxn3b 2

1 2 * *

Relative mRNA expression 0 0

B B II B II B - - -BI T no eu -BI B T -BI T re B N e - re Neu e re Neu -Pro Pro Pre Mo Pro-B Pr re tu Mono Pro-B Pr re-B tu Mono e atu r M e P Ma e P Ma P Pre-Pro-B rg Pre-Pro-B rg Large Pre-BII La La Rag1 250 Ubxn3b+/+ 200 Ubxn3b-/-

150

100 50 *

Relative mRNA expression 0 * I I -B T o e-B -BI re B Neu Pr re Mono Pr atu e P M Pre-Pro-B rg La b Cd19 +/+ Il7ra 90000 Ubxn3b 5 Ubxn3b+/+ 80000 -/- Ubxn3b -/- 70000 4 Ubxn3b 60000 3 50000 40000 2 30000 20000 1 10000

Relative mRNA expression 0 0

I I I I o I II u B T B B B T n eu B B T e -BI e B ro- ro- -BI re B N ro- -B re B N ro-B r Neu re re u Pro-B P Pre- u Mono P P Pre- Mo P Pre- t Mono - Pre at P P e M Matu Ma Pre Pre- rge Pre-Pro-B rge Larg La La Supplemental Fig. s9 qRT-PCR quantification of transcription factors and surface markers in steady-state Ubxn3b+/+ and Ubxn3b-/- littermates. The bone marrow cell compartments were sorted by flow cytometry and the mRNA expression of a) transcription factors and b) cell surface markers was quantitated by qRT-PCR and normalized to a house keeping gene, Actb. Each symbol=one animal. The horizontal line indicates the mean of the results. *, P<0.05; **, P<0.01, ***, P<0.001 (two-tailed Student’s t-test).